È, JO if4
CONSELHO NAC'ONALDE DESENVOLVIMENTOCIENTÍFICO E TECNOLÓGICO
IV JAPAN-BRAZIL SYMPOSIUMON SCIENCE AND TECHNOLOGY
Vol. II — NEUROPHYSIOLOGY - IMUNOLOGYBIOTECHNOLOGY - LIMNOLOGYCITRUS PESTS - BIOMEDICAL ENGINEERING
Academia de Ciências do Estado de S. Paulo1984
The pr int ing of t h i s Proceedings wassupported by:CÕN5EIH0 NACIONAL DE DESENVOLVBCNTO
CIBnTFICO E flEOJOLOGICOAnd
ASSOCIATION OF THE JAPAN SHIPBUILDINGINDUSTRY FOUNDATION
PREFACE
Started in 1979, the Japan-Brazil Symposium on Science
and Technology, the fourth to be met this year, has fulfilled
its main aim, namely, to strengthen the bilateral cooperation
between scie*\ s ts of both countries.
In fa.* ; everal research groups develop joint coopera-
tive proje / is consequence of the above Symposium.
The fourth Japan-Brazil Symposium on Science and Tech-
nology gathrs large number of participants, with equally la_r
ge number T invited papers and contributed papers indicating
an increa• ng interects in the meeting. A keen expectation to
interchange recent developments in Science and Technology
with visit, rs is quite apparent.
The Symposium Committee express its gratitude to every
one that contributed for the success of the Symposium.
or *
CONTENTS
PREFACE
NEUROPHYSIOLOGY
DUPLEX SYSTEM IN THE RETINA OF THE TERRESTRIAL AND MARINEGASTROPODS - K. Tasaki, S. Suzuki and T. Nakaye - Dep. ofPhysiology, Tohoku University School of Medicine 1
INTRACELLULAR RECORDINGS AND MARKINGS IN THE EYE OF THEBRAZILIAN BEE MELIPONA QUADRIFASCIATA - Dora Fix Ventura,Randolf Menzel, John Manuel de Souza and Luiz C.M.Joaquim,USP and Freie Universitat Berlin 2
INTERRECEPTOR JUNCTION IN DOUBLE CONE OF CHICKEN RETINA -Ricardo L. Smith, Yozo Nishimura and Giuseppina Raviola ,Dep. de Morfologia, Esc.Paul.Medicina, School of MedicineKeio University, Tokio, Boston University Boston, USA .... 4
PECULIAR EFFECTS OF TEMTERATURE ON RETINAL SPREADING DE-:PRESSION (SD) - H.Martins Ferreira and R.J.do Carmo, Ins.de Biofísica, UFRJ, Rio de Janeiro 6
PHYSIOLOGICAL AND MORPHOLOGICAL PROPERTIES OF THE EXPAN-DED IPSILATERAL RETINOGENICULATE PROJECTION IN NEONATALLYONE-EYE-REMOVED ALBINO RATS - Yutaka Fukuda, Tesuya Shirokawa and Chie-Fang Hsiao, Dep. of Neurophysiology, Insti-tute of Higher Nervous Activity, Osaka University MedicalSchool, Kita-ku, Osaka, 530, Japan 9
NORMAL AND ABNORMAL DEVELOPMENT OF RETINAL GANGLION CELLPOPULATIONS - Rafael Linden, Dep. de Neurobiologia, Inst.de Biofísica, UFRJ, Rio de Janeiro 17
QUANTITATIVE ANALYSIS OF THE DISTRIBUTION OF THE RETINALGANGLION CELLS IN AMAZON RODENTS - Luiz CL.Silveira,Cristovam W.P.Diniz and Eduardo Oswaldo Cruz 19
THE NASOTEMPORAL DIVISION OF THE OPOSSUM'S RETINA - J.N.Hokoç and L.G. Gawryszewski, Dep. de NeuTobiologia, inst.de Biofísica, UFRJ, Rio de Janeiro 24
BIOTECNOLOGY
BRAZILIAN MEASLES CAM-70 VACCINE PRODUCTION: A TECHNICALCOOPERATION TASK - A.Homma and J.R.S.Chaves, T.Otsuka ,Oswaldo Cruz Foundation, Diseases of Osaka University.... 26
AN APPROACH TO MALARIA VACINE - JOINT INTEREST BETWEEN PARASITE IMMUNOLOGY AND CANCER IMMUNOLOGY, S.Suzuki, S.WakaáS. Nakazawa, T.Miyagani, I.Igarashi, M.Adachi.S.Asakura ,A.Sakanoue, Gunna University School of Medicini, JapanImnuno Res. Laboratories Co.Ltd 30
EXPRESSION AND SECRETION OF Bacillus sp. o-AMYLASE GENEIN Hscherichia coli and Basillus subtilis - C.J.Ulhoa, M.B.N.S. Souza, S.M.R. Teixeira, S.L.B. Silva, I.S.Pereira,M.C.N.D. Menezes, V.M.Q.G. Lima and S.Astolfi Filho, Dep.de Biologia Celular, Un. de Brasilia 32
SYNTHESIS AND SECRETION OF MOUSE PANCREATIC aAMYLASE BYYEAST - S.Astolfi Filho, E.Vicente Galembeck, A.C.Schen-berg Frascino, Inst. de Ciências Biológicas, Un. BrasiliaInstituto de Química, Un. de São Paulo 33
GENETICS OF AMYLOGLUCOSIDADE PRODUCTION IN Aspergillus ni-ger and Aspergillus awamori, R.Bonatelli Jr, G.Umbuzeiro ,M.Masiero, A.Vialta e M.R.Calil, Dep. of Genet, and Evolu-tion, Inst. of Biology, UNICAMP VJ4
PREPARATION OF 3SS-METHIONINE - E.Rondinelli, N.MoussatchiJ.F.Oliveira Carvalho, M.G.C.Carvalho, R.S.Moura Neto, D.S.Lobo, I.C.Frugulhetti, R.Silva e M.A.Rebello - Inst. deBiofísica da UFRJ
MOLECULAR BIOLOGICAL APPLICATIONS TO VIRAL DIAGNOSIS - H. ^•G. Pereira, Dep. of Virology, Inst, Oswaldo Cruz (43
RECOMBINANT DNA TECHNOLOGY FOR PRODUCTION OF USEFUL SUBS-TANCES BY MICROORGANISMS - Teruhiko Beppu, Fac. of Agric.University of Tokyo 44
A BRAZILIAN EXPERIENCE ON PLANT CELL AND TISSUE CULTURE:A TOOL FOR PLANT IMPROVEMENT - O.J.Ci-ocomo, ESALQ/USP; A.Natal Gonçalves, Dep. de Silvicultura, ESALQ/USP 54
PURIFICATION AND CHARACTERIZATION OF NEW .RESTRICTION ENDO-NUCLEASES, A.K.Cruz, G.Kidane, M.Q.Pires and CM.Morel,Inst. Oswaldo Cruz, R.Janeiro SS
SURFACE ANTIGENS OF TRYPANOSOMA CRUZI RECOGNIZED BY MONO-CLONAL ANTIBODIES, M.J.M. Alves, G.Abuin and W.Colli, Dep.de Bioquímica, Inst. de Química, USP 56
DEFINITION OF RELEVANT SURFACE ANTIGENS INVOLVED IN THE INTERIORIZATION OF TRYPANOSOMA CRUZI IN HOST CELLS, M.V.Ar- .-">ruda, B. Zingales and W-Colli, Inst. de Química, USP ... [ 577
A COMPARATIVE BIOCHEMICAL ANALYSIS OF ISOLATES OF LEISHMA-NIASIS FROM THE STATES OF BAHIA AND RIO DE JANEIRO - R.S.Pacheco, H. Momen, G. Grimaldi Jr. M.C.A. Marzochi and C.M.Morel, Inst. Oswaldo Cruz, FIOCRUZ, R. Badaro, Un.Fede-ral da Bahia 58
PERMANENCE AND VARIATION OF ENZYME POLYMORPHISM IN CLONESOF Trypanosoma cruzi STRAIN Y*- Amilcar Tanuri, Darcy F.de Almeida, Inst. de Biofísica, CCS, Un.Federal do R.Janei^ro 64
PURIFICATION OF HUMAN AMNION INTERFERON BY CIBACRON BLUE-AGAROSE CHROMATOGRAPHY - C.Gonzaga, R.R.Golgher, P.C.P.Ferreira, E.G.Kroon, Dep. of Microbiology, Int.CiênciasBiológicas da UFMG 71
CHARACTERIZATION OF BOVINE LEUKEMIA VIRUS IMMUNOGENS - A.L.T.O. do Nascimento and T.Higuchi, Inst. de Química/USP.. 72
CHARACTERIZATION AND PURIFICATION OF STRUCTURAL COMPONENTSOF AVIAN LEUCOSIS (EXOGENOUS AND ENDOGENOUS) VIRUS - J.M.M.S. Felippe, Inst. Química, USP, C.Romero, EMBRAPA, SC. ,T. Higuchi, Inst. de Química, USP 73
IMMUNOLOGICAL CHARACTERIZATION OF STRUCTURAL PROTEINS OFTHE AVIAN TUMOUR VIRUS - J.M.M.S. Felippe, T.Higuchi, Dep.of Biochemistry, USP 74
OBTENTION OF SHEEP ANTI-RABBIT IgG : PRECIPITATING SYSTEMOF RADIOIMMUNOASSAY - H. Ogata, Inst. de Química, USP, T. -Higuchi, Inst. de Química, USP ( 75
PERPECTIVES ON AVIAN AND BOVINE LEUKEMIA VIRUS IMMUNOLOGI-CAL STUDIES - Tomoko Higuchi, Julia M.M.Souza, Zélia MariaNogueira, Hiroe Ogata, Dep. of Biochemistry, USP f 76
REGULATION OF TREHALOSE METABOLISM IN Saccharomyces - ANAPPROACH TO THE IMPROVEMENT OF TECHNOLOGICAL PROCESSES, A.D.Panek, V.L.A. Paschoalin, A.C. Panek, Inst. of Chemistry,UFRJ, Rio de Janeiro / 87
NITROGEN FIXATION RESEARCH IN BRAZIL - AN OVERVIEW - Joha-nna Dobereiner, Unidade de Apoio ao ProgTama Nacional dèPesquisa de Biologia do Solo- EMBRAPA ; 96
NITROGEN FIXATION RESEARCH IN BRAZIL - ON OVERVIEW -Johanna Dobereiner, UAPNPBS- EMBRAPA, Rio de Janeiro 97
IMUNOLOGY - PARASITIC DISEASES
SKIN TEST WITH PURIFIED SAWADA ANTIGEN IN BRAZILIAN INDIANSWITH OCULAR ONCHOCERCIASIS - Milton M.Hida, Fac. Med. de 'Botucatu, José J. FeTraroni, Un. do Aaazonas, JCumiko SatoSchool of Medicine, Japan 106
CORRELATION BETWEEN MICE ACUTE TRYPANOSOMA CRUZI INFECTIONAMD PLASMATIC LEVEL OF LAPID PEROXIDE - Paulo E.Soares Pa-lhares, Inst. Oswaldo Cruz, Pedro Fontana Jr., Inst. Nacional de Cancer, Rio de Janeiro 113
GRANULOMATOUS FORMATION IN MICE LIVER FOR ABSORTION OF S.MANSONI WORMS, AFTER TREATMENT WITH OXAMINIQUINE - A. Magalhães, M.E.Bezerra Melo, Fundação Oswaldo Cruz, N.TellesPontes, Un. Federal de Pernambuco 119
MALARIA IN HUMAITA COUNTRY, AMAZONAS STATE, BRAZIL, XXIXSOME COMPARATIVE EPIDEMIOLOGIC ASPECTS IN 1976, 1979 AND1983 - D.A.Meira, P.R. Curi, B.Barraviera, Fac. de Medici-na de Botucatu 136
MALARIA IN HUMAIRA COUNTRY, AMAZONAS STATE, BRAZIL, XXXV-INQUIRY INTO THE EPIDEMIOLOGY OF THE GENERAL POPULATIONUSING PASSIVE HEMAGGLUTINATION - D.A. Meira, J.Marcondes ,R.P.Mendes, Fac. Med. Botucatu, UNESP, A.B.El-Khoury, V.Cu-ry, Hosp. do Serv.Público, P. Rui, SUCAM de Humaitá, Minis.da Saúde 148
MALARIA IN HUMAITA COUNTRY, AMAZONAS STATE, BRAZIL, XXXIV-IMMUNE RESPONSE OF PATIENTS WITH Plasmodium Falciparumaccording to gametocytes - D.A.Meira, P.R.Curi, J.Marcon-des, Fac. de Med. Botucatu, E.S. Matsuoka, M.A.Fabrin, A.B.El Khoury, Hosp. do Serv. Público, N.G.S. Motta, Inst. Blsi."co de Biologia Médica e Agrícola da UNESP 152
MALARIA IN HUMAITA COUNTRY, AMAZONAS STATE, BRAZIL, XXXII-FREQUENCE OF THE HLA ANTIGEN IN THE GENERAL POPULATION ANDPATIENTS - D.A. Meira, J.PelegrinO'Jr., J. Marcondes, Fac.Medicina de Botucatu, UNESP, K.Tsuji, Tokai University, E.S.Matsuoka, E.E.Haida, A.B. El-Khoury, Hosp. do ServidorPúblico 162
MALARIA IN HUMAITA COUNi'RY, AMAZONAS STATE, BRA7XL, XXXVIIIDETERMINATION OF HLA ANTIGENS IN THE FAMILY OF PATIENTS PRONE TO CEREBRAL MALARIA - D.A.Meira, J.Pellegrino Jr., J.Marcondes, Fac. de Med. Botucatu, UNESP, K.Tsuji, Tokai Un.,E.S.Matsuoka, A.B.El-Khoury, E. Haida, Hosp. do Servidor Público 172
LIMNOLOGY
JAPAN-BRAZIL COOPERATIVE STUDY ON BIOLOGICAL PRODUCTIVITYOF RIO DOCE VALLEY LAKE SYSTEM - Y. Saijo, Nagoya Univ. J.G. Tundisi, Un. Federal de São Carlos 177
JAPAN-BRAZIL COOPERATIVE STUDY ON BIOLOGICAL PRODUCTIVITYOF RIO DOCE VALLEY LAKE SYSTEM - Y.Saijo, Water Res. Inst.Najjoya Un., J.G.Tundisi, Un. Federal de S.Carlos 179
DESERTIFICATION AND DEFORESTATION - Jiro Kondo, Universityof Tokyo 193
SPATIAL DISTRIBUTION OF A BIVALVE POPULATION (Diplodon de-locontus expansus) IN A SMALL TROPICAL RESERVOIR WITH EM-PHASIS ON DISTRIBUTION NEAR THE BASES OF TREES - R.Henry,C A . Si mão, Dep. of Zoology, Un. Est. Paul., UNESP 210
THE ECOSYSTEM LAGOA CARIOCA: MAJOR CONSEQUENCES OF ITSTHERMAL BEHAVIOR DURING THE WINTER AND SUMMER - Y.A.R.Bar-boa, Dep. of Gen.Biology, UFMG, J.G.Tundisi, Dep. Biolog.Sciences, UFSCar 227
FILTER-FEEDING RATES AND SELECTIVITIES OF TWO CICHLIDS ONTHE ZOOPLANKTON OF BROA RESERVOIR - " X.Lazzaro, Laborato-ry of Limnology, UFSCar 228
EUTROPHICATION IN SAO PAULO STATE RESERVOIRS - J.G. Tundi-si, T.Matsumura, Lab. of Limnology, UFSCar 231
LIMNOLOGY AND ECOLOGY OF FURNAS RESERVOIR - J.G.Tundisi ,Kozo Hino, Raoul Henry, J.G.Gentil, Dirceu M.Ribeiro, UFSCar,UNESP, Botucatu, Furnas Hydroelectric Company 232
ZOOPLANKTON COMPOSITION OF FURNAS RESERVOIR - T.Matsumura,O.Rocha, Laboratory of Limnology, UFSCar 233
THE LIFE CYCLE AND PRODUCTION OF Daphnia gessneri - 0.Ro-cha, T.Matsumura, Laboratory of Limnology, UFSCar 234
GEOMORPHOLOGICAL AND LIMNOLOGICAL PROCESSES AS A BASIS FORLAKE TYPOLOGY - MIDDLE RIO DOCE VALLEY - J.G.Tundi?;, Laboratory of Limnology, UFSCar, M.Regina M.de Meis, UFRJ ... 235
ECOLOGICAL MODELS AS A WAY FOR ORGANISING CONDENSING ANDTRANSMITING KNOWLEDGE - Mauricio V.Kritz, Labor, de Comput.Científica, Rio de Janeiro 236
THE ECOLOGICAL DISTRIBUTION OF FISH AND THEIR FOOD IN THEESTUARINE R2GI0N OF ÕHTA RIVER IN HIROSHIMA PREFECTURE, JAPAN - Tetsuo Sunaga, Fac. of Education Kagawa University.. 238
CITRUS PESTS
PARASITISM OF PUPAE OF Anastrepha spp. (Dip.: Tephritidae)BY Doryctobracon areolatus (Szêpligeti, 1911) (Hynu: Braconidae) IN CITRUS AND TROPICAL FRUITS - A.S.Nascimento, A.L.M. Mesquita, Centro Nacional de Pesquisa de Mandioca eFruticultura, EMBRAPA, R.A. Zucchi, Dep. of Entomology,ESALQ/USP 239
EFFECT OF STERILANTS ON Ceratitis capitata (WIEDEMANN.1924)(DIPTERA TEPHRITIDAE), ITS SYMBIOTES AND THE PREDATOR Chry-soperla externa (HAGEN, 1861) (NEURCPTERA:CHRYSOPIDAE) - C.Alberto Perez, Dep. of Entomology, O.Nakano, ESALQ 247
STUDY OF CONTINUOUS FERMENTATION FOR OBTAINING BACTERIAL INSECTICIDE TO CONTROL AGRICULTURAL PESTS - D.M.F. Capalbo,1.0. Moraes 248
EFFECT OF AVERMECTIN IN THE CONTROL OF Phyllocoptruta 01ei_vora (ASHMEAD 1879) (ACARI-ERIOPHYIDAE) AND Brevipalpusphoenixis (GEIJSKES, 1939) (ACARI-TENUIPALPIDAE) ON CITRUS-O.Nakano, C.Omoto, M.J. Fornazier, Dep.Entomology, ESALQ.. 256
BIOENGINEERING
DEVELOPMENT OF EMISSION COMPUTED TOMOGRAPHY IN JAPAN - E.Tanaka, National Inst. of Radiological Sciences / 266
ELECTRICAL STIMULATION IN EXPERIMENTAL SPEUDARTHROSIS - A.E. Rodriguez Fuentes, S. Mascarenhas , Ir.st. de Física eQuímica de S.Carlos, USP 278
SPACE SCIENCE AND TECHNOLOGY
DATA PROCESSOR UNIT DESIGN AND DEVELOPMENT FOR JAPANESESCIENTIFIC SATELLITE - T.Hayashi, Inst.of Space, H. Oya,Tohoku University 279
PROGRAM MANAGEMENT, SYSTEMS DESING AND INTEGRATION OF JAPANESE SCIENTIFIC SATELLITES - Toaonao Hayashi, Inst. of Space and Astronautical Science, Harumitsu Yaaaaoto, HironoriHara, Space Developaent Division, NEC Corporation 298
RECENT DEVELOPMENT OF ATTITUDE CONTROL SYSTEMS FOR JAPANE-SE SCIENTIFIC SATÉLITES - Toaonao Hayashi, Ryojiro Akiba ,Keiken Ninomiya, Inst. of Space and Astronautical Science,Hironori Hara, Haruaitsu Yaaaaoto, Junichi Aoyaaa, SpaceDevelopment Div., NEC Corporation 315
HIGH SPEED SATELLITE IMAGE PROCESSING SYSTEM FOR OPTICAL
SENSOR IMAGE'S DISTORTION CORRECTION AND SAR DATA PROCESSING
Nobuhiko Mori, Space Dev. Dev. NEC Corpor. , JAPAN, Hi tosh i
Nohmi, Guidance and E lec tro -Opi tc s D i v i s i o n , NEC Corpora-
t i o n , Japan 332
RECENT ACTIVITIES ON THE SCIENTIFIC BALLOONING IN JAPAN -J. Nishimura, H. Hirosawa, Institute of Space and Astro-nautical Science, Tokyo, Japan 344
OVERVIEW OF SPACE RESEARCH PROGRAM IN JAPAN - Kunio Hirao,Tamiya Nomura, The Inst. of Space and Astron. Science ... 359
NEUROPHYSIOLOGY
Coordinator
Dr. HISS MARTINS FERREIRA
Brasilian Academy of Sciences and
Biophysics Institute, Fed. Univ.
of Rio de Janeiro
DUPLEX SYSTEM IN THE RETINA OF THE TERRESTRIAL AND MARINE
GASTROPODS
K. Tasaki, S. Suzuki and T. Nakaye
Department of Physiology, Tohoku University School of Medicine
The ERG and optic nerve discharge were recorded from the
optic nerve of nine species of land gastropods and six species
of marine gastropods. The intensity-amplitude curves and dark
adaptation curves of all land gastropods and four species of
marine gastropods were found to consist of two sections separated
by a sudden change of gradient. Intracellular recordings from the
retina of an African snail revealed twc types of photoreceptor
cells, sustained (S) and transient (T) types. Although the both
types of cells initiate spike potentials superimposed upon graded
receptor potentials, these two types of cells behave differently
in many respects: (1) The S cells generate sustaining impulses
which last as long as stimulus continues, but the T cells generate
only several impulses. (2) The threshold for impulses generation
is about 3 log units lower in S cells than in T cells. (3) S cells
recovers much more slowly from light adaptation then T.cells.
From these results the duplex system, which is equivalent to the
rod and cone systems in the vertebrate, has been suggested in the
gastropod visual system.
.1.
INTRACELLULAR RECORDINGS AND MARKINGS IN THE EYE OF THE BRAZILIAN
BEE NELIPONA QUADRIFASCIATA
Dora Fix Ventura, Randolf Menzel*, John Manuel de Souza and Luiz
Cláudio Martins Joaquim
Universidade de São Paulo and *Freie Universitat Berlin
Spectral sensitivity in the eleetrophysiological responses
of photoreceptors has been studied in a variety of arthropods. But
it is only in the fly and the bee that the identification of
spectral sensitivity classes with anatomically defined types in
the ommatidium has been established. It is also only in those two
species that the wiring of these receptor types with the cells in
the optic ganglia has been studied. In the case of the European
bee, there is, in addition, a large body of knowledge regarding
its complex behavior, with many studies of color and form vision.
No information, however, is available on how the highly developed
color vision system of the European bee compares with those of
phylogenetically related species and how it might have evolved
within the higher hymenopterans. The existence of over 200 species
of native stingless bees in Brazil living in a variety of
ecological conditions offers a wide range of possibilities for
comparative research.
The studies to be presented are concerned with the
determination of photoreceptor types in the Brazilian bee Melipona
quadrifasciata. This was done by means of electrophysiological
recording from individual retinular cells and morphological
identification of their axon terminals through marking with the
.2.
I i
í
fluorescent dye Lucifer yellow. A general morphological description
of optic lobes and brain of this bee, as well as of its neuron
types was done by means of staining methods and of Golgi
impregnation.
Xntracellular recordings were obtained in 224 photoreceptors.
The results show ultraviolet, green, and blue sensitive cells,with
peaks that fall close to those observed in the European bee. Apis
mellifera. Many recorded units also exhibited two and even three
sensitivity maxima, which points to the existence of strong
interactions between receptors. These interactions are most likely
due to electrical coupling. The morphological results show great
differences between the two species in the much larger corpora
pedunculata found in the Brazilian bee. The stainings of the
electrophysiologically identified units followed the pattern known
for the other species, i. e, green and blue receptor axons ending
in the lamina, and ultraviolet ones in the medulla. Many multiple
markings were also found, sometimes corresponding to the
electrophysiological results, other times without correspondence
to those identifications. The existence of dye coupling in the
absence of electrical coupling might explain this.
.3.
INTERRECEPTOR JUNCTION IN DOUBLE CONE OF CHICKEN RETINA
Ricardo Luiz Smith*, Yozo Nishimura** and Giuseppina Raviola***
•Departamento de Morfologia, Escola Paulista de Medicina, S.Paulo;
**Anatomy Dept., School of Medicine, Keic University, Tokyo, Japan
and * "Anatomy Dept., School of Medicine, Boston University,Boston,
USA
Until the past decade, it was assumed that photoreceptor cells
operate as individual independent light transducers whose signals
interact only at the level of the external plexiform layer. Quite
recently however, several workers showed that physiological
coupling occurs in various vertebrate species, between rods, rods
and cones, and cones. The anatomical substrate of this interaction
was mostly described near the receptor terminals. Some of these
interreceptor contacts have characteristics of gap junctions
(Raviola and Gilula, 1973s. Gap junctions between receptors
located in the inner segment were recognized in the rods (Gold and
Dowling, 1979), and some attachments between double cones of
chicken were evidenced by some of us (Nishimura, Smith and Shimai,
1981).
With conventional electron microscopy and freeze-fracture
technique we localized a peculiar junction between the two elements
of the double cones of the chicken retina. This contact is composed
of gap and intermediate junctions. In thin sections the plasma
membranes of the principal and accessory cones are seen to approach
each other closely, being separated by a cleft of 3 to 11 run; focal
gap junctions are interposed. A layer of fine filamentous material
.4.
is constantly present on the cytoplasmatic aspect of the plasma
membrane. The junction is located at the level of the myoid of the
principal cone and the perikaryon of the accessory cone, in close
proximity to the outer limiting membrane. In replicas of freeze-
fractured retinas the junction appears as a fascia composed by 8
to 9 ran junctional particles clustered as polygonal aggretates or
disposed as linear arrays. The particles remain associated with
the inner leaflet of the plasmalemma (P-face). The linear gap
junctions are straight, angular or branching, usually associated
with the polygonal gap strands.
In close proximity to the gap junctions the membrane matrix
appears devoid of intramembrane partidos.
The presence of gap junctions suggests that the two elements
of the double cones are electrically coupled.
References;
Gold G.H. and Oowling J.D. J. Neurophysiol. 42:292-310, 1979.
Nishimura Y., Smith R.L. and Shimai K. Cell Tissue Res.218:113-116,
1981.
Raviola E. and Gilula N.B. Proc. Nat. Acad. Sci. 70:1677-1681,1973.
.5.
t
-. k
HBÜUUMt EFFECTS CF UUWWIOC GN FEXDAL SPfEMttNG EBfESSXEM (SD)
H.Martins-Ferreira and R.J.do Can»
Instituto de Biofísica, UFRJ, Rio de Janeiro
Changes of temperature of the retina influence the velocity
of propagation and duration of various concomitants of SD such
as fields potentials, ionic translocations and light scattering
changes.
Usualy the decrease of temperature slows the various
parameters and angmting it, the effects are in reverse direction.
On the other hand we have shown that an abrupt decrease of
temperature is a very potent stimulus of the reaction while
slowly lowering it, leads to a progressive increase of threshold
for optical and mechanical stimuli.
However, when the variation of temperature is varied at
a slower rate, peculiar effects are observed which will be
described therein.
The experiments were performed in chick eye cups superfused
with Ringer solutions at 30°C,containing (in mM): NaCl 100,KC1 6,
MgS04 1.0, CaCl2 1.0, Na HcO3 30, NaHP03 1.0 and glucose 20. SD
was evoked by mechanical or optical stimuli. Double-barreled
microelectodes were used to measure field potentials and extra-
celular ionic activity in the various depths of the retina. As
it was shown (1), the wave shape and amplitude of the slow
voltage variations accompanying SD vary as the microelectrode is
.6.
displaced radially through the retinal layers,the characteristic
negative shift being of higher amplitude in the inner plexiform
layer but, in the distal part of the retina, the field potential
being positive and with smaller voltage. The study of the depth
profile of extracelular ionic activity changes duriny SD (2; have
shown decrease of Na+, Cl~ and Ca2+, but increase of K+, in fie
extracelular milieu, the changes of all these ions beiiv: maxima
in the inner plexifonn layer. Studies using extracelular markers
as a - naphthalenesulfonate and tetraethylanunonium ions(3),showed
mainly an increase of activity during SD, indicating a diminution
of extracelular space during the reaction and, again, such
variations being higher at the inner plexiform layer and almost
undetected in the distal region of the retina.
When the temperature is decresed at a rate of approximately
2°C every 15 minutes, from 30°C to 23°C, the experiments showed
that, during SD: a) the negative shift in the inner plexiform
layer, decreases progressively and, in the distal retina, the
voltage changes becoming negative and with higher amplitudes, b)
the Cl~ extracelular diminutions in the proximal retinal are less
intense but appears in the distal retina; on the other hand,the
K+ increase is smaller in the inner plexiform but augments in the
distal regions. Similar findings where detected with the extra-
celular marker ions which have less variation of activity in the
inner plexifonn but appears more intense in the distal regions.
All these different types of effects were reversible when
the temperature is returned to 30°C.
These experiments show differential actions of cooling
upon the nervous processes that form the various layers of the
.7.
retina. These influences are in the sense to halt* the full
development of SD in the proxiaal plexus but favouring its
appearance in distal parts- of the retina. How much such effects
are due to membrane characteristics, Metabolic or sinaptical
differences of the processes involved with SD, have to
be cleared up in future experiments.
R E F E R E N C E S
1 - Martins-Ferreira,H., do Cars», R.J., and Oliveira Castro, G.
de. An Acad.brasil.Cienc. 52, 188-189 (1980).
2 - Martins-Ferreira,H. In; The Brain and Behaviour of the fowl
ed. by T.Ookawa, Japan Scientific Press, Tokyo 1983, pp.317-333.
3 - do Can», R.3. and Martins-Ferreira,H. An.Acad.brasil.Cienc.,
52; 432-433 (1980).
.8.
PHYSIOLOGICAL A » MMFMLOGICAL PROPERTIES OF THE EXPANDED IPSILATERAL
RETIKOCEMICULATE PROJECTION 1* NEONATALLY ONE-EYE-REHOVED ALBINO RATS
Yutaka Fukuda, Tetsuya ShliJkawa and Chie-Fang Hsiao
Department of Ncttrophyslology, Institute of Higher Nervous Activity,
Osaka University Medical School, Klta-km, Osaka, 530, U
After unilateral eye renovai at birth the retiaogeuiculate projection
to cover «hole extent of the ipsilateral lateral genleulate nucleus
(LCM) la rats (1). lhe «panéid Ipsilateral projections have been shown to
make synaptic contacts with relay cells of the LCN. The present series of
experinents consists of three parts. First, In order to know how tbene
synapses function we nade single unit studies on response properties of relay
cells, especially on their receptive field properties and responses to single
optic chiasa shocks (2). Second part is concerned with which part of the
reanining retina the Ipsilateral projection originates from and in which size
classes are the ganglion cells as its origin (3). Thirdly, we counted
electron microscopically the number of optic fibers which project to the
ipsilateral optic tract (4). Ibis series of experiments led us to conclude
that the expanded ipsilateral retlnogeniculate projection functions to son*
extent and that about one fourth of the total number of optic fibers arc
recruited to join the ipsilatcral projection. Furthermore, it was suggested
that axons of medium-sized to large cells have rerouted lpsllsterally during
the course of development after unilateral eye enudcatlon.
I. PHYSIOLOGICAL PROPERTIES OF THE EXPANDED RETINOGENICULATE PROJECTION
In urcthan anesthetized albino rats with one eye enucleated shortly
.9.
after birth, single unit recording was made from relay cells of the LGN.
Response latencies to single optic chi?sm shocks were compared between the
LGNs ipsi- and contralateral to the remaining eye in enucleated rats (Fig.
1A). As a control, similar data are presented for normal rats in Fig. IB.
In contrast with a quite rare occurence of ipsilaterally activated cells in
the LGN of normal rats, in enucleated rat's LGN such cells were easily
sampled. Thus, most of these cells can be assumed to be innervated by the
expanded projection and their chiasm latencies were distributed widely in a
range from 1.2 to 7 msec. This latency distribution is quite similar to that
of cells in the contralateral LGN of both enucleated and normal rats, though
the mean latency was slightly longer.
In contrast with relatively normal range of optic chiasm latencies, the
relay cells that were innervated by the expanded projection had the following
properties in their visual responses. First, responses to stationary light
spots were significantly weaker than those of ceils innervated contra-
laterally. In Fig. 2 visual responses of three representative types of
receptive fields (OFF-phasic, ON-tonic and OtJ-OFF phasic) are compared
between the cells sampled from ipsi- and contralateral LGNs. Secondly,
receptive field center sizes were significantly larger than Chose of cells in
contralateral LGN. In Fig. 3 the size and location of receptive field centers
are plotted for each cell on a tangent screen placed in front of the
remaining eye. Furthermore, as can be noticed in Table 1, the sampling
frequency of various receptive field types in the ipsilateral LGN cells of
enucleated rats became similar to that of the samples in the contralateral
LGN of both enucleated and normal rats. This is in quite contrast with the
sampling frequency observed in the ipsilateral LGN cells of normal rats.
This change is probably because of the increase in sampling frequency of
phasic type cells in the ipsilateral LOW of enucleated rats.
.10.
Table 1.
Receptive field type
OFF-phasic
OFF-tonic
ON-phasic
ON-tonic
ON-OFF-phasic
total
12
5
31
16
4
68
Enucleated ratr
IPSI CONTRA
\
(17.6)
( 7.4)
(45.6)
(23.5)
( 3.6)
(100)
39
4
40
19
10
112
I
(34.8)
( 3.6)
(35.7)
(17.0)
( 8.9)
(100)
2
4
5
6
0
17
[jQriT.3 1
IPSI
I
(11.8)
(23.5)
(29.4)
(35.31
( 0 )
(100)
rats
contra
36
7
80
29
11
163
1
(22.1)
( 4.3)
(49.1)
(17.8)
( 6.7)
(100)
II. IPSILATERALLY PROJECTING GANGLION CEILS
After »•ssive injections of HRP into the ipsilateral optic tract near
the LGN we studied retrogradelly labeled ganglion cells in whole-mounted
retinas. Fig. 4 compares the distributions of HRP-labeled ganglion cells in
enucleated and normal rats. Two features were noticed in enucleated rats.
First, though the labeled cells tend to be concentrated in the ventrolateral
crescent area, they are also distributed in other retinal areas. Secondly,
the density of ganglion cells in the ventrotemporal crescent is much higher
in enucleated than in normal rats.
After localized injections of HRP within the LGN, soma areas of ganglion
cells were measured for the contra- and ipsilateral projections (Fig. 5). In
each case, the mean soma areas of cells sampled in lower nasal and temporal
retinas are compared between normal and enucleated rats. In the ipsilaterally
projecting cell group the soma size was consistently larger in thr
enucleates, whereas in the contralaterally projecting cell group it was
consistently smaller. These differences were statistically significant.
.1 1.
III. NUMBERS OF IPSI AND CONTRALATERALLY PROJECTING FIBERS
Using light and electron microscopy on the cross sectioned areas of the
optic tract, we estimated the number of ipsi- or contralaterally projecting
fibers in neonatally enucleated (HE) rats (Table 2). For comparison, the two
groups of fibers were also counted for rats with one eye enucleated in adult
(AE rats). The cross section area of the ipsilateral tract increased in
enucleated rats with a concomitant decrease of area in the contralateral
tract. In the ipsilateral tract of the enucleates, fiber density also
increased by three to four times so that the total number increased by about
35,500 fibers. On the contrary, «bout the same number of fibers were lost in
the contralateral tract.
Table 2.
Preparations Laterality Cross section Density
of OT to area of of fibers
remaining eye OT (an2) (M*~2)
Total number
of
fibers
AB rats
ME rats
CONTRA
IPSI
CONTRA
IPSI
0.354
0.365
0.110
0.093
0.254
0.294
0.1*7
0.151
330,600 (403,500) 117,000
(472,300)
•4,«00 (104,600) 9,300
(106,600)
336,000 (400,500) 15,300
(465,300)
261,100 (335,200) 44,100
(397,500)
REFERENCES
1. R.D. Lund, T.J.Cunningham and J.S. Lund, Brain Behav. Evol. 8 (1973) 51.
2. Y. Fukuda, I. Sumitomo and C.-F. Hsiao, J. Reurophyslol. 50 (1983) 46.
3. C.-F. Hsiao and Y. Fukuda, Brain Res. 301 (1984) 1.
4. T. Shirokawa, Y. Fukuda and T. Sugimoto, Exp. Brain Res. 51 (1983) 172.
.12.
A EVE ENUCLEATED
CONTRA IPSI
O 1 2 3 « S I 7 O 1 2 3 * S « 7
LATENCY FROM OX ( M K ) LATENCY FROM OX (mwc)
Pig. 1. Distribution of optic chlasa latencies in relay cells of the LGNs
Innervated by the contra- and ipsilateral projections in eye
enucleated and normal rats.
. 1 3 .
1PSI CONTRA
B
Lit - .1.1. • kl [
ON OFF ON OFF
'*« I sec
Fig. 2. Responses of LGN relay cells to stationary light spots.
Fig. i. Plots of receptive field centers of LGN relay cells on a tangent
screen.
.14.
EYE ENUCLEATED
HRP 51 V 5mm HRP 54
B
HRP 52 V
NORMAL
5 mm HRP 53
Fig. 4. Distributions of retrogradely labeled ganglion cells after HRP
injections into the ipsilateral optic tract near the LGN.
.15.
CONTRA(.•Nasal L
D Normal
Eye-Enucleated
Fig. 5. Comparisons of th* aaan soaa areas of contra- and ipsilaterally
projecting ganglion cells between normal and eye enucleated rats.
.16.
NORMAL AND ABNORMAL DEVELOPMENT OF RETINAL GANGLION CELL
POPULATIONS
Rafael Linden
Departamento de Neurobiologia, Instituto de Biofísica, UFRJ, Rio
de Janeiro, RJ.
This paper reviews a series of studies of normal retinal
development and of the consequences upon the retina of neonatal
lesions to the central nervous system in pigmented rats. In normal
rats, a marked reduction in the number of ganglion cells occurs
following birth; most if not the whole cell loss is due 'to
naturally occurring neuronal death. Histochemical evidence
indicates that monocular phagocytes inhabit the vitreal surface of
the normal retina during the neonatal period of cell death,
presumably concurring for the clearance of degeneration products.
• The ganglion cell population can be reduced further by retrograde
\ degeneration induced by neonatal midbrain lesions,with behavioural
! consequences. The population of ganglion cells with uncrossed
I projections is increased following neonatal removal of the other
\ eye; this has been attributed to reduced cell death as a
] consequence of diminished competition among developing axons for
\ terminal space. Enhanced uncrossed projections also follow
depletion of contralaterally-projecting ganglion cells within the
same retina, induced by neonatal lesions to the contralateral optic
tract; this is attributed to reduced cell death as a result of
diminished dendritic competition. The orientation of ganglion cell
dendrites in adult rats is strongly dependent on the presence of
neighbouring ganglion cells at critical stages of development.
.17.
Studies of the single and combined effects of enucleation and
optic tract lesions suggest a topographic and cell-type dependence
of retinal plasticity. It is proposed that retinal ganglion cell
populations are patterned by interactions of terminal and
dendritic competitive processes which regulate developmental
neuronal death.
(Supported by various grants from CNPq, FINEP, CEPG, NRC-UK)
.18.
QUANTITATIVE ANALYSIS OF THE DISTRIBUTION OF THE RETINAL GANGLION
CELLS IN AMAZON RODENTS
Luiz Carlos L. Silveira*, Cristovam W. Picanço-Diniz* and
Eduardo Oswaldo-Cruz**
* Departamento de Fisiologia, Centro de Ciências Biológicas,
Universidade Federal do Pará and ** Departamento de Neurobiologia,
Instituto de Biofísica, Universidade Federal do Rio de Janeiro
The heterogeneity of the species of Rodentia with their
adaptative diversity remains a large and productive area for
systematic work in Comparative Neurology. These species are largely
representated in Brazil, filling the whole range of habitats and
ways of life (Moojen, 1952).
We have made the quantitative analysis of the retinal ganglion
cells distribution of terrestrial rodents of the suborder
Hystrichomorpha which are filogenetically related but present
major differences in diuturnal cycles and others behavioural
aspects: the agouti, Dasyprocta agutl aguti, a largely diurnal
animal with a well developed praxic behaviour; the capybara,
Hydrochoerus hydrochaeris hydrochaeris, a gigant crepuscular rodent;
the spotted paca, Cunlculus paca paca, and the cayenne spiny rat,
Proechimys guyannens'- oris, nocturnal animals that spend daylight
hours in their burrows (Heinemann, 1975).
In order to study the ganglion layer, whole mounted retinae,
vitreous surface upward, were stained with cresyl violet and
examined under microscope (Stone, 1981). Ganglion cells were
identified following the criteria established for other species
(Stone, 1965, 1976; Hughes, 1975, 1981) with additional verification
.19.
by horseradish peroxidase retrograde labelling (Silveira et ai., in
preparation). Ganglion cell density was evaluated by counting, under
500X Magnification, the number of cells included in 200pa side
squares separated by one «111*1 ter intervals, or by half this
distance in regions of density higher than 1,500 ganglion cells/sai .
The total area of each preparation was determined from the enlarged
outline drawings and measurements were aade by Means of a HP 9864A
digit!ter coupled to a HP 9630A aicrocoaputer.
Retinal areas range froa 75s*2 to 85s»2 in the spiny rat, fro*
440sm2 to 580MB 2 in the agouti, froa 480aa2 to 580aa2 in the paca
and froa 1,000a*2 to 1,100a*2 in the capybara. Based on the local
densities at the various points sampled, we estimated the total
number of ganglion cells to be 90,000-116,000 for the spiny rat,
230,000 for the paca, 370,000 for the capybara and 517,000-530,000
for the agouti.
In theses species the distribution of ganglion cells shows
striking differences and similarities. These are common features:
1) the existences of an elongated, horizontally oriented region of
relatively higher cell density, the visual streak; 11) the lower
segment of retinal periphery attains higher density values as
compared with equivalent points in the upper retinal segment; ill)
the isodensity lines surrounding the streak are more spaced in the
temporal than the nasal retina. However, these species differ in
the development of the visual streak. The agouti has a retina with
steep centropripheral density gradient (CPDG) at about 24X and its
visual streak is sharp with a peak count up to 7,000gc/am2. The
capybara has means values for retinal CPDG at about 16X and visual
streak peak count of l,500cg/nm2. The paca and the spiny rat have
retinae with little CPDG of 3X and their visual streaks are
correspondents rudimentary, the peak count values being respectively
.20.
750cg/mm2 and 2S00cg/aa2. The paca has its visual streak overlaying
a region of the fundus entirely subserved by tapetu*.
Stone (1983) raised the hypothesis that the visual streak is a
general feature of maamalina retina,inherited from a phylogenetic
ancestor, and developed in the various species according to visual
needs originated from particular environmental pressures. Our data
and those obtained by Choudhury (1978) with guinea pig, which is
also a hystrichomorph, agreed with this view showing that over the
basio ground-plan in the topography of the retina some modifications
were imposed related mainly with the sharpness of the visual streak
which seems to be more developed in predominantly diurnal forms.
The visual streak being a specialized retinal structure,
enables the animal to scan a wide segment of its visual field
without the need of developing sophisticated eyes and head movements
(Hughes, 1977; Stone, 1983). This can be shown taking the agouti as
an example. The retinal magnification factor (RMF) can be derived
from the relation RMF * 0.011 L, where L is axial length of the eye
(Hughes, 1977). In the agouti L • 15.68mm (s.d. » 0.54mm, n • 24),
then BMP » 0.172mm/degree. From this value and applying Shannon's
sampling theorem we obtain a spatial resolution of 7.2 cycles/degree
for the region where the ganglion cell density is higher. When
eating, agouti uses the forelimbs to bring food to its mouth or to
hold nuts and small fruits while gnawing, maintaining a typical
stance, hunched on its hindlimbs, with the head in a fixed position
(Pimentel-Souza et al., 1980). He compared pictures of head position
during feeding with other pictures taken while the retina of an
anesthetized and curarized animal was labelled with photolitic
lesions, and.found that the image of the horizon was projected on
the visual streak. Taking into account the eye position (at about
20cm above the ground, as photographically evaluated) and the width
.21.
of the streak (3.2ram as definided by the l,500gc/nm2 isodensity line)
we may assume that the visual streak deals with images of ground
objects from infinity to a distance 120cm away from the animal.
Taking from granted that during feeding the animal is more vulnerable
to its predators, these behavioural and structural visual features
are distinctively evolutive resources of survival value.
Preliminary reports of this work has been published (Silveira
et ai., 1981, 1983).
Supported by research grants from FINEP/FADESP (n9 3.3.82.0237.00),
CNPq (n9 302213/82, 302216/82 and 402332/83) and UFPa-PRODESP
(n9 09871/80). He thank A. Gadotti for assistance in the editing of
manuscript and the Lab. de Matemática Aplicada e Computação (NCGG,
UFPa) for computer facilities.
REFERENCES
Choudhury, B.P. (1978) Retinotopic organization of the guinea
pig's visual cortex. Brain Res. 144; 19-29.
Heinemann, D. (1972) The cavies. In Animal Life Encyclopedia
(Edited by Grzimek, B.). Van Nostrand Reinhold Company, New York.
Hughes, A. (1975) A Quantitative analysis of the cat retinal
ganglion cell topography. J. Comp. Neurol. 163; 107-128.
Hughes, A. (1977) The topography of vision in mammals of
contrasting life style: comparative optics and retinal organization.
In Handbook of Sensory Physiology Vol. VII/5 The Visual System in
Vertebrate» (Edited by Crescitelli, F.). Springer.Verlag, Berlin.
.22.
Hughes, A. (1981) Population Magnitudes and distribution of the
major aodal classes of cat retinal ganglion cells as estimated from
HRP fillings and a systematic survey of the soma diameter spectra
for classical neurones. J. Comp. Neurol. 197: 303-340.
Moojen, J. (1952) Os Roedores do B sil. Instituto Nacional do
Livro, Rio de Janeiro.
Pimentel-Souza, F., Cosenza, R.M., Campos, G.B. t Johnson, J.I.
(1980) Somatic sensory cortical regions of the agouti, Dasyprocta
agutl. Brain Behav. Evol. 17: 218-240.
Silveira, L.C.L., Picanço-Diniz, C.W. ft Oswaldo-Cruz, E. (1981)
Quantitative analysis and distribution of ganglion cells in the
retina of the agouti (Dasyprocta aguti). An. Acad. brasil. Ciênc.
53: 633.
Silveira, L.C.L., Botelho, A.C., Picanço-Diniz, C.W. ft Oswaldo-
Cruz, E. (1963) Distribuição das células ganglionares retinianas do
Proechimys. Ci. e Cult. 35 (supl.): 615.
Stone, J. (1965) A quantitative analysis of the distribution
of the ganglion cells in the cat's retina. J. Comp. Neurol. 124:
337-352.
Stone, J. (1981) The Whole Mount Handbook. Ma it land Publications,
Sydney.
I Stone, J. (1983) Parallel Processing in the Visual System.
I Plenum Press, New York.
.23.
II
THE NASOTENPORAL DIVISION OF THE OPOSSUM'S RETINA
J.N. HOKOÇ and L.G. GAHRYSZEHSKI
Departamento de Neurobiologia, Instituto de Biofísica, UFRJ, Rio
Janeiro, RJ
The distribution of crossed and uncrossed ganglion cells in
the retina of the opossum (Didelphis marsupialis ; urita) was
revealed by retrograde degeneration (surgical section of one optic
tract) and the retrograde transport of HRP (multiple unilateral
injections in the brain). The cells of origin of the ipsilateral
projection were restricted to the temporal retina while those of
the contralateral were distributed over the entire retina.
By associating photocoagulation it was possible to determine
the projection of the vertical meridian on the retina (Vo)
relative to the position of the region with highest ganglion cell
density (area centralis) and, the region of overlap between
crossed and uncrossed ganglion cells (median strip).
Taking the projection of the vertical meridian (Vo) as 0
degrees of eccentricity, the median strip was found to be 20
degrees wide, stradling the vertical meridian from about 7.5
degrees nasal to about 12.5 degrees temporal. A plateau of highest
cell density was found to be localized between 0 and 5 degrees of
nasal eccentricity.
Visuotopographic studies of the superior colliculus and
striate cortex in the opossum have lead to the definition of
axes of 0 disparity (central axe* of binocularity) which project
on the retina approximately 2.5 degrees temporal to Vo. This
.24.
physiological reference is located at the center of the Median
strip as determined in the present investigation.
1. Rocha-Miranda, C.E.; Mendez-Otero, R.; Ranôa, A.S.; Volchan, E.;
Gawryszevski, L.6. In: Development of the visual pathways in
«MB—li. Stone, J.; Dreher, B.; Rapaport, D. (Eds.). Alan R.
Liss, 1984, in press.
2. Volchan, E. Personal comunication.
(Financial support: CNPq; PINEP; CEPG/UFRJ)
.25.
BIOTECHNOLOGY
Coordinator
Dr. CARLOS MEDICIS MOREL
Oswaldo Cruz Foundation
Rio de Janeiro, RJ
BRAZILIAN MEASLES CAM-70 VACCINE PRODUCTION: A TECHNICAL
COOPERATION TASK
A. HOMMA and J. R. SALCEDO CHAVES
Bio-Manguinhos/Oswaldo Cruz Foundation
T. OTSUKA
The Research Foundation for MicrobialDiseases of Osaka University
A project for establishment of Measles vaccine
production plant at Oswaldo Cruz Foundation, which is part
of Ministry of Health, was undertaken for a period of 4
years since August 1980, within the Technical Cooperation
Agreement between Brazilian and Japanese Government.
For the accomplishment of the proposed target were
considered the activities as specific training of brazilian
technical staff at The Research Foundation for Microbial
Diseases of Osaka University, providing the Oswaldo Cruz
Foundation of basic installations and laboratory facilities
(for three main areas: quality control , virus suspension
production, filling and lyophilization) which fullfill the
existing international requirement for vaccine manufacturing
areas, donation of measles attenuated virus strain - Biken CAM-70
developed and established by Prof. Y. Okuno et al. of
University of Osaka in 1970 ?nd the participation of Japanese
experts in the development of all steps of the implementaion
of the project.
.26.
Among several aspects it was considered also very
important the use of products produced in Brazil such as
equipments, raw materials, glassware and other material,
whenever possible, in the production of the vaccine.
The fertile SPF (specific pathogenic free) eggs
which are used for making the substrate for virus production,
the chicken embryo fibroblastic cells are obtainned from two
brazilian private breeding stattion, specialized and with a
monitoring schedule of laboratory control for the major avian
pathogen, for chicken SPF eggs production.
The methodology of cell cultivation was standartized
after series of studies which included the selection of the
method of embryo collection, fragmentation, improvement of the
operations of trypnization and cell suspension steps resulting
in the 10 viable cells per each chicken embryo incubated for
9 days. These cells are enough for seeding a Roux bottle of
460 cm* with 100 ml of 1x 10 cell concentration which will
complete the monolayer after incubation at 379Cfor48 hours.
The better relations of virus particle of inoculum
per each cell (MOD was find to be 0,1 to 0.03 virus per cell9 0resulting in average of virus titer of 10 ' TCID^Q per each
Roux bottle with 100 ml of medium, after 14 days of
incubation at 279C. This virus concentration enables the
manufacturing of at least of 100 dosis of final vaccine
containing 10 ' TCIDgg virus per human dose.
-page II-
.27.
Ás virus thermal stabilizer a combined solution of
hidrolisate of gelatin, saccharose and arginine is added to
the virus suspension before lyophilization. These freeze-
dryed vaccine when submited to the accelerated thermal
degradation test shows virus titer drop less than 1 log and
more than 10J TCID50 of virus per dose, fullfilling the
requirement for vaccine thermal stability.
The production of first five experimental vaccine
lots produced in 1982, only two years after the begning of
activities related to the project, was submited to the field
trial study in north and northest of Brazil, The results of
these field trials were then compared with the results of two
similar studies made in the same region, in a similar children
population, but immunizing them with a vaccine Biken CAM-70
produced in Japan and a Moraten strain vaccine. The latter
was used in a study made two years before the beginning of
this project and the imported Japanese Biken CAM-70 was used
at the same time when the activities of the project started.
The results of sero-conversion measured by the HI
test showed comparable results for all three studies. More
than 326 children were included in the study using brazilian
Biken CAM-70 vaccine and reached more than 951 of sero
conversion in the children above 9 months old with a
geometric HI mean titer of 25'28.
-page III-
,28.
The side reaction observed for Hoth Biken CAM-70
studies showed less symptomatology than usually observed for
other measles attenuated strains. For that brazilian Biken
CAM-70 field trial showed prevalence of fever higher than
37.59C in less than 5.51 of immunized children.
In 1983, after routinizing the activities of vaccine
production, it was possible manufacturing 10 million dosis
of vaccine all furnished to the National Immunization
Program of Ministry of Health. For 1984, the production
program of 15 million dosis is established a. goal.
The consolidation of. measles vaccine production
program will strenghthen the brazilian biologicals production
capability and enables further development of other viral
vaccine.
.29,
AN APPROACH TO MALARIA VACCINE
-JOINT INTEREST BETWEEN
PARASITE IMMUNOLOGY AND CANCER IMMUNOLOGY-
M.Suzuki, S.Waki, S.Nakazawa, T.Miyagami, I.Igarashi
(Gunma University School of Medicine)
M.Adachi, S.Asakura, A.Sakanoue
(Japan Inununo Research Laboratories Co.Ltd)
ABSTRACT
Since detrioration of global malaria eradication programme,
new countermeasures against the disease which is still most
notorious in the tropics have been strongly desired. New
insecticides, new drugs, and especially safe and effective
vaccine have been studied at laboratory as well as at field
levels. Among all those researches, development of anti-
malaria vaccine is most attractive not only for those who
are engaging in control actions, but also to laboratory
workers. Malaria is caused by infection with a protozoan
organisms, therefore the «vents are interplays between
animal host and eukaryotic animal cells, hence the studies
may share some common interests with cancer research. From
such background in mind, several approaches to malaria
vaccine worked by the authors will be presented.
In Plasmodium.berghei-Balb/C mice system, a live type
vaccine using permanent attenuated parasite obtained by X-
ray irradiation created 100% -survival against lethal
virulent P. berghei challenge for 4 months since vaccination
One year after vaccination, however, protection in terms of
survival rate was reduced, still 40% animals survived the
inoculation with the same lethal parasite. Recent advances
in antigen analysis of cancer cells revealed that at least
in certain systft» glycoprotein make surface structure which
distinguish cancer cells from normal host cells, and attempt
of immunological attack at the sugar target of cancer cell
.30.
surface have presented hopeful results for future cancer
therapy. Hinted by such kind of informations, we isolated
glycoprotein from Aotus erythrocytes infected with
P.falclparum and used the material in vaccination experi-
ments in Aotus-P.faleiparum system. Considerable effects
were observed, and in some monkeys, only 10 microgrammes of
the separated glycoprotein worked for the suppression of
parasitemia of inoculated P.faleiparum. Boiling of the
material gave no harm to the effects. Both rodent and Aotus
systems will be discussed from view point of joint research
between cencer and parasite immunology.
.31.
1
EXPRESSION AND SECRETION OF Baci l lus sp. a-AMYLAS7 GENE IN
Escherichia col i and Baci l lus s u b t i l i s .
C.J. ULHOA; M.B.N.S. SOUZA; S.M.R. TEIXEIRA; S .L .B. SUVA;
I . S . PEREIRA; M.C.N.D. MENEZES; V.M.Q.G. LIMA and S. ASTOL
FI FILHO.
Departamento de Biologia Celular
Caixa Postal n9 153081
Universidade de B r a s í l i a
70.910 - B r a s í l i a , DF
BRAZIL
The a-amylase gene of a Bacillus >p. ( isolated
in Brasi l ia ) was cloned using an £ . col i - B. subti11s b i -
] functional cloning vector pATUB5 (Ap r , T c r , Km r-derivative
from pAT153 and pUBHO).
The DNA of the amyiolytic bacteria was p a r t i a l l y
digested with Saji 3A and 11 gated to the 6am HI site ofpATUB5,
and this material was used to transform E_. coll 5K ce l ls .
From about 8500 £ . coll recombinant clones, I t was possible
to Isolate one that has the capacity of hydroiyse starch
making a typical halo in L agar plates with 0,5% starch when
stained with iodine.
From this amyloiytic E_. coll clone, a piasmiddenominated pAKAl (Ap r
r Kmr, ANY4) was Isolated, that has; the ab i l i t y to transform B_. subt i l is (SB 202) increasing Us
\ digcsti on of starch.
I Since the product of the cloned gene can be
I expressed and secreted in bo$h jram - positive and negative
I bacteria, a perspective Is opened for construction of an
* expression - secretion b1funct1ona1 cloning vector. The
biotechnoiogical value of the recombinant Bacillus a-amylase
production 1s presently beeing Investigated.
t This work was supported by CNPq - BRAZIL.1I . 3 2 .
SYNTHESIS AND SECRETION OF MOUSE PANCREATIC aAMYLASE BY YEAST
S.ASTOLFI FILHO
Department of Cellular BiologyInstituto de Ciências BiológicasUniversidade de BrasiliaBrasilia - Brazil
E.VICENTE GALEMBECKA-.C.SCHENBERG FRASCINO
Department of BiochemistryInstituto de QuimicaUniversidade de São PauloSão Paulo - Brazil
Mouse pancreatic ct-amylase cDNA p-eviously clonedafter Bal31 treatment in the HindIII site of the shuttle vec-tor pAAH5 (1) was purified from low melting point agarose af-ter Hindlll complete digestion of the plasmid followed by gelelectrophoresis.
On the other hand, plasmid p69A carrying the struc-tural gene for Saccharomyces cerevisiae pheromone a (2) wascompletely digested with Hindlll and ligated again in orderto eliminate the structural MFa region. This new plasmid waslinearized with Hindlll and subsequently ligated to the a-amy_lase cDNA fragment.
Transformation of E.coli 5K to ampicilin-resistancewith the ligation mix yielded 1200 clones, 11 of which werefurther analyzed for insertion through plasmid minipreps fol-lowed by Hindlll digestion and agarose gel electrophoresis.The observed insertion frequency was c.a. 1/10.
The plasmid DNA pool from the bacterial transfor-mants was used to transform Saccharomycet, cerevisiae DC6 andC7RFI8 strains to leucine prototrophy. 600 yeast transformantswere screened on starch containing medium: 16 transformantswere able to generate clear zones on the plates after iodinestaining. These transformants are presently beeing furtherinvestigated.
(1) Schenberg Frascino, A.C., Galembeck, E.V. e S.Astolfi Fi-lho, Abst. 99 Congresso Latino Americano de MicrobiologiaSão Paulo, SP (1983) p.304.
(2) Kurjan, 3. and I .Herskowitz; Cell 30_:933 (1982).
.33.
GENETICS OF AMYLOGLUCOSIDASE PRODUCTION IN Asperqiilus
niger and Aspergillus awamori.
R. BONATELLI JR., G. UMBUZEIRO-VALENT, M. MASIERO, A. VIALTA
e M.R. CALIL.
Department of Genetics and Evolution,
Institute of Biology, UNICANP.
ABSTRACT
This paper reports the results obtained with the
isolation and characterization of mutants altered in the
accumulation of amyioglucosidase (E.C. 3.2.1.3), the aiielic
interactions of these mutations and the inhibitory effects
of glycerol.
The enzyme was at first identified by using limit
dextrin and an inhibitor kindly donated by Dr. S. UEDA(Dpt.
of Food Science and Technology, Kyushu University,Fukuoka).
Several raw starch sources were tried as Inducers and in all
but one a great variation of the yield was observed.
In some cases dipioids were overproducers and some
segregants from heterozygous dipioids produced more enzyme
than the parental strains. All mutants isolated were
recessive and segregated as single nuclear genes.
Glycerol is a strong inhibitor and work is now In
progress to isolate derepressed mutants.
.34.
Amyloglucosidase (E.C. 3.2.1.3) is capable of producing glucose from
stardi with or without cooking. It is of considerable interest to Brazil,
because of cassava root starch is one of the most abundant carbohydrate re-
sources that can be used to obtain ethanol .
There are several reports about increased amyloglucosidase production in
Aspergillus by the use of mutant strains but little is Know about the genetic
components involved on enzyme production and improvement .
In the past years we have studied several genetic aspects of Aspergillus
niger and A. awamori *• *• *• 7*and now our interest is centered on the
isolation and characterization of mutants altered on the accumulation of this
enzyme, the allelic interactions of these mutations, the inhibitory effects
of glycerol and on recombination experiments between these two species.
MATERIAL AND METHODS
Strains and culture media, were described elsewhere * '
ENZYME PRODUCTION, spores from agar slants were inoculated into 5 or 25 ml ofliquid medium (MAC) consisting of raw starch from cassava root, 20 g; NaNOj ,2.0g; KH2PO4, 1,0 g; MgSO4 . 7H2O, 0,5 g; distilled water, 1,000ml at pH 5.0in flasks F., F2 or F, or F. (125 ml erlenmeyer), respectively and incubatedat 30°C for 4 days. In some cases glycerol ( H , v/v) replaced raw starch ascarbon source (MAG medium). Amyloglucosidase activity of the culture fil-trate was determined by incubating a mixture of equal volumes of 11 (p/v)soluble starch in 0,1 M citrate buffer (pH 4.0) and culture filtrate con-veniently diluted, at 60°C for lh. Reducing sugars were estimated by o-toluidine method in the culture filtrate and after enzyme reaction. One enzymeunit is the amount of enzyme that will'catalyze the production of 10 mg ofglucose on the conditions cited above.
ENZYME CHARACTERIZATION: Culture filtrates from A. niger strain were assayedagainst soluble starch and TAKA-DIASTASE limit dextrin with or without enzymeinhibitor. Limit dextrin and the inhibitor were kindly donated by Dr. S. UEDA(Dpt, of Food Science and Technology, Kyushu University, Fukuoka - Japan).
M n W T S ISOLATION: Mutants were induced irradiating 5-8 days old spores with
.35.
ultraviolet light from Mineralight UVSL-25. Survivors were assayed for enzyme
production on MAC or MAG or for auxotrophy on MM (mniaal mediua).
RESULTS AND DISCUSSION
Aspergillus ni£er strains accumulated reasonable amounts of anyloglu-
cosidase - AG and only trace amounts a-arnylase (Table 1 and 2; S. Ueda, per
sonnal conaunication, at least in the conditions utilized during the present
research. It could be noted from Table 1 that higher levels of AG are corre-
lated with low levels of reducing sugars in the culture filtrate.
When glycerol replaced raw starch as carbon source it was observed a
strong AG inhibition (80-981) similar to the results related by others authors
'b' This effect was determined only on A. niger because A. a*amori cannot gro>
with glycerol as a carbon source and consequently it is easy to explain why
glycerol inhibits only AG accumulation on A. -niger in solid media (Table 3) .
Another result evident on Table 3 is that the use of Triton X-100 (0.U, v/v)
enhances the halo of hydrolisis of starch in A. niger. If it is a result of
AG activity it remains to be stated. At present we isolate a possible derepre
sed mutant and work is now in progress to determine AG levels on MAC.
Several mutants altered on AG accumulation were isolated. Out of 268
assayed colonies, 3(1.2%) were law and 5(1.9$) were medium producers. By
using our selection procedure it was not possible to isolate high producers.
When some of these mutants were crossed with parental strains they behaved as
recessive (Table 4) and it was not possible to see effects of ploidy, except
in only one occasion . Effects of ploidy are related for several authors '
foT others metabolites. A diploid strain when induced to segregate exhibited
sectors with higher production than parental strains.
The use of the A. awamori strain in the present research is because our
intention is to obtain hybrids and recombinants with A. niger and so we had
to study several biological aspects of the former species. We observed a high
spontaneous frequency of auxotrophic spores (2-204) requiring proline or
.36.
arginine for growth on W. It was possible to stabilize only auxotrophic strains.
Trying to.determine the origin of the spontaneous segregation we observed that
Benlate (fungicide - 0,12 ug/ml) enhances the frequency of sectors as observed
in diploid strains of Aspergillus and that these sectors were not resistant
strains. Furthermore these sectors showed a large variation of multinucleated
conidia compared to the parental strains (Table 5).
Trying to get auxotrophic mutants by using UV light as inducer it was
observed that the frequency is very low (0.1J) and it raises 10 fold when
the filtration enrichment technique was used. Work is now in progress to make
reconstruction experiments trying to establish even better conditions to
isolate auxotrophic mutants from this strain.
REFERENCES
1. UEDA, S.; ZANIN, C.T.; MDNTEIRO, D.A. 5 PARK, Y.K. (1981). Biotechnol
Bioeng., 23: 291.
2. NEVALAINEN, K.M.H. 5 PALVA, E.T. (1979). J. Çhem. Tech. Biotechnol, 29: 390.
3. BALL, C ; LAWRENCE, A.J.; BUTLER, J.M. 5 MORRISON, K.B. (1978). Euro. J.
Appl. Microbiol. Biotechnol., 5: 95.
4. BONATELLI JR., R., AZEVEDO, J.L. 6 VALENT, G.U. (1982). Braz. J. Genet., j>:
483.
5. BONATELLI JR., R.; AZEVEDO, J.L. § VALENT, G.U. (1983). Braz. J. Genet., 6:
399.
6. BONATELLI JR., R. 5 AZEVEDO, J.L. (1982). Biotechnol. Utters 4: 761.
7. VIALTA, A. § BONATELLI JR., R. (1983). Ciência e Cultura, tt: 687.
8. BARTON, L.L.; GEORGI, C.E. $ LINEBACK, D.R. (1972). J. Bacteriol., Ill: 771.
9. VALENT, G.U. § BONATELLI JR., R. (1982). Ciência e Cultura, 34: 749.
.37.
TABLE 1. Aayloglucosidase and reducing sugars (RS) on culture filtrates of several types of flasks
(aean of 4 replicates) :
FLASKS
1
2
3
4
48
AG(U/*1)
1.8
3.4
3.6
1.4
RS*
230
485
448
511
72
AG(U/ml)
6.3
13.4
14.5
8.7
H
RS
260
167
73
243
O U R S
96
AG(U/ml)
0.6
15.3
15.8
9.0
RS
274
22
6
29.
120
AG(U/ml)
1.7
16.8
16.2
11.7
RS
566
25
10
6
TABLE 2 - Amyloglucosidase (AG) activity assayed on soluble starch
(SS) and limit dextrin (LD) with or without inhibitor (I),
I (ug)
0
6.6
11.4
22.8
SSAG(U/al)
9.0
4.4
3.6
2.1
1 inhibition
-
51
60
76
AG (U/ml)
9.73.8
3.5
2.1
LDI inhibition
-
61
64
78
TABLE 3 - Inhibitory effect of glycerol (41) on AG accumulation by
Aspergillus in solid culture media.
TREATMENT
Control Glycerol (41) Triton X-100 (O.lt)
A. niger 1.1* 1.0 1.?
A. awamori 1.9 1.9 nd.
* AG index - it is the relationship between the diameter of the
halo and that of the colony.
nd, not done
.39.
-TABLE 4 - AG production of the mediu* (M) and ION (L) producers,
parental strains and diploids obtained from them.
1.
i. •
5.
4 .
5 .
6 .
7.
8 .
9 .
1 0 .
1 1 .
HAPLOIDSSTRAIN N'
nic-, o l v , (NO)
£âíll íüüi (pp)NO 3 M
NO 1 6 6 M
NO 1 1 1 M
NO 1 9 9 M
NO 1 0 0 M
NO 38 L
NO 33 L
NO 73 L
PF 61 L
AG(U/ml)
1 1 . 3 ( 2 4 ) *
14.4 (19).
6.9 (5)
7.2 (9)
6 .5 (5)
6 .1 (5)
6.0 (9)
2 . 0 ( 1 3 )
0.0 (5)
1.5 (15)
0 .3 (10)
DIPLOIDS
STRAINS IN THE CROSS**
1 x 2
7 x 2
4 x 2
3 x 2
8 x 2
8 x 11
10 x 2
10 x 11
11 x 1
AC {
9 . 9
1 2 . 7
1 6 . 6
1 0 . 5
13.2
10.7
12.6
9 . 2
10.3
:u/«(S )
( 4 )
( 4 )
(5)
(5)
(5)
(5)
(5)
(5)
*, number of replicates.
, see first row.
.40.
TABLE 5 - Frequency of aultinucleated conidia of the parental
strains and sectors isolated on Benlate - containing
•edia.
NUMBER OF NUCLEI PER CONIDIA
1 2 3 4 5
NRRL 3112 58 41 1 - -
ANB 3» 9 54 27 8 2
ANN 1 15 48 32 4 1
Sectors** 1 1 - 6 2 3 4 - 7 0 3 - 45 0.3 - 3.3
*, AWB e AWW - Brown «utant and white Mutants, respectively.
**, seventeen sectors isolated.
PREPARATION OF 35S-METHIONINE
E.RONDINELLI, N.MOUSSATCHÊ, J.F.OLIVEIRA CARVALHO,
M.G.C.CARVALHO, R.S.MOURA NETO, D.S.LOBO,
I.C.FRÜGULHETTI, R.SILVA e M.A.REBELLO
Department of Molecular Biophysics
Instituto de Biofísica da UFRJ
In this abstract is described a simple method forit r • 35
obtention of S-methionine using Na_ SO prepared by
CNEN/SSo Paulo-Brazil. Cultures of E.coli B grown in a
rich medium (L-Broth) are submitted to sulfur starvation
by two subsequent passages in culture media containg low
sulphate concentration. Under these conditions the bac-Q
terial growth stops in a density of 2 x 10 cells/ml. At
this point radioactive Na_ SO. is added and the culture
allowed to grow for 17 hours. The cells are harvested by
centrifugation and processed for protein hydrolysis. The
material is then dried under vacuum and the S-methionine
is separated by descending paper chromatography on Whatman
3 MM paper. The regions containing the radioactive amino-
acids were identified and the S-methionine eluted with
5 mM dithiothreitol and stored in liquid nitrogen. The
yeld of S-sulphur incorporated in methionine is 30 to
40% of the added radioactivity. The obtained 35S methio-
nine is under current use in our laboratories for in vivo
and in vitro studies of protein synthesis.
He believe that this method is feasible to be
developed in laboratories where the technology described
is of current use.
MOLECULAR BIOLOGICAL APPLICATIONS TO VIRAL DIAGNOSIS
H.G. PEREIRADepartment of VirologyInstituto Oswaldo Cruz
ABSTRACT
Methods traditionally used to detect and identifyviruses in pathological materials are based on theirpropagation in experimental animals or cell culturs, or onthe direct demonstration of their presence by imnunoassays.
New techniques derived from molecular biology makepossible a new approach to viral diagnosis, namely the detectionof viral nucleic acids. Electrophoretic analysis of viralnucleic acids has been successfully applied and shown to beof value as a rapid diagnostic test for rotaviruses (Pereiraet ai., 1983). The technique is feasible in routine diagnosticlaboratories with moderate resources and has, among others ,the advantage of requiring only readily available, chemicallydefined reagents.
Other methods applicable to viral diagnosis arebased on the detection of nucleic acids by hybridization ininfected cells or on filter membranes (dot hybridization)The presence of viral genomes is revaled by hybridizationwith probes consisting of nucleic acids homologous to theviruses being searched for. Probes used most frequently arelabelled with " P and detected by autoradiography. The useof non-radioactive probes labelled with biotin (Brigati etal,, 1983) and detected by enzvme-avidin-biotin complexes(Leary et al., 1983) is an important development in bringingdot hybridization assays to the level of routine diagnosis.
Brigati, D.J.; Myerson, D.; Leary, 3.3.; Spalholz, B.;Travis, S.Z.; Fong, C.K.Y.; Hsiung, G.D. a Ward, D.C.(1983), Virology, JK26, 32.
Leary, J.J.; Brigati, O.J. ft Ward, D.C. (1983) Proc. Nat.Acad. Sci., USA, 80, 4045.
Pereira, H.G.; Azeredo, R.S.; Leite, J.P.G.; Barth, O.M.;Sutmoller, F.; de Farias, V. & Vidal, M.N.P. (1983) Mem.Inst. Oswaldo Cruz, 21» 483-
.43.
RECOMBINANT DNA TECHNOLOGY FOR PRODUCTION OF USEFUL
SUBSTANCES BY MICROORGANISMS
Teruhiko Beppu
Department of Agricultural ChemistryFaculty of. AgricultureThe University of Tokyo
Recombinant DNA technology has provided new possibilities
in applying microorganisms to produce various useful substances.
One of the most important application of this technology is
microbial production of human, animal and viral polypeptides,
several of which are now in the final stage of the industrial
development. Another is its application to improve microbial
strains used in fermentation industries through which more rationj.
approaches than those hitherto adopted'in the traditional strain
improvement might become possible.
Here I will describe our works in these lines, i.e., product-
ion of chymosin (calf rennin) in microorganisms and possible
improvement of Streptomyces strains for antibiotics production.
I. Cloning and expression of chymosin cDNA in microorganisms
= Chymosin, also known as calf rennin, is a milk-clottingi
! enzyme essential for cheese manufacturing. It is secreted from
I the mucosal tissue of the 4th stomach of suckling calves as an
i inactive enzyme precursor, prochymosin, which contains 365 amino
| acid residues with molecular weight of approximately 41,000.\
I .44.
Its N-terminal peptide of 42 amino acid residues is cleaved auto-
catalytically under the acidic condition to form the active enzyme.
The mRNA for prochymosin was found to be abundant in the
mucosal tissue of calf stomach. By using phenol-extraction and
fractiónation by poly(U)-sepharose affinity chromatography and
sucrose density gradient sedimentation, we obtained an mRNA fracton
which encoded almost entirely for prochymosin in a cell-free
protein synthesizing system. Starting from the enriched mRNA
for prochymosin as a template, double-stranded cDNA was prepared
by standard reverse transcription techniques and was cloned in
Escherichia coli using pBR322 as a vector (Fig. 1). Several
clones each carrying a part of the coding sequence for prochymosin
were obtained by two-step screening by colony hybidization
and hybrid-arrested translation assay, from which the total nucleotide
sequence of 1095 bp. for prochymosin was determined (1,2).
Several expression plasmids were constructed by inserting
bacterial promotors at upstream of the cloned cDNA. pCR301
contains lac UV5 promotor and a fused gene in which the coding
sequence for the N-terminal 4 amino acids of prochymosin are
replaced by the N-terminal 10 amino acids of 6-galactosidase (3)
(Fig. 2). pCR501 and pCR601 contain trp promotor and fused genes
having similar short N-terminal replacement with trp E and trp
L polypeptides, respectively. pCR701 is a plasmid coding direct
expression of fMet-prochymosin under the control of trp promotor.
All these plasmids, especially pCR301 and pCR501 caused marked
production of protin reactive with anti-prochymosin antibody.
Lower expression efficiency of pCR701 was improved by changing
the SD - ATG distance from 14 bp. to 3 bp.
Prochymosin polypeptide synthesized in E. coli cells by
these plasmids was found to sediment by low-speed centrifugation
.45.
of the disrupted cells. Electron microscopic obs'.-rva». ion 'A
the cells harvoring these plasmids showed the presence of largo
inclusion bodies composed of prochymosin. Quantitative extraction
of bacterially synthesized prochymosin was achieved with 8 M
urea. Dialysis of the extracts in the presence of 0.5 M NaCl
at pH 10 ccaused almost complete refolding of the denatured pro-
chymosin (Fig. 3), which could be converted autocatalytically
to chymosin by incubating at acidic pH. Thus substancial product-
ion of chymosin in E. coli cells has become possible by use of
recombinant DNA technology 14).
II. Cloning of pleiotiropic and positive regulatory gene for
secondary metabolism in streptomycetes
Streptomycetes are the highly differentiated prokaryotes
characterized by their versatile capability to produce diversed
secondary metabolites including a large number of antibiotics.
A close relationship between the secondary metabolism and the
morphological cell differentiation is well recognized. A better
understanding of regulatory mechanisms for these complexed metabolic
processes will provide a new vista for developing rational
approaches to increase antibiotic productivity in streptomycetes.
Our recent studies on the streptomycin-productivity in
StrepLomyces griseus led us to confirm Khokhlov's finding of
A-factor (Fig. 4) (5) as an essential chemical regulatory factor
for both the antibiotic production and morphological differentiat-
ion in this organism (6, 7). Mutants of S. griseus deficient
for A-factor biosynthesis simultaneously lost the antibiotic
productivity and spore-forming ability, both of which were recovetd
146.
by addition of extremely low amounts of A-factor to the cultures
of the mutants. A-factor seems to be a bacterial hormone regulat-
ing multiple physiological functions in this organism. Genetic
analyses suggested that A-factor gene of S. griseus was located
on a plasmid or a highly transmissible genetic determinant (8).
On the other hand, A-factor productivity was also found
in S. coelicolor A3(2), genetically the most intensively studied
Streptomyces strain. Using the well developed gene exchange
systems of this organism through conjugation, two A-factor genes
(afsA and B) were determined to locate on the chromosome but
not on a plasmid (8). A chromosomal DNA fragment containing
afsB gene was cloned in a S. lividans strain using pIJ41 as a
vector plasmid and size of the cloned fragment was trimmed down
to less than 2,000 bp. (Fig. 5). Introduction of the cloned
afsB gene into the host caused marked production of not only
A-factor but also two pigments with antibiotic acitivity, i.e.,
actinorhodin and prodigiosin (Fig. 6).
At the bigining, this typically pleiotiropic feature of
afsB gene seemed to be a consequence of the multi-functional
activating effect of A-factor whose synthesis was induced by
afsB. However, exogenous addition of A-factor to the afsB mutans
of S. coelicolor caused no recovery of the pigment production,
which clearly indicated that A-factor itself was not involved
in the afsB function. The minimum size of the cloned af sB
gene allows to code for only one polypeptide, while the three
metabolites (A-factor, actinorhodin and prodigiosin) have entirely
different chemical structures and biosynthetic pathways. From
these considerations, it is probable that afsB gene is a regulatoy
gene which positively controls various genes involved in the
secondary metabolism in S. coelicolor A3(2) and S. lividans.
.47.
The fact that ' introduction of afsB gene into S. lividans
activated production of "new" antibiotics actinorohdin and prodi-
giosin in this organism suggests a practical significance to
improve antibiotic productivity or to obtain new antibiotics.
Screening of new antibiotics by introducing afsB or similar
positive regulatory genes through recombinant DNA technology
seems to be an useful approach which will be exploited in future.
References
1. K. Nishimori, Y. Kawaguchi, M. Hidaka, T. Uozumi and T.
Beppu. J. Biocheru. (Tokyo), 90, 901 (1981)
2 K. Nishimori, Y. Kawaguchi, M. Hidaka, T. Uozumi and T.
Beppu, J. Biochem.(Tokyo), 9±, 1085 (1982)
3 K. Nishimori, Y. Kawaguchi, M. Hidaka, Y. Uozumi and T.
Beppu, Gene, VS., 337 (1982)
4 T. Beppu, Trends in Biothechnology, _1, 85 (1983)
5. A. S. Khokhlov, in Overproduction of Microbial Products,
FEMS Symposium No.13, pp.97, Academic Press, N.Y.(1982)
6. O. Hara and T. Beppu, J. Antibiot. 35, 349 (1982)
7. O. Hara and T. Beppu, J. Antibiot. 35, 1208 (1982)
8. 0. Hara, S. Horinouchi, T. Uozumi and T. Beppu, J. Gen.
Microbiol. 12J?, 2939 (1983)
9. S. Horinouchi, 0. Hara and T. Beppu, J. Bacteriol. 155,
1238 (1983)
.48.
pBR322
Sal I
TCGAC•
31 G
DNA poly-merase 1
dNTPs
TCGAC-AGCTG-
Terminaltransferase dCTP
3 TCGAC-„ CCCCAGCTG
Sal 1
G 3 >
CAGCT
GTCGACAGCT
GTCGACCCCCAGCT
mRNA3> AAAA 5'
Reverse dNTPstranscriptase I oligo dT
\AAAA
5, TTT r
Alkaline Ihydrolysis
\5 > T T T
DNA poly-merase I
5'
3
3'
dNTPs
TTT-, AAA-
Nuciease SI
TTT-AAA-
Terminaltransferase dGTP
3 TTT-, GGGGAAA-
GGGG
51
3>
Anneal ingTransformat ion
GTCGACCCCTTT-CAGCTGGGGAAA•
GGGGTCGACCCCCAGCTG
Fig. 1. Synthesis of double-stranded cDNA and insertion into
a plasmid vector.
.49.
v
iT4-Xltan* l t i 1 S * 7 i $ l O 5 t 7 t
—ACCTATGAGCATGTTAACGGATTCACTGGAATTCCGGATCCCTCTG-
t-çalacto*idatê prochy«o«ln
tz
prochymoiln cONA sequence.
l. Eco RI ; S , Sal I ; B , Btm HIK, Kpn I ;
Pig. 2. Construction of expression plasmid pCR301 for prochymosin.
. 5 0 .
E» 40c .3
r 30>oCO20
o
1 10 f..#^j 1 1 1 1—o
4 5 6 7 8 9 10 11 12pH
Pig. 3. Renaturation of bacterially synthesized prochymosin
extracted with 8 M urea.
Fig. 4. Chemical structure of A-factor.
.51.
Megadaltons
PLASWD 0 1 2 3 *
"P« RI Xhc Bel
5 6 6.4
Pst Sac Bà™ KP"
PHENOTYPE
A. FACTOR RED
P!JI?1-AP1
PIJÜ1-AP3
Cl l
PlJUl-Nl
PIJW-P1
Sph
*-*Pvu
Bel
•Pst
"Sph
• •Bel
mmSac
SphSvuPst
MBSph p ¥ U
•1z±
Pst
Sac
• •Sac
maSac
Bam>
Bam
Sph
Kpn
Sph
Kpn
•Sph
Kpn
•
YES
YES
YES
NO
YES
YES
YES
NO
Bd Sph
NO NO
PIJW-N6
PIJ11-AP11
PIJW-AP15
PIJ41-AP17
Kpn
hiKpn
hi
RI
Cla
RI
ripCla
Xho
Xho
warnSph
warnSph
Bel
Bel
•ÉH
Pst
tapiSph
Pst
máSph
Pst
• •Sph
Bel
""Is
Sac
Sac
wmiPvu
Sac
m
Bam>
Bam
smá
Kpn
Sph
Kpn
Sph
NO
YES
YES
YES
NO
YES
YES
YES
Fig. 5. Summarized results of subloning of afsB gene. "Red"
phenotype indicates stimulation of the pigment production in
the host S. lividans strain.
. 5 2 .
CH3 OH O O OH CH3
OHOOC coon
OH O O OH
B OCH3
Fig. 6. Chmical structures of actinorohdin (A) and prodigiosin
.53.
A BRASILIA» EXPERIENCE ON PLANT CELL AMP TISSUE CULTURE:
A TOOL FOR PLANT IMPROVEMENT
O.J.CROCOHODqpt. of Cha*istry/ESNQ/USP ;CB4A/USP
and Canter Cor Agricultunl Biotechnology (CEBIBC/FEKQ) .
A.NATAL GONÇALVES
Dept. of Silvicultura/ESAIfi/USPand Canter Car Agricultural Biotechnology (CEBTEC/FEALQ)
13.400 - PIRACICABA - SP - BRASIL
ABSTRACT
Cells, tissues and organs of plants can be cultured
under asseptic conditions in culture media that allows for
their growth and proliferation. This technique is basic for
micropropagation and an excellent tool in the breeding of
economically important plant species. This can be approached
through the ability to apply -in vitio cellular selection for
recovering useful genetic variants, use of anther culture to
speed the attainment of homozygosity, somatic hybridization
for recombining genomes of sexually incompatible plant
species and also the possibility to use this technique
coupled with the recombinant DNA techniques. In our labora-
tories fruit crops, such as citrus, passion fruit, grapes,
and ornamentals, as well as forestry species, such as
eucaliptus, are micropropagated using protocols developed
by our group. Sugarcane genetic variants tolerant to herbiei
des can be recovered using our protocols. To by pass the
problem of abortion in interespecific crosses in Phaie.olu&
species, we developed a protocol to culture in vitxo immatu-
re embryo axis, recovering intact and fertile bean plants.
.54.
PURIFICATION AND CHARACTERIZATION OF NEW RESTRICTION ENDO-NUCLEASES
A. K. CRUZ, G. KIDANE, H. Q. PIRES and C. M. Morel
D*pt. of Biochemistry and Molecular BiologyInstituto Osvaldo CruzAv. Brasil 4365 - Manguinhos21040 Rio de Janeiro, RJ - Brazil
ABSTRACT
A large number of restriction endonucleases, endodeoxy^
ribonucleases which recognize and cleave the DNA at a specific
site, has been purified and characterized since the last
decade. Their usefulness in the analysis and restructuring of
DNA molecules has been a major reason for the search of new
specificities (Roberts, R. J. Nucleic Acids Research Il:rl35-
rl68, 1983).
He have screened four hundred different strains of
Bacillus sp for the presence of restriction enzymes. Of these
six were found to be good producers of type II restriction
endonucleases.
In this paper we describe the purification and characte
rization of the first three we have studied in more detail:
- Bee 243, an isoschizomer of Sau 3A, which recognizes the site
/GATC and cuts as indicated by the bar, cleaving both normal
and N6-methyladenine free DNAs;
- an isoschizomer of Hae III isolated from a thermophilic
Bacillus;
- an endonuclease which leaves a 5' terminal T nucleotide
residue after cleavage and is still being characterized.
This work was supported by CNPq and FINEP through grants to
C. Morel.
.55.
SURFACE ANTIGENS OF TRYPANOSOMA CRUZI RECOGNIZED BY
MONOCLONAL ANTIBODIES
M. J. M. ALVES, G. ABUIN and W. COLLI
Departamento de Bioquímica, Instituto de Química
Universidade de São Paulo, C. P. 20780, São Paulo, Brazil
ABSTRACTCirculating antibodies exist in humans and animals
infected with T. cruzi, the etiological agent of Chagas'
disease. These antibodies have been used as an instrumental
for the definition of relevant antigens on the surface mem-
brane of the parasite (Andrews et al., Eur. J. Bioçhem., in
press). The development of monoclonal antibodies (McAb)
ag linst these antigens is an obligatory step in order to pro-
be ti,..iir function, particularly at the level of the host-pa-
rasite interaction.
Thirteen McAb have been raised against the surface
o£ infective forms of T. cruzi via immunization with 8-metho-
xypsoralen-treated intact trypomastigotes. These antibodies
belong to.different immunoglobuH n classes: IgM (5), IgG,(3),
IgG- (4), anã IgG3(I). t'ive of the McAb recognize common api-
topes to all T. cruzi stages, except amastigotes, an intra-
cellular stage. The others recognized only infective forms
of the parasite.
One McAb, specific for the infective form, immun'o-
precipitated a surface glycoprotein (85 kDa, pi 6.3-7.5)
found only on the surface of trypomastigotes and previously
described to have affinity for WGA (Katzin and Colli, Biochim.
Biophys. Acta 722, 403,1983). This, as well as other McAb
which recognize specific antigens, partially block T. cruzi
interiorization into LLC-MK2 cultured cells.
Supports UNDP/World Bank/WHO, CNPq, ÍINEP and FAPESP
.56.
DEFINITION OF RELEVANT SURFACE ANTIGENS INVOLVED IN THE
INTERIORIZATION OF TRYPANOSOMA CRU2I IN HOST CELLS
M. V. ARRUDA, B. ZINGALES and W. COLLI
Departamento de Bioquímica, Instituto de Química
Universidade de São Paulo, C P . 20780, São Paulo, Brazil.
ABSTRACTIn a previous report (Zingales et al., Mol.Biochem.
Parasitol., £,111, 1982) the surface antigens of the infec-
tive stage of T. cruzi have been identified. Since T. cruzi
is an obligatory intracellular parasite, blocking of the pe-
netration step could lead to the abortion cf the infection.
The definition of relevant antigens involved in penetration
(most likely plasma membrane borne) and the molecular cloning
of the corresponding genes are necessary steps towards the
production of an efficient immunizing agent.
Polyclona1 antibodies have been raised against
some T. cruzi antigens isolated by preparative SOS-PAGE. The
antisera prepared against the trypomastigote polypeptides
with molecular masses ranging from 30-90 kDa and 50-60 kDa
have been found to inhibit up to 85% the parasite interiori-
zation into LLC-MK2 cells. In contrast, antisera prepared
against epimastigote (non infective form) polypeptides, obtai-
ned from gel regions equivalent to those used for the isola-
tion of trypomastigote antigens, had a much less pronounced
inhibitory effect (up to 35%), These antigens were identified
by immunoprecipitation of surface radioiodinated and S-me-
thionine labeled parasites. The antisera thus obtained will
be used for the screening of recombinant clones.
Support: UNDP/World Bank/WHO, CNPq, FINEP and FAPESP
.57.
A COMPARATIVE BIOCHEMICAL ANALYSIS OF ISOLATES OF VISCERAL LEISHMANfASISFROM THE STATES OF BAHIA AMD RIO PE JANEIRO
R. S. PACHECO; H. MOMEN; G. GRIMALDI J r . ; M. C. A. NARZOCHI and C. M. MORELf Instituto Oswaldo Cruz (FIOCRUZ)
Caixa Postal 92620.000 Rio de Janeiro, RJ - Brasil
R. BADAROUniversidade Federal da Bahia40.000 Salvador, BA - Brasil
Leishaaniasis 1s a parasitic disease caused by protozoa of the genusLeishmania. The disease Is transmitted to man by the bite of thephiebotomine sandfly and has a worldwide distribution with most casesoccuring within the tropics. In recent years there has been a resurgence ofthe disease In the endemic areas and cases of 1e1shman1as1s have beenreported In countries where the disease was previously unknown, such asJapan (OKANO et a i . , 1977). Leishmaniases 1s divided Into two major forms,cutaneous- and visceral, of which the la t te r 1s the more serious form of thedisease and can be fatal I f untreated.
Visceral Ie1shman1as1s, also called Kaia-azar, Is well known fromancient times 1n the Old World where I t Is caused by the parasite speciesLeishmania donovani. The f i r s t known case of American Visceral Le1shman1as1s(AVL) was probably acquired 1n Brazil and reported by MIGNONE In 1913. Laterthe causative agent of AVL was given the name Leishmania chagasi (CUNHA &CHAGAS, 1937). Extensive studies since then have revealed ImportantInformation on the epidemiology, pathology and clinical aspects of thedisease In Brazil (DEANE 4 GRIMALDI, 1984) where more than 90% of recordedcases of AVL occur. The disease Is now recognised as a serious public healthproblem with more than 1000 new cases being reported annually to theBrazilian Ministry of Health In recent years. The main endemic focus of thedisease occurs In the rural area of the N.E. of Brazil while more recentlyfoci of the disease have become established In urban areas. Dogs arefrequently found with the Infection 1n areas where human visceral1e1shman1as1s occurs and their role as a reservoir for AVL Is wellestablished.
The Identity of the causative agent of AVL has been the cause of somecontroversy (reviewed by LAINSON, 1983). On the one hand the parasite Isthought to be Indigenous to the New World and 1s considered as a distinctspecies L. chagasi or separate subspecies L. d. chaoasi. On the other handsome authors consider that the parasite has been introduced Into America Inrecent times by Europen colonists, African slaves or accompanying dogs andthat the parasite should be classified as L. d. Infantum, the etiologicalagent of visceral leishmaniases In the Mediterranean Basin. A third view Isthat more than one parasite may be Involved.
We decided therefore to Investigate using biochemical techniques the4 Identity of the etiological agent of AVL. For this purpose we obtained
Isolates from cases of the disease from the state of Bahia In the mainnortheastern endemic focus as well as isolates from human 4 canine casesfrom a perl urban focus In the municipality of Rio de Janeiro (MARZOCHI et
. 5 8 .
a l . 1983).
LeishMania parasites possess a mitochondriai organdie known as thekinetoplast which contains 20-30% of the total cell DNA and protozoacontaining this organeile are classified in the order Kinetoplastida.Restriction-endonuclease generated fragments of this kinetoplast DNA (kDNA)can be analysed by gel electrophoresis in a technique known as sdrizotfowanalysis (MOREL et a l . 1980). The electrophoretic pattern obtained is abiochemical marker at the organelle genotype level that allows theclassification of Isolates and strains of parasites into schizodemes -populations having similar kDNA sequences. In recent years this techniquehas been used in taxonomic and epidemiological studies involvingkinetopiastid protozoa (see GONÇALVES et a l . , 1984; LOPES et a l , 1984).
Enzyme electrophoresis is a more widely used technique where enzymevariation between parasite populations are analyzed. The tecnnique measuresa phenotypic characteristic of the organism, the ami no acid sequence of theenzyme. As this character 1s a direct expression of the genome i t Is verystable and unlike some other phenotypic characters i t is unaffected byextrinsic factors. Using these two biochemical techniques, onecharacterizing the organism at the genotype level and one at the phenotypelevel, we analysed the isolates of AVL and compared them with referencestrains Isolated fro» cases of visceral leishmaniasis from the Old World.
Materiais I Hetftads
Details about the strains of Lei shut ant a used in this study arc shown inTable I . Isolation and culture of parasites, preparation of sample,conditions for enzyme electrophoresis and staining of gels was as describedpreviously (LOPES et a l . 1984). Schizodeme analysis was performed asdescribed by GONÇALVES et a l . (1984).
tcsmits
All isolates from both Rio de Janeiro and Bahia produced the sameenzyme variants when analysed by enzyme electrophoresis with the f o r wingenzymes: Aspartate aminotransferase IE.C.2.6.1.); Alanine andnotrans;.rase(E.C.2.6.I.2.); Mai ate dehydrogenase (E.C.I, 1.3.7.); Giucose-o-phospha,.*dehydrogenase (E.C.I.1.1.49.); Phosphogiucomutase (E.C.2.7.5.I.); and
; glucosephosphate Isomerase (E.C.5.3.I.9.).
The enzyme prof i le of these Isolates was the same as that of the two'; Old World strains and is the same as the enzyme profi le of Isolates of AVL\ from Rio de Janeiro published previously (LOPES et a l . 1964).
I In the state of Rio de Janeiro, 6 Isolates (4 human, 2 canine) were\ examined from the d is t r i c t of Campo Grande, 6 isolates (3 human, 3 canine)
from the d is t r ic t of Realengo and two human Isolates from the d is t r ic t ofBangu. The Isolates were examined by the restriction enzymes Msp I and MboI . The Isolate: from Campo Grande, Bangú and the canine Isolates fromRealengo gave Identical kDNA restr ict ion profiles with both enzymes whilethe remaining Isolates from Realengo were very slightly different. Fig 1shows the Mbo I restrict ion prof i le of these strains.
.59.
í
Nine Isolates of AVI from the state of Bahia as well as one Isolatefrom the state of Maranhão were also analysed using the sane enzymes (seeFig 2). The Isolates fe l l Into two schizoderaes the larger group containing 7Isolates from Bahia and the Isolate from Maranhão.
The remaining Isolate from Bahia (IOC-L72) has a restr ict ion prof i ledifferent from al l the other Isolates but similar to a strain (IX-L95)labelled L. chagaSI of uncertain origin but possibly Isolated from the foxCerdocyon thous. In Fig» 3 the two strains are I l lustrated together with thetwo strains from the Old World.
OlsaissiM
The main endemic area for AVL in Brazil stretches from the states ofBahia to Maranhão. The Isolate from Maranhão had a restr ict ion prof i lesimilar to that of the principal schizodeme found among the Isolates fromBahia and suggests that this schizodeme may have a wide spread distributionin the N.E. of Brazi l . Among the Isolates from Rio de Janeiro the sameschizodeme was found 1n Isolates from dogs and humans confirming theImportance of the dog as a reservoir host for this disease.
The results from the eiuyne electrophoresis analysis suggests that theetioiogical agent of AVL Is the same as that causing the disease 1n the OldWorld confirming previous studies (SCHNUR et a l . , 1981). Other workers(LAINSON et a l . , 1981 and MAAZOUN et a l . , 1981) have also been unable todistinguish L. chagasi from the agent of viceral 1e1shman1as1s 1n theMediterranean basin) L. d. Infantum using enzyme el ectrophoretic techniques.
The results from the schizodeme analysis however are more complex.Although some similarity was noted between the Isolates from R1o de Janeiroand the strain of L. d. Infantun, other strains Including the ones fromBahia had a different prof i le in particular Isolates IOC-L72 and IOC-L95.The distinct restr ict ion prof i le of those later two strains support thesuggestion that more than one etiologfcal agent may be causing AVL inBrazi l .
This work was supported by grants from the UNDP/WORLD BANK/MHOSpecial Programme for Research and Training 1n Tropical Diseases as well asthe Brazilian research funding agencies CNPq and FINEP.
.60.
1 . CUNHA, * . N. ft CHAGAS, E. (1937). Nova espécie de protozoan"o do gêneroLeishaania patogênico para o hoaem, Leishnania chagasi s.p. Hospital (RJ), 11:3-9.
2. DEANE, L. N. ft GRIMALOI, G. J r . Leishnaniasis in Brazi l . In: Chang K. P.* Bray R.S. (eds) leishmania. Amsterdam, Elsevier Biomedical Press ( InPress).
3. GONÇALVES, A. M., NEHME, N. S. ft MOREL, C. M. (1984). Trypanosonatidcharacterization by schizodeae analysis. In: C. M. Morel, ed.. Genes andAntigens of Parasites. A Laboratory Manual. Second edition (revised andtoryenlarged). Fundação Oswaldo Cruz, Rio de Janeiro, pp 95-109.
4. LAINSON (1983). The American Leishmaniases: some observation on theirecology and epidemiology. Trans. R. Soc. Trop.. Med. Hyg., 77:569-596.
5. LAINSON, R.; MILES, M. A. ft SHAW J . J . (1981). On the identification ofviscerotropic le i shmani as. Ann Trop. Med. Parasit. 75:251-253.
6. LOPES, U. G.; HOMEM, H.; GRIMALOI, G. J r . ; MARZOCHI, M. C. A.; PACHECO,R. S. ft MOREL, C. M. (1984). Schizodeme and zymodane characterization ofLei shmani a In the Investigation of foci of visceral and cutaneousle i shmani asis. J . Parasito!. ( In Press).
7. MAAZOUN, R.; LANOTTE, G.; PASTEUR, N.; RIOUX, J . A.; KENNOU, M. F. ;PRATLONG, F. (1981a). Ecologie des leishmaniases dars l e sud de laFrance. Ann. Parasito!. 56:131-146.
8 . MARZOCHI; M. C. A.; TOLEDO, L. M.; MARZOCHI, K. B. F.; COUTINHO, S. G. ftTRAMONTANO, *N. C. (1983). Lei shmani ose visceral no Rio de Janeiro.Aspectos epidemiologicos. XlXth. Cong. Soc. Bras. Med. Trop. 20-25thFebruary 1983. Abstract p. 60
9. MIGNONE, L. E. (1913). Un caso de kala-azar a Assuncion (Paraguay). Bul l .Soc. Path. Exo.. 118-120.
10. MOREL. C. M.; CHIAR I , E.; MATTEI, D. M.; ROMANHA, A. J , ft SIMPSON, L.(1980). Strains and clones of trypanosoma cruzi can be characterized bypattern of restriction endonuclease products of kinetoplast DNAminicircles. Proc. Nat!. Acad. Sci. (USA). 77:6810-6814.
11 . OK ANO, K.; SHIMAOA, M.; GO S.; MIYANO Y.; TANAKA, M.; WATANABE, Y.;TAGANI ft INOKI, S. (1977). A probable case of Ieishman1as1s (espundia)acquired in Japan. Southeast Asian J . Trop. Med. Pub. Helth. 8:546-551.
, 6 1 .
Table I - L i s t o f I s o l a t e s from human and canine cases o f AVL
Ourstock code
1 . I0C-L222 . I0C-L243 . IOC-L284 . IOC-L335 . J0C-L386 . I0C-L397 . I0C-L418 . I0C-L469 . I0C-L47
1 0 . I0C-L481 1 . I0C-L491 2 . I0C-L721 3 . I0C-L731 4 . I0C-L751 5 . I0C-L1201 6 . I0C-L1211 7 . I0C-L1471 8 . I0C-L1511 9 . I0C-L1522 0 . I0C-L1532 1 . IOC-LI702 2 . I0C-L1712 3 . I0C-L1722 4 . I0C-L1732 5 . I0C-L1742 6 . I0C-L1752 7 . IOC-LI76
OtherDesignations
HCG5HCG7ETS(MO)ACD0B19CCG3HR4CR9CRIORSCLSWP(BA-l)
Va1de11no,BA-5Ednaei ,BA-2
LRC-L52LRC-L47ImperatrizHR3HR5CR11BA-3BA-4BA-7BA-8BA-9BA-10BA-11
Host
ManNanManManDogDogManDogManManManManManManManManManManManDogManManManManManManMan
Place Isolat ion^)
Canpo Grande, RJCanpo Grande, RJCampo Grande, RJCampo Grande, RJCampo Grande, RJCampo Grande, RJRealengo, RJRealengo, RJRealengo, RJBangú, RJBangú, RJIrecê, BAJacobina, BAE. da Cunha, BAIndiaFranceImperatriz, MARealengo, RJRealengo, RJRealengo, RJJacobina, BAJacobina, BAR. Jacuipe, BACamaçari, BAIpi ra , BAIp i rá , BAM. Souza, BA
(*) Abbreviation of states of Brazi l :RJ - Rio de Janeiro; BA - Bahia; MA - Maranhão.
. 6 2 .
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.63.
PERMANENCE AND VARIATION OF ENZYME POLYMORPHISM IN CLONES OF
Trypanosoma unuzi STRAIN Y
AMILCAR TANURI and
DARCY F. DE ALMEIDA
Instituto de Biofísica, CCS, Universidade Federal do Rio de
Janeiro, Cidade Universitária, 21.941 Rio, RJ
Bloodstream trypomastigotes obtained from SW55 albino
mice experimentally infected with Trypanosoma cruzi strain Y
were cloned on 40% blood-agar medium. Twenty individual
clones were examined for their enzyme profiles by starch gel
electrophoresis; enzymes studied included glucosephosphate
isomerase (GPI), alanine aminotransferase, aspartate amino-
transferase, glucose-6-phosphate dehydrogenase, phosphogluco
mutase and 6-phosphogluconate dehydrogenase. All clones
presented the zymodeme A pattern (Romanha, A.J. et al., Comp.
Biochem. Physiol., 62B:139, 1979). To study the zymodeme
stability, one of these clones (Y12) was kept in the labora-
tory for one year, either through albino mice inoculation
(50 passages) or in NNN40% biphasic medium (25 passages) or
in LIT medium containing 10% calf serum (LIT1O%) (22 passa-
ges) . in every case zymodeme A pattern was preserved. Sub-
cloning of Y12 on agar-LIT10% originated ten subclones, all
of which presented the zymodeme B pattern. This was stable
for 22 passages in LIT10% medium. Further subcloning was
achieved by plating on agar-LIT5%: 10 subclones thus obtain-
ed showed the zymodeme C pattern. GPI isoenzymes extracted
from clones belonging to zymodemes A or C had identical
optimum pH (8.0) and Km values (2.5 x 10~ M).
r.64.
INTRASPECIFIC ISOEMZYMB DIVERSITY IN Trvpanosoma cruzi.
A. Tanuri and D.F. de Almeida. Instituto de Biofísica, univer-sidade Federal do Rio de Janeiro, Cidade Universitária, 21941,RJ, Brasil.
.Understanding the biology of T. cruzl and the transmission
of Chagas V disease depends to a large extent on the knowledge of
the intrinsic characterization of the parasite. This should
provide the basis for the investigation and analysis of important
properties of the parasite, such as, among others, the
pathogenicity and its variations as regards the animal hosts,
the tissue troplsm, the epidemiology, and the relationship host-
-parasite.
The occurrence of isoenzyme differences between T. cruzl
stocks (Toyi, P.J. 1974. Trans. Roy. Trop. Med. Hyg. 68, 147)
prompted several authors to attempt a definition of the
characteristics of isolated stocks of the parasite (Godfrey,
D.6. 1979. Brit. Soc. Parasitol, Synsp. 17: 31-53; Miles, M.A. et
al. 1980. Trans. Roy. Soc. Trop. Med. Hyg. 74: 221-237; Romanha,
A.J. et al. 1979. Comp. Biochem. Physlol. 62B: 139-142; Romanha,
A:J. 1982. Thesis, UFMG, Brazil; Lanham, S.M. et al. 1981. Trans'.
Roy. Soc. Trop. Med. Hyg. 75: 742-750; Tibayrenc, M. * Miles,
M.A. 1983. Trans. Roy. Soc. Trop. Med. Hyg. 77: 76-83; Ebert, F.
1982. Tropenmed. Parasitol. 33: 140-146; Ebert, F. 1983.
Tropenmed. Parasitol. 34: 84-88). As a result of such efforts,
three zymodemes could be identified on the basis of the
electrophoretic patterns of enzynic extracts of T. cruzl
(Romanha et al. 1979; Romanha, 1982; Miles et al., 1980).0
The significance of enzymic characters for the purposes
defined above depends on their stability (Miles et al., 1980).
Romanha et al. (1979) described changes of lsoenzyme patterns
upon prolonged subcultures of uncloned T. cruzl strain Y.
.65.
Cultures initiated from blood cultures evolved progressively
from symodeme A to B and to C, as indicated by the
electrophoretic patterns of six enzymes. Questions arising from
these studies are typically related to the origin of the
variations. Are they primarily due to genetically determined
variations, or do they result from the peculiar constitution of
different cell lines?
We have investigated the stability of clones of various
strains (Y, CL and Colombiana) of T. cruzl, talcing advantage of
the cell cloning method developed by Tanuri et al. (J. Parasitol.,
submitted for publication). More particularly, we examined the
pattern exhibited by clones of strain Y kept under various
culture conditions. Here we present the results obtained with
clones of strain Y (Silva, L.H.P. and Nussenzweig, V. 1953.
Folia Clin. Blol. 20: 191-208). In one case we studied further
the enzyme kinetics of alternative forms of a single enzyme.
Materials and Methods. T. cruzl strain Y has been provided
by Z. Brener. Bloodstream trypomastigotes were obtained from
infected SK55 albino mice weighing about 20 g and were cloned on
401 blood-agar solid medium (Tanuri et al. 1984. J. Parasitol.,
submitted for publication). Eplmastigotes were from cultures
in LIT (liver infusion-tryptose)-agar containing 5 or 10t calf
serum (Goldberg, S.S. et al. 1976. J. Protozool. Z3t 179-186).
The activity of the following enzymes were examined through
thin-layer starch-gel electrophoresls» glucosephosphate
isomerase (GPI, E.C.5.3.1.9), alanlne aminotransf erase (ALAT,
B.C.2.6.1.2), aspartate aminotransferase (ASAT, Z.C.2.6.1.1),
glucose 6-phosphate dehydrogenase (C6PD, E.C.I.1.1.49),
phosphoglucomutase (PGM, E.C.2.7.5,1), 6-phosphogluconate
dehydrogenase (6PGD, E.C.1.1.1.44), malate dehydrogenase
(oxaloacetate decarboxylatlng) (KADP+)(ME, B.C.1.1.1.40), and
.66.
ma late dehydrogenase (MDH, B.C.1.1.1.37). General procedures
ver* aa described by Romanha (1982). Ca-11 counts were performed
in a ZBI Bodel Coulter counter. Activity of GPI in extracts of
T. crmi was determined according to Noltmann (J. Biol. Chem.
239t 1545, 1964).
Besults.
1. Symodeme». Twenty individual clones have been obtained
from bloodstream trypomastigotes by plating on solid medium
(Tanuri et al., 1984). They were all identified as zymodeme A.
One of these clones. Y12(A), was initially kept for one year in
the laboratory, either by weekly passages in albino mice (total
of 50 passages), or else by serial transfer to fresh biphasic
Hl»40t medium (Tanuri, A. it al. 1981. An. Acad. brasil. Ciinc.
53t 842) (25 passages) or to LITlOt medium (LIT containing 10%
calf serum) (22 passages). Isoenzyme patterns for the eight
enzymes were not influenced by the various maintenance routines,
zymodeme A being detected in extracts of Y12(A) in every case.
Subsequently, cultures kept in LIT10% liquid medium have
been sub-cloned on LIT10%-agar. Ten sub-clones have been
isolated; they were all identified at zymodeme B, and remained
as such after two-day passages (total of 22 passages) in LITlOt
medium. These sub-clones are designated Y12(B).
Y12(B) sub-clones have been further sub-cloned on LIT5I-
-agar (solid LIT medium containing 51 calf serum). Ten sub-
-dones have been isolated, all of which belonged to zymodeme
C, as revealed by the electrophoretic pattern of eight chosen
enzymes.
2. Km and optimum pH« GPI isoenzymes have been examined
for Km and optimum pH values in extract* prepared from organisms
identified as zymodemes A or C. Identical optimum pH (8.0) and
Km (2.5 x 10"*M) have been found.
.67.
3. Infectivity. Organisms from the clone Y12(A) Inoculated
in mice (105 parasites/animal) produced parasitemia with a peak
of 5 x 106 parasites/ml. Organisms from clones Y12(B) and Y12
(C) inoculated in mice (up to 10 parasites/animal) did not cause
patent parasitemia (Brener, Z. 1962. Rev. Inst. Med. Trop. S.P.
±: 389-393). Infectivity of these clones could be restored by
three successive passages In baby mice, originating clones Y12
(B)ri and Y12(C)ri, respectively, which have been identified as
zymodeme A.
The results described point to the occurrence of enzyme
variation within clones of T. crnzl strain Y. The utilization
of cloned organisms eliminates the possibility of a
heterogeneous parental stock, as detected by other authors
(Goldberg, S.S. and Pereira, A.A.S. 1983. J. Parasitol. 69_: 91-
-96). The finding of unaltered kinetic characteristics of an
enzyme (GPI) from different zymodemes Is Intriguing. This kind
of study should be extended to other characteristics and to
other enzymes, in various T. cruzl strains.
Stability of isoenzyme patterns seems thus to be a function
of the environment provided for the organisms. Romanha et al.
(1979) have described changes in isoenzyme patterns of T. cruzi
upon prolonged sub-cultures. Therefore, the time factor is
also relevant for zymodeme stability in culture. Our data,
while supporting these findings, add another important variable
to the problem, namely the environment to which the parasite
is submitted. Zymodeae changed in parallel with modifications
introduced in the composition of culture media. Besides, our
experiments on the pathogenicity of clones exhibiting zymodemes
B, or C serve to further Illustrate this point. Recovery of
Infectivity of these clones, obtained through passages in baby
mice, was accompanied by a return to zymodeme A. Bearing in
.68.
í
11
mind the complex life cycle of the parasite, successively exposed
to changing environments within its respective hosts, we are
Inclined to consider enzyme polymorphism as one, among several
indicators, of the genetically determined behaviour of T. cruxl.
The most prominent of these indicators is cell differentiation,
which could represent a fitting response to specific changes in
the environment.
(Supported by grants from FIHEP, CHPq and CEPG/UFRJ. D.F.A. is
a Research Fellow, CNPq).
,69.
T. cruzi as a model system for studying cell differentiation :
Differential expression of the genes coding for surface antigens.
S. Goldenberg, V.T. Contreras, J.M. Ealles, M.c. Bonaldo, M.P.Amaral
de Lima Franco, D. Valle, J. Linss & CM. Morel
Deptí Bioquimica e Biologia Molecular - FIOCRUZAv. Brasil 4365 -• 21040 - Rio âc Janeiro - Brasil
The transformation of epimastigotes (the non-infective form)
to trypomastigotes (the infective form) is a crucial step within
T. cruzi life cycle. The transformation process, known as meta-
cyclogenesis, occurs naturally within the invertebrate- triatomine.
We have developed an in vitro differentiating system
allowing T. cruzi metacyclogenesis. The study of gene expression
products during the transformation process has shown that specific
sets of genes are switched on and off during the process. Among the
in vivo translation products primarily expressed during T. c>jzi
cell differentiation, we have detected trypomastigote stage specific
surface antigens. The appearance of trypomastigote stage specific
surface antigens has been correlated to the acquisition by
differentiating cells od trypomastigote stage specific biological
properties.
CNPq
UNDP/WORLD BANK/WHO- Special Programme for Research and Trainingin Tropical Diseases.
PURIFICATION OF HUMAN AMNION 1NTERFERON BY CIBACRON BLUE-AGAROSECHROMATOGRAPHYC.GONZAGA, R.R. GOLGHER, P.C.P. FERREIRA and E.G.KROON
Virus Laboratory - Department of MicrobiologyInstituto de Ciências Biológicas da UFMG.
ABSTRACTHuman amniop interferon (IFN) produced by amniotic membranes
infected by Senc'ei virus can be purified by several affinity ligandsafter concentration with trichloroacetic acid. Trp-trp-agarose, trp -tyr-agarose, cccanavalin A-agarose, bovin plasma albumin-agarose andcibacron blue F Gi-agarose were employed and the best preliminaryresults were cbtaited with this last ligand (GONZAGA et ai., I EncontroNacional de Vi-olora, p. 34, 1982).
Further experiments vere performed in order to stablish optimalconditions for the system. In chromatography columns, IFN was retainedby the ligand when it «as washed with phosphate buffered saline, pH7,2 (70 to 100%) and i small proportion (9 to 34%) eluted when the NaClconcentration was raised to 1M. The majority of the biological activityof IFN was recovered by displacing it with hydrophobic solventcontaining 50% ethylene glycol (EG). Recoveries with EG were in therange of 57 to 93% and the bindig of IFN was 17 thousand units per mlof gel. Under the same conditions but using batch preparation,quantity of IFN eluted with EG buffpr raised to 90% and 48 thousandunits of IFN per ml of gel were boundet.
pH dependence on the binding was investigated, varying the pH from3 to 7. It could be shown that the hydrophobicity of this IFN wasoptimal in pH 5.4. In higher pHs more IFN could be recovered in thewash through (low salt) fractions and with lower pHs, in the high saltfractions.
With batch applications in pH 5.4, recoveries of IFN wereconsistently higher than 100%. The average number of IFN units retainedper ml of gel was 36 thousand, with a peak of 60 thousand. Thepurification factor with this single step varied from 22 to 36.
These purified preparations were shown to be compatible with humanclinical trials in viral eye infections.
Work supporteo by FIPEC (Fundo de Incentivo ã Pesquisa Técnico -Científica do Banco do Brasil) and FINEP (Financiadora dé Estudos eProjetos) grant B.76.81.237.00.00.
.71.
CHARACTERIZATION OF BOVINE LEUKEMIA VIRUS IMMUNOGENS
A.L.T.O.do NASCIMENTO and
T. HIGUCHI
Dept Biochemistry - Instituto de Química - USP - São Paulo.
The bovine leukemia virus (BLV) was identified in pe-
ripheral blood lymphocytes from infected cattle (FERRER et al-
Cancer Res.. }2_ : 1864-70, 1972). Their finding was confirmed
by several av.thois based on other criteria. There are variety
of pathological < ffects due to the viral infection and the vi-
rus particles have been found in bat,sheep,mite and fleas. Mor
phologically, BLV is very similar to other known type C tumour
virus : budding process, density 1,15 - l,17g/cm , 70S-RNA and
reverse transcriptase. In the other hand, the viruses have pe
culiar properties; they seem to have different determinant
groups. Similarly to other tumor viruses antigens, these immu
nogens seem to be cleavage products of precursor, in this case
p65. The structural component p25 was purified in Sephadex G-
150 column in two steps; 5.7 ig of p25 was recovered from 14.4
mg of extract.- The glycoprotein gp51 was separated using affi_
nity column - LH-Sepharose, the bound glycoprotein was e lu-
ted witho(-methyl manopiranoside lOOmM. Improvements in the
particles recovery in sucrose gradient is needed. The unders-
tanding of the antigen-antibody relationship should provide us
of ways of detection of viral particles in lymphocytes, secre-
tions, cellular extracts; regulation of expression of p25. The
immunogology of the system could give indications of the possj_
bility of some protein be used for vaccination and/or produc-
tion of antibodies for therapy.
SUPPORT : FAPESP and CNPq.
.72.
CHARACTERIZATION AND PURIFICATION OF STRUCTURAL COMPONENTS OF
AVIAN LEUCOSIS (EXOGENOUS AND ENDOGENOUS) VIRUS
J.M.M.S. FELIPPE
Department of Biochemistry
Instituto de Química - USP - São Paulo
C. ROMERO
Department of Animal Patology
EMBRAPA - Concórdia - Santa Catarina
T. HIGUCHI
Department of Biochemistry
Instituto de Química - USP - São Paulo
Structural proteins of tumour viruses are located
in the core and outer membrane, they are the result of suces-
sive cleavages of 180,000 moleculaT weight precursor. Virus
extract obtainned from inactivaded culture of chicken embryo
fibroblast infected with ALV or AMV (exogenous) and derived
from culture fluid of normal embryo fibroblast of line 1515 e
7j (endogenous virus) after proper purification were applied
into Sepharose-6B column saturated with guanidine hydroclori-
de and eluted with acetate buffer and guanidine-HCl 6M. Seven
components were separated, identified as p27, p!9, pl5, pl2
and plO, the structural proteins. The purification steps were
followed by PAGE and protein determination. The properties of
each component are under investigation. It is highly desira-
ble the knowledge of physico-chemical properties for a better
understanding of these unique immunogens. The regulation, ex-
pression of these proteins, function in the embryogenesis and
the trigger of tumor process are some of the problems to be
solved. The significance of endogenous virus activation should
be considered by the detection of determinants groups. The im
portancc of the expression of structural proteins in brazi-
lian flocks deserve attention.
.73.
w
IMMUNOLOGICAL CHARACTERIZATION OF STRUCTURAL PROTEINS OF THE
AVIAN TUMOUR VIRUS
J.M.M.S. FELIPPE
Department of BiochemistryInstituto de Química - USP - São Paulo
T. HIGUCHI
Department of Biochemistry
Instituto de Química - USP - São Paulo
The arrangement of retrovirus genes has been propo-
sed as being : 5'-R-U5-PB~-gag-pol-env-src-PB -Uj-LTR-3'. The
gag region ("v2Kb) is translated into 75K poliprotein which
upon sucessivo cleavages give rise to structural antigens. The
p27 is the major component, arrangements of the subunits com-
poses cne core shell. The pl9 is a phosphopTotein found in
association with viral RNA; pl2 is highly basic antigen and
plO is the component found in the smallest amount. The pl5 is
supposed to be 1ocated between the core and inner envelope
with the remarkable enzymatic activity of cleaving the precur
sor into virions proteins. The immunogens p2 7, pl9 and pl5 w£
re characterized by RIA, using -apToximately l.Ong of label-
led antigen the antiserum (1:2,000; 1:2,000 and 1:1,000 res-
pectively) were determinated. Competition assay were carried
out with chicken embTyo fibroblasts, several tissue extracts
of adult birds and structural proteins derived fTom endoge-
nous virus. Some expression were found in certain tissues.
These immunogens are an exciting subject foT further analysis
in order to help the understanding of the complex phenomenon
of cellular differentiation and interaction of factors lea-
ding to the loss of cellular equilibrium. It is highly desi-
rable to choose an antigen that afteT propeT treatment could
be used to prevent the disease as well for large scale antibo
dy indue ion.
.74.
OBTENTION OF SHEEP ANTI-RABBIT IgG : PRECIPITATING SYSTEM OF
RADIOIMMUNOASSAY
H. OGATA
Dept of Biochemistry - Instituto de Química - USP-S.Paulo.
T. HIGUCHIDept of Biochemistry - Instituto de Química - USP - S.Paulo.
Two classes of methods are generally used in antibo-
dy purification. The inespecific techniques are based on the
physicochemical properties of globulins. We have been purify-
ing rabbit IgG by serum precipitation with cold ammonium sul-
phate to 501 saturation, followed by DEAE-cellulose chromato-
graphy. Further on, gel filtration in Sephadex G-200 and G-
100 were carried out in order to eliminate globulin aggregates.
All the purification steps were followed by polyacrylamide so-
dium dodecyl sulphate electrophoresis and protein content de-
termination. The purified IgG carefully suspended in comple-
te Freíínd adjuvant was injected in sheep three times. The de-
gree of immunization was followed by immunodiffusion test-Ouch
terlony. The animal showed response three months after injec-
tion. The system rabbit IgG vs anti-rabbit IgG is used as precini
tatiiig sysrem of radioimmunoassay. Booster injections are un-
derway in order to increase the titre of immune serum and then
higher sensitivity could be achieved besides the reduction of
reaction volume. The immunoglobulin studies iswof significant
importance not only because of its ability to neutralize the
antigen determinant groups as well as because of several uses
in detection of the presence of particular macromolecule in
precipitation systems with or without labelled components.
.75.
PERPECTIVES ON AVIAN AND BOVINE LEUKEMIA VIRUS IMHUNOLOGICAL STUDIES
TOHOKO HIGUCHI; JULIA M.M.OE SOUZA; ZÉLIA MARIA NOGUEIRA AND HI ROE
OGATA - Dept of Biochemistry - " Inst i tuto de Química" - Universida^
de São Paulo - Cx. Postal, 20.780 - 01498 - São Paulo - Brasi l .
All eucaryotic organisms might go through al terat ion in the
cel l growth pattern and d i f f e ren t i a t ion , giving rise to localized
or disseminated process. Several agents have being described as
responsible factor for tumour and cancer, some by i t s e l f and others
by combination of two or more factors. Among the et io logical agents
our attention is focused on the RNA vi rus, retrovirus or oncornav_[
rus. The retrovirus have been isolated in f i s h , domestic animals,
primatas and recently among human beings (HINUMA et a i . , 1982; P0I_
ESZ et a i . (1982). One of the feature of this virus is the preseii
ce of the enzyme reverse transcriptase or DNA polymerase RNA depen
dent described simultaneously by TEMIN & MIZUTANI (1970) and BALH
MORE (1970) , this enzyme catalyzes the synthesis of DNA from RNA
template, i t is encoded in the v i ra l genome, composed of twto sub-
units a and $. The replication of viruses need not k i l l the host
cel l and the maturation of particles does not necessary leaves cy-
tophatic e f fec ts . There are three main knids of interaction betwe
en viruses-host cel l : transformed productive, transformed-non-pro
ductive and non-transformed-productive.
The genome of a competent RNA tumour viruses has been proposed
as follows : 5'-R-U5-PB(-)-L-gag-pol-env-src-PB(+)-U3-R, where R
1s a short sequence repeated at both ends, supposed to be Involved
In the transfer of DNA polymerase; U5 and U3 separate the R from
PB, I t s function 1s unknown; PB(-) 1s the binding site of the tRNA
for DNA negative strand synthesis; L * leader sequence; might con-
tain the sequence necessary for the element of RNA packing; gag-
.76.
encodes al l the internal structural proteins; pol-encodes the re-
verse transcriptase; env-encodes the surface glycoprotein; src-co-
des for the factor responsible for transformation and not required
for replication; one-codes for the component leading to transform^
tion and induction of tumour; PB(+)-initiation site of the positi-
ve strand; U.-might interfere in the transcription of the genome
(WEISS et a i . , 1982).
The structural proteins are synthesized as precursor of 180.000
K, which is sucessively cleaved by proteolytic enzymes (SHAPIRO &
AUGUST, 1976; VOGT et a l . , 1975). The origin of a l l the proteases
are not known, but in the avian retravirus the pi5 protein seems
to have catalytic activity.
The retrovirus are composed of an internal core structure sur-
rounded by an outer membrane with knobs and spikes and ribonucleo-
protein complex. The biochemical nature of the structural compo-
nents is poorly understood. In the avian tumour viruses, p27 is
the major component, being rich in group antigenic determinants,
p19 is hydrophobic; pl2 is highly phosphorilated and basic in natu
re; pi5 1s the carboxiterminal residue with remarkable proteolytic
activity.
Structural proteins from avian myeiobiastosis, avian leukemia
and bovine leukemia viruses have being Investigated. The avian and
bovine leukemia viruses cultures are done by Dr C.H. Romero (Cen-
tro Nacional de Pesquisas de Suínos e Aves EMBRAPA, Concórdia) in
conditions written elsewhere, and are kindly, generously supplied
whenever necessary.
The culture fluids from tumour viruses are clarified by low
speed centHfugation. followed by high ultracentr1fugat1on(P.I = 10)
then, the pellets are submitted to sucrose gradient centrifugation
.77.
In the avian leukosis virus system, the extract prepared un-
der controlled conditions is submitted to a Sepharose-4B column sa
turated with guanidine hydrochloride. The elution pattern is shown
in the Figure 1 , sample eluted with sodium acetate buffer pH 8.5
containning B-ME and GuHCl. The second peak is the glycoprotein
plus some contaminating proteins, the foilowings peaks are the an-
tigens p27, p l9 , p l5 , pi2 and plO. The protein renaturation is do
ne by dialysis against buffer with 8ME and EDTA pH 8 .0 , giving ap-
proximatelly 60X recovery. The protein content and polyacrylamide
sodium dodecyl sulphate eiectrophoresis (PAGE) are done af ter dia-
lys is . The TABLE I shows the protein determination of the p u r i f i -
ed components according to BREADFORD method. (1976). The y ie ld can
be improved by treatment of the diaiysed supernatant, neverthless
the amount of time used might be quite high.
The react iv i ty of the immunogens; class of determinants groups
and a f f i n i t y are usually evaluated by rath oimmunoassay in homolo-
gous and heterologous competition assays. (HANAFUSA et a l . , 1972;
HIGUCHI, 1975; HIGUCHI & AUGUST, 1977; GUPTA & FERRFR, 1980) due
to high s e n s i t i . t y and spec i f ic i ty of the experimental procedure.
The t i t r a t i o n of antisera is presented in Figure 2, the labelled4
antigens show the following specif ic ac t iv i t ies : 1.96 x 10 cpm/ng
p27; 2.16 x 10 cpm/ng p19 and 2.12 A lü4cpm/ng p!5 and give us
hints about the meaning of the expression. The shape of the cur-
ves allows us the assertion that pl5 is more reactive than p27 and
p19, however the phenomenon depends on the class of determinants
groups and/or the number of exposed reactive residues in the mole-
cule under the experimental conditions used (HIGUCHI, 1977 and JOHN
SON et a l . , 1981). Competition assays are carried out with ex-
tracts from cardiac muscle, bra in , wing and tights of adult chicken
and embryo fibrobiasts with protein concentration ranging from 300ug
.73.
to 3.0mg,surprisin!y, high expression is found in wing muscle -
4ng of p15 equivalents in 3.8rag,of ex t ract ; moreover, 2.5ng plS/mg
cardiac muscle (SOUZA & HfGUCHI, 1981, 1983; HIG'CHI &SOUZA,1983.
The assay system is composed of 0.03ml NRS d i l u t i o n ; 0.02ml Immu-•joc
ne serum; 0.02ml I-P (= 55,000 cpm) and 0.03ml Goat anti - r a b -
b i t , Pel let collected by centrifugation and radioact iv i ty measu-
red in gamma counter. Determinations of the structural compo-
nents in flocks of several regions in f ibroblasts and embryos in
dif ferent stages of d i f ferent ia t ion are in progress in order to
have more signif icant results . The endogenous viruses are of h i -
ghly relevance, excite the curiosity and i t is a grapple of seve-
ral groups of investigators. (HANAFUSA et a l . , 1970) fount, in u-
ninfected chicke-n embryos expression of a helper factor , now cha-
racterized as glycoprotein, product of env gene. The presence of
determinants groups of avian tumour virus and other viruses in
normal cells might be an Indication of the i r functional role in
the embryogenesis and even in expontaneous induction of tumour.
The virus genomes can be transmitted from one generation to
another, without expression of the i r products. In these condi-
t ions, the v i ra l genome seems to be Integrated in the host cel l
under certain regulation process (GROUDINE et a l . , 1981). The di£
tribution and expression of endogenous provi ruses can very grea-
t l y from Individual to Individual ranging from complete silence
through production of par t ic les . The endogenous viruses are of
h i jh iy relevance (JENKINS et a l . , 1981; ROWE & KOZAK, 1980; DENI-
SON & WEINER, 1982; HOLLIS et a l . , 1982). Complete v i ra l p a r t i -
cles were Isolated by VOGT & FRISS (1971), hybridization and clo-
ning experiments have shown the structure and specific functional
competence (HISHIMURA et a l . , 1981; BAKER et a l . , 1981;. The
expression of some endogenous virus 1s controlled by
. 7 9 .
gene V-E7, not related to £S and chf. Dr. Rf.ro provided us'with
two endogenous lines 7? and 151 5 . The p. ' ication of both lines
have shown identical structural compcn * t s , althrough in sucrose
gradient the prof i le is s l ight ty n i e r e n t from exogenous viruses.
The endogenous part icles seem •. nave smaller density coeff icient
(SOUZA & HIGUCHI, 1984). T - ' .ature, function as well as other
biological aspects are n< understood,questions have been asked,
l ike : are there benefi t* or dangers to a host cel l to carry endoge
nous provirus, where do they come from? for some authors the endo
genous provirus is a sucessful l i f e - s t y l e (WEISS et a l . , 1983).
The bovine leukaemia virus (BLV) was found in association
with lymphosarcoma, a malignancy of lymphoreticular system but
might have heart, uterus and other organs damages. The BLV is a
type C part ic le as far as morphology and size but have some pecu-
l i a r i t i e s in the reverse transcriptase propert ies, envelope glyco
protein, antigen plG (WUU et a l . , 1977; DEVARE I STEPHENSON, 1977);
induces syncytia formation in EP-BESP cells (non producer mouse
sarcoma virus transformed, fel ine t e l l s ) . The BLV is not related
to other known cat t le viruses; the infected lymphocytes do not
produce part icles unless a f ter cult ivation (BALIGA & FERRER, 1977).
A protein of 65K has been ident i f ied as blocking factor in the ex
pression of v i rus , i ts level might undergo changes leading to v i -
ral par t ic le maturation.
The pur i f icat ion of p25 and glycoprotein have been done by a
modification of the procedure written by ONUNA et a l . (1976) PHIL.
LIPS et a l . (1978) and KONNO (1983). The pe l le t of the f lu id u l -
tracentri fugation was used for glycoprotein pur i f icat ion and the
soluble fraction for p25 Iso la t ion . The Figure 3 shows the e lu -
tion pattern of glycoprotein when 22mg of protein was passed
through LH-Sepharose and la ter washed with a-D-manopiranoside 100
.80.
mN at pH 7.4 (NASCIMENTO & HIGUCHI, 1983, 1984). The p25 compo-
nent was puri f ied from the supernatant of the c l a r i f i e d culture
f lu id af ter ammonium sulphate precipitation and Sephadex 6-150 co-
lumn. The polyacrylamide gel eiectrophoresis showed the presence
of impurities in the prep besides the structural antigens p15 and
plO. Improvements in the y ie ld of virus pe l le t was gotten by a se
cond sucrose gradient (30-50%) centrifugation for 18 hours (KONNO,
personal communication).
Surely, a l l antigens studies require the complementation with
antibodies research. The immunoglobulins are induced molecules, se_
creted by activacted lymphocytes (plasmocytes), being highly spec^
f i e , having two combining sites Fat> located in the heavy and l ight
chains. The study of the IgG molecule need obviously, the p u r i f i -
cation of the molecule from immune serum. The pur i f icat ion is car
ried out by ammonium sulphate precip i ta t ion, followed by DEAE-cel-
luiose chromatography, f i l t r a t i o n on Sephadex G-200 and eventually
in Sephadex G-100. The Figure 4 shows the prof i le of elution in
DEAE-cellulose and Sephadex G-200 of rabbit IgG puri f icat ion (Expe
Mmentai detai ls written elsewhere (ZERBINI et a i . , 1980; OGATA &
HIGUCHI, 1984). In the TABLE I I the protein content of each step
is presented.
. In order to study the Fa_b fragment, more precisely the CDR's
(Complementary determining region) and Fr's (Framework region), the
heavy and l ight chains of the molecule was separated. lOOmg of IgG
was reduced in the presence of 0.75M 6-ME, the reaction was k i l l e d
at 49C with lodoacetamide 75mM. After proper dialysis the mixture
1s f i l t e r e d 1n Sephadex G-75 column eluted with propionic acid IN
(Figure 5 ) . Reduction of IgG 1n the presence of 10,30 and 50mM
cysteine 1s under way.
. 8 1 .
The knowledge of immunoglobulins, the size and nature of the i r
active sites are of relevance In the proper therapeutic and p r o f i -
l a t ic measures in public health.
The determination of the kinetics of v i ra l structural proteins
expression in the course of tumour development; the detection of
the.role of some antigens in the d i f ferent ia t ion process or embry£
genesis; ways of spoting the presence of viruses and quantitative
measurement in f luids and organs of chicken and animals are the g£
ais of our projects and undoubtedly the pier of further steps. I t
is desirable the def in i t ion of at least one antigen which is able
to induce protective antibodies to be used in vaccination and the-
rapy. The significance of immun o logical defense in the role of tit
mour control requires a better understanding of the nature of the
viruses antigens and i t s immunoglobulins.
ACKNOWLEDGMENTS : We are deeply grateful to Dr. C.H. Romero(EMBRAPA)
for providing us with the cultures and he is co-author in some of
our studies. Prof Dr A.A. Pupo for le t t ing us use his gamma counter
our sincere appreciation.Mrs. A.L.O.N. was involved in early sta-
ges of BLV project. Research and fellowships support from : FAPESP,
CNPq, CAPES and F. Bunka.
REFERENCES : BALTIMORE, D. - Nature (London); 226 : 1209, 1970
BREADFORD, M. - Anal. Bi ochem., Jl '• 2 4 8 » 1 9 7 6 - DENISON & WEINER -
Mol .Cel l .B io . , 2 : 815, 1982 - GREEN,R.W. & BOLOGNESI, D.P. - Anal.
Biochem., 57 : 108, 1974 - GROUDINE, M. & WEINTRAUB, H. - Proc.
Nati .Acad.Sci.(USA)., Jl (9) = 5351-54, 1980, and Nature. , 292 :
311, 1981 - GUPTA, P. & FERRER, J.F. - J .Gen.V i ro l . , 47 : 311,
1980 - GUPTA, P. & FERRER, J.F. - Science., 215 : 405, 1982 - HANA
FUSA, H.; MIYAMOTO, T . ; HANAFUSA, T. - Proc.Natl.Acad.Sci.(USA).,
.82.
66 : 314 , 1970 - HANAFUSA. T . ; HANAFUSA, M . ; MIYAMOTO, T . ; FLEIS-
SNER, E. - V i r o l o g y . , 47 : 4 7 5 , 1972 - HIGUCHI, T. - Tese de L i v r e
Docência, 1975 - HIGUCHI, T. ft AUGUST, J . T . - Rev.Bras.Pesq.Med.
B i o l . , H) : 1 , 1 9 7 7 - HIGUCHI, T . ft AUGUST, J . T . - An .Acad.bras .
C i ê n c , 49 ( 2 ) : 3 3 7 , 1977 - HIGUCHI, T. & SOUZA, J.M.M.de - 5 —
I n t l . C o n g r e s s . I m u n o l . , 1983 - HINUMA, Y . ; NAGATA, K. ; HANAOKA, M.;
NAKAI, M. ; MATSUMOTO, T . ; KINOSHITA, K . I . ; SHIRAKAWA, S. ; MIYOSHI,
I . - P r o c . N a t 1 . A c a d . S c 1 . ( U S A ) . , 78 ( 10 ) : 6476 , 1981 - JENKINS, N.
A. ' f t COOPER, G.M. - J . V i r o l . , 36 ( 3 ) : 6 8 4 - 9 1 , 1980 , and N a t u r e . ,
293 : 370 , 1981 - JOHNSON, J . C . ; HIGUCHI, T . ; GEISSLER, E. - Can .J .
M i c r o b i o l . , 2_7 ( 2 ) : 2 3 8 , 1981 - NASCIMENTO, A .L .O . & HIGUCHI, T. -
A r q . B i o l . T e c n o l . , 26 ( 2 ) : 3 0 9 , 1983 - NASCIMENTO, A .L .O . & HIGU-
CHI , T. - A r q . B i o l . T e c n o l . , 27 ( 2 ) : 104, 1984 - POIESZ, B . J . ; RUS
CETTI , F.W.; MIER, J . W . ; WOODS, A . M . ; GALLO, R.C. - P r o c . N a t i . A c a d .
S d . ( U S A ) . , 77 : 6815 , 1980 - ROWE, W.P. & KOZAK, C E . - P r o c . N a t l .
A c a d . S c i . ( U S A ) . , 72 ( 8 ) : 4 8 7 1 , 1980 - SHAPIRO, S .Z . ft AUGUST, J.T.
- B1och.B1oph.Acta . , 458 : 375 , 1976 - SOUZA, J.M.M.de & HIGUCHI,
T. - A r q . B i o l . T e c n o l . , 24 ( 1 ) : 155, 1981 - SOUZA, J.M.M.de & HIGU
CHI , T . - A r q . B i o l . T e c n o l . , 26 ( 2 ) : 308 , 1983 - SOUZA, J.M.M.de &
HIGUCHI, T. - A r q . B i o l . T e c n o l . , 27 ( 2 ) : 104, 1984 - TEMIN, H.M. &
MIZUTANI, S. - Nature ( L o n d o n ) . , 226 : 1 2 1 1 , 1970 - VARMUS, I . &LE
VINE, A . J . - Readings in Tumour V i ro logy - 1983-CSHL - VOGT, P.K. ft
FRISS, R.R. - V i r o l o g y . , 43 : 2 2 3 , 1971 - VOGT, V . M . ; EISENMAN, R.;
DIGGELMANN, H. - J . M o l . B i o i . , 96 : 4 7 1 , 1975 - WEISS, R. ; TEICH.N. ;
VARMUS, H . ; COFFIN, J . - Mo lecu lar Biology of Tumour V i r u s e s . , 2 —
e d . , 1982 - Cold Spring Harbor Laboratory .
. 83 .
TABLE I - Protein Determination - BREADFORD Metnod
Fraction
I
II "
III
IV
V
VI
VII
V9
16.0
9.5
17.5
7.5
4.0
6.0
2.0
mg/ml
0.320
0.475
0.875
0.373
0.200
0.300
0.100
Total : ug
372
950
1 . 7 4 2
675
300
330
140
lOyi sample + reagent (Coomassie Blue-G; phosphoM e acid + ethanoi) - Incubation period * 2-5 minutes; absorbance at 595nm. Standard curve =0.1mg/ml of bovine serum albumin.
TABLE II - Rabbit IgG Purification Protein Determination
Fraction
SerumA.S.stepDEAE-coiumn6-200G-100
mg/ml
27.436.260.8
0.50.88
Vol.(ml)
105.025.06.8
222.064.0
Totalmg
2,877.0905.0413.4111.056.4
Yield
10031.4614.343.861.96
.84.
Of ftLV ifcru.ctu.roA.
as-
0.1 -
VI15 K) < 130 I5S
K
i240
FRACTION N°(ml)
0.C
ÍI «4 rt and
.85 .
50
TITRATION OF ANTI-SERUM: Anti-p27, Anti-pl9 and Anti-pl5
rose w
100
P27
1:100 I 10000Ami-scrum dilution
FIGURE I
Of BLV-pbS" ;
•» « • MO HO
.86.
REGULATION OF TREHALOSE METABOLISM IN Saaeharomyee» - AN APPROACH
TO THE IMPROVEMENT OF TECHNOLOGICAL PROCESSES
A.D. PANEK, V.L.A. COSTA-CARVALHO, C.H.D. ORTIZ, G.M. DELLAMORA-
ORTIZ, V.M.F. PASCHOALIN and A.C. PANEK
Dept. of Biochemistry, Inst. of Chemistry, CCMN, UFRJ, Brasil
Trehalose has been classified over the years as a storage ca±
bohydrate in yeast. We have proposed an additional function as a
regulator of the glycolytic flux during maltose utilization.
According to Suomalainen f, Pfãffli (1961) viability and act^
vity of pressed baker's yeast is guaranteed by high contents of
storage carbohydrates. Trehalose, specifically has been shown to
have effect on the baking properties of active dried yeast. Idea
lly, the feed of nutrients should be stopped at the end of the
production process to allow for trehalose accumulation and, there
fore, improve the leavening capacity and stability upon storage
of commercial baker's yeast (Oura et ai. 1974). It should be po
tencially possible, once we learn the mechanisms which regulate
trehalose metabolism, to construct strains with high trehalose
synthase activity. On the other hand, when the biomass is condi^
tioned for leavening, the hydrolytic enzyme, trehalase, should be
activated.
Our approach to the study of the regulation of trehalose meta
boi ism has been construction of mutants with specific lesions,
cloning of genes involved in the regulation of trehalose synthase
and of trehalase, as well as, isolation and purification of enzjr
mes from the various mutants constructed.
Cloning of a gene which affects trehalose synthesis
Trehalose-6-P-synthase was identified and partially purified
by Cabib ft Leloir (1958), however, no information is available on
allosteric effectors or on interconversion of forms similarly to
187.
yeast glycogen synthase. The substrates for the enzyme are G-6-P
and UDPG.
A mutant of S.carlsbergentie which does not grow on glucose,
fructose, mannose or sucrose was described by van de Poll et al
in 1974. High levels of fructose diphosphatase were proposed to
explain the observed phenotype designated as fdp. Later results
suggested that the defect could correspond to a secondary conse
quence of an unidentified primary lesion because glycogen metabo
lism is also affected (van de Poll 5 Schamhart, 1977). Moreover,
the strain proved also to be unable to accumulate trehalose in
spite of normal trehalase activity (Panek et al. 1979). Such wide
variety of effects indicates that the fdp mutation affects some
regulatory system which modulates many metabolic functions in the
cell. We have obtained a partial revertant of the original fdp mu
tant capable of normal growth on glucose which retained the inabjL
lity to grow on fructose and showed absence of UDPG-1inked treha
lose-6-P-synthase activity. Another mutant of Saceharomyce8 was
described (cif) with similar phenotypic characteristics to the
partial revertant we have obtained (Navon et al. 1979). Complemen
tat ion tests between these two strains indicated that genes fdp
and cif are allelic (Charlab et al. in press). Due to the pleio
tropic effects observed in these mutants we propose that the lack
of trehalose-6-P-synthase activity corresponds to one of the
effects of a lesion in a regulatory gene.
Two yeast genomic libraries constructed by K. Nasmith and
S. Reed (1980) in the E.ooli - yeast shuttle vectors YRp7 and
YEpl3, are being used to clone the FDP gene. Mutants with trpl
fdp lyeZ and Ieu2 fdp Iys2 genotypes were constructed from strains
Q6R2 (fdp) and KlglO2 (cif). These strains showed to be good r£
ceptor strains by transformation with DNA prepared from both plas_
.88.
mids. We envisage the isolation of the FDP gene and hope that an
overdosage will produce strains with high levels of trehalose syn
thase activity. This would confer to the strains improved stabili_
ty upon storage and better baking activity.
Maltose utilization linked to trehalose synthesis in yeast
Years ago I studied trehalose synthesis during growth on glu
cose in a strain of baker's yeast (Fanek, 1962). The observation
that significant accumulation occurred during the transition pha
se from fermentative to oxidative metabolism led to the hypothe
sis that the disaccharide would provide energy for respiratory
adaptation (Panek 5 Mattoon, 1977). However, when various unrela
ted strains were examined, only in very few cases, was trehalose
detected during diauxie. Therefore, the disaccharide should not
be considered as a significant source of energy for driving reac
tions associated with the biogenesis of mitochondria.
Some strains of brewer's yeast showed the same pattern of
accumulation than my first batch of baker's yeast. Genetic analy_
sis indicated that the common characteristic in these strains was
the presence of a constitutive MAL4 gene. Thus, 1 learned that
the accumulation of trehalose during growth on glucose is an ex
ception rather than a rule. However, when various strains of Saa_
charonycae which harbour MAL genes are compared, accumulation of
trehalose during growth on glucose, is not invariably associated
with genes that regulate maltose fermentation. The simple presen
ce of a MAL6 or a MAL2 gene does not elicit trehalose accumulation
during growth on glucose. These genes are inducible while the
MAL4 gene, in the batches I had used, is always found in nature
in constitutive form. When we mutated a MAL2 strain to its consti.
tutive form,trehalose synthesis was seen during diauxie (Oliveira
et ai. 1981). The product of the gene is, therefore, a pre-requ_i
,89.
site of the phenomenon of trehalose accumulation. Obviously, any
strain harbouring a MAL gene would accumulate trehalose when grown
on maltose because then the gene would be expressed.
In the presence of galactose, another fermentable carbon sour
ce or of glycerol, a non fermentable carbon source, strains harbo
ring MAL indueible genes behaved as in the presence of glucose:
no trehalose accumulation occurred (Panek et al. 1979). This was
good evidence for the existence of a specific, unique, relation
ship between maltose utilization and trehalose accumulation.
Oi<r first attempt at elucidating this problem was to construct
a strain with a lesions for UDPG-trehalose-6-P-synthase activity
(fdp) as well as, for uncontrolled maltose uptake (hex2) resulting
in intracellular maltose accumulation (Entian, 1980). Since gene
HEX2 appears to be specifically involved in regulating the forma
tion or activity of the maltose uptake system, our rationale was
to study trehalose synthesis linked to maltose uptake in a mutant
in which that regulatory activity is abolished and in which no
UDPG-trehalose synthase activity can be detected. When cells were
incubated with maltose in non proliferating conditions, intrace^
lular maltose was constant at 15 yg/mg in a control strain, where
as, in the hexZ mutant a 3-fold increase was observed. Trehalose
accumulation followed exactly the same pattern corroborating our
hypothesis of a maltose-linked synthesis. Partial inhibition of
hexokinase by xylose (DelaFuente, 1970), during growth on maltose,
reduced glycogen synthesis in 70S and trehalose in 491. On the
other hand, NaF blocked glycogen synthesis to the same extent as
xylose, however, trehalose accumulation was not affected. In all
cases calculations were made considering the ratio between carbo
hydrate formed and maltose utilized. Trehalose should be determi
ned after destruction of other sugars by alkali (Elbein, 1967)
.90.
whereas maltose assessed after hydrolysis by the glucose oxidase
method. The anthrone reaction for trehalose in the presence of
maltose gives misleading values.
These preliminary results indicate that G-6-P, impaired by xy_
lose but not by NaF, plays a role in trehalose synthesis by this
specific system. Reduced levels of UDPG by both inhibitors affec
ted glycogen accumulation. However, it seems premature to analyze
the role of UDPG in the synthesis of trehalose by this alternate
ve pathway. We propose that a product of the MAL gene would serve
as a common positive regulator for the expression of the genes co
ding for maltose permease, a-glucosidase and some component of
the trehalose synthesis system. The obligatory passage through
trehalose would slow down the glycolytic flux allowing for dere
pression of mitochondria which in turn, are a pre-requisite for
efficient maltose utilization. Not only malt but many partial
starch hydrolysates contain maltose, therefore, this alternative
system for trehalose accumulation has to be focused for ethanol
production, as well as for enriching baker's yeast. In this case
high levels of accumulated trehalose should be rapidly depleted
when the biomass is used for leavening. This leads us to the next
aspect of our research.
Trehalase - a specific substrate for cAMP dependent protein kinase
In the sixties I showed that trehalose was very rapidly bro
ken down at the onset of a growth cycle (Panek, 1963). Years la
ter, van der Plaat § Solingen (1974) demonstrated that this pheno
menon coincided with a peak in cAMP and then obtained a
partial purification of trehalase and of its "activating factor"
(Solingen § van der Plaat, 1975). We have pursued in the invest^,
gation of the regulatory mechanism of trehalase activation in
S.aerevieiae by a cAMP-dependent protein kinase.
.91.
The bulk of trehalase, in wild type Saccharomycea is present
in an inactive form and can be activated by incubation with ATP
Mg in che presence of cAMP and of a protein kinase present in
the same homogenate (Ortiz et al. 1983). It was also possible to
demonstrate the conversion of active trehalase to its cryptic
form by a protein phosphatase. The kinetic data obtained suggest
that interconversion between cryptic and active forms of trehala
se is regulated by a cAMP-dependent nonocyclic cascade system.
This hypothesis was corroborated by studies with a glcl mutant of
S.aereviaiae described as defective in the conversion of the inac
tive, phosphorylated, form of glycogen synthase into the more
active, unphosphorylated enzyme (Rothman-Denes 5 Cabib, 1970). In
consequence, the mutant is unable to store glycogen. The same ina
bility was also observed for trehalose (Padrão et al. 1982) and
the reason for lack of trehalose accumulation is being presently
investigated. Preliminary results indicate that glcl strains con
tain a protein kinase with substantially decreased dependence on
cAMP while dephosphorylation occurs at the same rate as in the pa
rental strain (Ortiz et al. 1983). It appears likely that the glcl
mutation alters the affinity of the regulatory subunit for the ca
talytic subunit so that low levels of cAMP suffice for full disso
ciation and activation. In consequence, both glycogen synthase and
trehalase would be maintained in their phosphorylated forms. The
former would then be less efficient and the latter active: neither
glycogen nor trehalose would accumulate in such a mutant.
Cryptic trehalase can be purified by DEAE-cellulose chromato
graphy at pH 6.2 followed by Sephadex G-200 gel filtration (3,000
fold purification). In such a preparation protein kinase is exclu
ded in the flow through fraction and can be used to determine tre
halase activity. The active form of trehalase can then be purified
.92.
by DEAE-cellulose chromatography at pH 7.5. This procedure allowed
us to demonstrate that cryptic trehalase could be activated by
incorporation of y- P mediated by a c-AMP protein kinase (Delia
mora-Ortiz et al. in press). We then proceeded to purify the pro
tein kinase. The usual CM-cellulose chromatography described in
the literature produced an enzyme with similar affinities for both
cAMP and cGMP. Introducing DEAE-cellulose chromatography at pH 7.5
prior to the CM-cellulose step, we were able to obtain an enzyme_Q
preparation with a Ka of 3.7 x 10 M for cAMP and a Ka of 5.17 x_g
10 for cGMP. Applying both purification procedures to the pro
tein kinase preparation from the glal mutant the Ka values for
cAMP were 10-fold higher irrespective of the methodology, thus
.confirming our hypothesis that the lesion affects the cAMP binding
site of the regulatory subunit.
Protein kinase activity from S.cerevieiae can be determined32either by incorporation of y- 'P from ATP into histone IIA in the
presence of 0.2 pM cAMP or by using cryptic trehalase as substra
te for phosphate incorporation. Furthermore, its activity can be
assessed simply by a determination of trehalase activity coupling
the assay to the determination of glucose derived from the hydr£
lysis of trehalose. This aspect is economically important because
it allows for the use of cryptic trehalase, a very stable enzyme,
as substrate for protein kinase instead of labelled ATP. It also
opens the possibility of using trehalase as substrate for protein
kinases from different origins.
From the point of view of the quality of bakers yeast, a mu
tant which rapidly breaks down trehalose when placed in leavening
conditions would be of great technological interest. In the case
of the glal mutant, addition of glucose would trigger the activa
tion of trehalase by a protein kinase. A good possibility would
.93.
be to construct a mutant which would lack the regulatory subunit
altogether (Uno et ai. 1983) but would be able to store the disac
charide by the constitutive maltose-linked synthesis system.
I am convinced that construction of useful models of yeast
strains and selection for specific mutants coupled to the cloning
of structural and regulatory genes, will provide a more precise
understanding of the role trehalose plays in yeast cells, and by
so doing, will, certainly, lead to new developments in yeast tech
nology.
Acknowledgments - Parts of this work are being undertaken in col_
laboration with C M . Morel from Fundação Oswaldo Cruz and J.C.C.
Mala from Universidade de São Paulo. We are grateful to J.R. Mattoon
and M. Grunstein for fruitful discussions and to CNPq, CEPG, FUJB
and FINEP for financial support.
References
Cabib, E. and Leioir, L.F. (1958) J. Biol. Chem. 231, 259-275
Charlab, R. , Oli'/eira, D.E. and Panek, A.D. (in. press)
DelaFuente, G. (1970) Eur. J. Biochem. 16, 2.40-243
Dellamora-Ortiz, G.M., Ortiz, C.H.D. and Panek, A.D. (in press)
Elbein, \.D. (1967) J. Biol. Chem. 242, 403-406
Entian, K.D. (1980) Mol, Gen. Genet. 179, 169-175
Oliveira, D.E., Rodrigues, E.G.C., Mattoon, J.R. and Panek, A.D.
(1981) Curr. Genet. 3, 235-242
Ortiz, C.H.D. , Maia, J.C.C. , Tena/2, M.N,, Braz-Padrão, G.R.,
Mattoon, J.R. and Ppnek, A.D. (19S3) J. Bacteriol. 153, 644-651
Nasmyth, K. and Reed, S. (1920) Proceed. Natl. Acad. Sci. 77,
2119-2123
Navon, G., Shulman, R.G., Yamanc, T., Eocleshall, T.R., Lam, K.B.,
Baronofsky, J.J. and Marmur, J. (1979) N'MR of S.cereviaiae 18,
4487-4499.94.
Oura, E., Suomalainen, H. and Parkkinen, E. (1974) Proceed. Fourth
Internatl. Symp. on Yeast, Vienna, 125-126
Padrão, G.R.B., Malamud, D.R., Panek, A.D. and Mattoon, J.R.
(1982) Mol. Gen. Genet. 185, 255-261
Panek. A.D. (1962) Arch. Biochen. Biophys. 98, 349-355
Panek, A.D. )1963) Arch. Biochen. Biophys. 100, 422-425
Panek, A.D. and Nattoon, J.R. (1977) Arch. Biochem. Biophys. 183,
306-316
Panek, A.D., Sampaio, A.L., Braz, G.C., Baker, S.J. and Mattoon,
J.R. (1979) Cell. Mol. Biol. 25, 345-354
Rothman-Denes, L.B. and Cabib, E. (1970) Proc. Natl. Acad. Sei.
66, 967-974
Suoaalainen, L. and Pfáffli, S. (1961) J. Inst. Brewing 67, 249-
254
Uno, I., Matsumoto, K., Adachi, K. and Ishikawa, T. (1983) J.
Biol. Chem. 258, 10867-10872
van de Poli, K.W., Kerknaar, A. and Schamhart, D.H.J. (1974) J.
Bacteriol. 117, 965-970
van de Poli, K.W. and Schamhart, D.H.J. (1977) Mol. Gen. Genet.
154, 61-66
van der Plaat, J.B. and van Solingen, P. (1974) Biochem. Res.
Conm. 56, 580-586
van Solingen, P. and van der Plaat, J.B. (1975) Biophys. Res.
Coram. 62, 533-560
.95.
NITROGEN FIXATION RESEARCH IN BRAZIL - AN OVERVIEW
JOHANNA DOBEREINER
Unidade de Apoio ao Programa Nacional de Pesquisa deBiologia do Solo/EMBRAPA
ABSTRACT
Due to the availability of large areas for agriculturein Brazil, the partial or complete replacement of nitrogenfertilizers by biological fixation has a much large scopethan in areas with intensive agriculture. Brazilian soybeanshave bean bread for nitrogen fixation and the 16 million tonsproduced obtain all nitrogen from fixation. Phaseolus beansonly recently are being bread in this direction but yields upto 1200 kg/ha are already obtained without nitrogenfertilizer. Research concentrates now on Rhizobium strain-plant genotype interactions in relation to photosyntheticefficiency and transference if the fixed nitrogen to thegrain. Tolerance to soil acidity and Al toxicity as well assome biological imbalance problems in soils recently takenunder culture (the central highland savannas called cerrados)are under investigation. A large number of tree legumes usedin Brazilian forestry have been found to nodulate and fixnitrogen, 63 of the specie; being described for the first,time as such.
Research on nitrogen fixation in cereals and foragegrasses in the last 10 years has been lead by Brazilianlaboratories. Quantification by the 15N dillution methodshowed amounts of 20 fo 40 kg N/ha (10 to 30% of the plantneeds) coming from biological fixation in forage grasses. Themechanism of the grass.Azospirilium associations is beingelucidated and three new Azospirilium spp have beendescribed. Establishment of certain inoculated Azospirillumstrains within roots has been observed under fieldconditions and yield increases were obtained.
.96.
NITROCTN FIXATION RESEARCH IN BRAZIL - ON OVERVIEW
Johanna Dflbereiner
UAPNPBS - EMBRAPA, Kn 47 23460 Seropédica, Rio de Janeiro
jüitroduçãb
Biological nitrogen fixation has assumed major importance in Brazil not only
because soybeans became the most important export product but mainly because the
availability of land is so for no limiting factor to the expansion of
agriculture and agricultural research envisages agricultural systems ard crop
rotations with minimum N fertilizer inputs. The Brazilian Agricultural Research
Organization (EMBRAPA) is giving increasing support to this field and a national
research program for Soil Biology is coordinating research on biological
nitrogen fixation. Also an international training center for legume inoculation
practices (MIRCEN) sponsored by the UNDP-ICRO ponel was localized in Porto Ale-
gre. All these developments helped to place Brazil into a leading position in
Latin-America and in certain fields in the developing world. In this paper we
will suttmarize the major research activities with outstanding examples and try
to transmit the most important results, in the various fields of biological
nitrogen fixation.
Grain Legumes.
Rhizobiun strain selection for Brazilian conditions was started for soybeans
in 1949 (Freire 1982). lhe Brazilian soybean cultivars,in contrast to the U.S.
and Japan were bread since the 1960'ies without nitrogen fertilizer and with
highly efficient Rhizobium inoculants. As a result this major export crop needs
no N fertilizer and competes better on the world market. Brazil's highland
edaphic savannas called "cerrados" comprizing 180 x 10 ha are being rapidly
taken into agriculture. Economically viable and highly productive farming
systems must relay on crop rotations with legumes as their major nitrogen
input and soybeans are one of the major crops (4.5 x 10 ha in 1981). For more
than 10 years the canmercial soybean inoculants did not work in new lands
until specifically adapted Rhizobium strains were found (Vargas & Suhet, 1980).
These strains were found to beresitantto high levels of streptotnycine a
characteristic later found to be a general feature of Rhizobium strains
isolated from cerrado soils (Scotti et al. 1982). Soils with similar problems
occur in the Colombian Llanos and also in newly cleared Amazon land planted to
cowpeas (DObereiner et al. 1981). The resistance to certain antibiotics however
is not the only cause of better establishment of certain strains under adverse
conditions. Tolerance to soil acidity problems (Mums ft Franco 1981) and
saprophytic competence (Vidor & Miller 1980, Peres ft Vidor 1980) play important
.97.
roles.
Phaseolus beans, the major food basis of Brazilian people until 3 years ago
have not been bread for nitrogen fixation and therefore needed N fertilization
for improuved yields. A large cooperative research project coordinated by CNPAF
in Goiânia has already yielded pranissing results os examplif ieâ with the
results of a chain experiment (Table 1) where it is shown that cultivars and
inoculants are already available which permit complete replacement of N
fertilizer. These data stress the importance of plant breeding for N2 fixation.
As inoculation beoanes a common practice the physiological factors affecting
nodule functioning and limiting seed production are of concern. Differences
were found among Phizohium strains in the efficiency of incorporation of the
fixed N into seeds which were correlated (r = 0.80 **) with the N transported
as ureide in the xylem sap (Hungria * Neves 1984). Similar differences
between Rhizobium strains were also observed in soybeans (Didonet et al. 1982,
A.D. Didonet, F.F. Duque t M.C.P. Neves, in preparation).
Cultivar
Carioca
Negro Argel
Venezuela
Rio Tibagi
Control
nodule
wt
(mg/pl)
4
46
3
1
grain
yield
(kg/ha)
379
. 494
378
316
Fertilizer100 kg
nodule
wt
(ng/pl)
10
22
5
29
N/ha
grain
yield
(kg/ha)
663
620
601
790
nodule
wt
(mg/pl)
123
155
39
17
Inculated
grain
yield
(kg/ha)
991
883
438
583
N 2 fixed
kg/haa
31.7
18.4
3.6
2.7
a As evaluated by 15N0, dillutlon
The interaction treatment x cultivar was significant for nodule weight(p = 0.01) and grain yields (p = 0.05).
Another important problem In the bean - Rhizobium symbiosis are excessive
soil temperatures. Possibilities of selecting heat tolerant strains are
indicated in Table 2. In tropical forage legumes nodulatlon and N2 fixation
was observed to be even more tolerant to high temperatures (up to 40°C) (Lee
4 Dfibereiner 1982).
Forest legumes
Brazilian reforestation projects, until recently did not consider one of
the Important characteristics of so many native legume trees s their ability
to fix N2. The most precious hard wood species and many native fast growing
trees are legumes but little is known about their capacity to ncdulate or fix
i*2. Surveys in the North eastern dry regions (Vasconcellos t Almeida 1979/.
1980) in the Amazon rain forests (Bradley et al. 1978, 1980, ftagalhães et al.
.98.
1982) and in South East Brazil (Faria et ai. 1983, 1984) revealed many
eoonanically important N_ fixing trees not known as such before (Table 3).
Mesquite (Proscpis juliflora) called algaroba in Brazil is being planted ii
large government projects in the North East dry regions and since 1982 is
inoculated with ocmnercially available inoculants developpsd by EMBRAPA.
TABLE 2. Selection of Rhizobium phaseoli strains for heat
tolerance (Oliveira et ai. 1984)
Rhi zriiiun
SEMIA 487
SEMIA 4021
F 413
F 413 1*1
BR 292
SEMIA 4002
CO 5
Teuperature
for Rhizobium
growth (°C)
2835
2835 .
2835
2838
3538
28
28
Nodule weight
Temperature for
ímbient
18.221.9
46.825.7
28.826.3
24.020.0
24.020.4
20.0
24.0
(mg/plant)
plant growtha
35°C
29.534.7
1.31.2
25.725.7
0.00.0
35.524.0
31.1
25.7
Plants were grown in sterilized jars placed into water-baths with eitiicr ambient tenperature or 8 h/day at 35PC
TABLE 3. Modulation of Brazilian forest legunes (Faria et ai. 1983,
1984; Bradley et ai. 1978, 1980; Magalhães et ai. 1982,
Vasconcellos & Almeida 1979/1980).
Subfamilies
N9 of species verified
N9 of species with nodules
N9 of species found forthe first time withnodules
N9 of genera found forthe first time withnodules
N9 of Rhizobivm strainsisolated
Himo-
soideae
60
51
25
0
257
Papilio
noideae
75
53
37
4
218
Caesalpi
noideae
72
9
8
2
62
Total
207
113
70
6
537
.99.
Cereals and Forage Ckasses
New approaches to the study of nitrogen fixation in the major cereals and
grasses have been started in the last decade (DObereiner t Day 1975, Neyra *
DObereiner 1977, DObereiner t De-Polli 1980). Several new N 2 fixing bacteria
have been described which associate with grasses and cereals (DObereiner 1966,
Tarrand et al. 1978, Barraquio et al. 1983, Magalhães et ai. 1983) and mora are
to come. Table 4 sinner izes the main characteristics of scne of them. The
process of infection of cereal roots has not yet been identified but root hair
deformations with specific Azospirillum brasilense strains could be associated
with plant responses to inoculation with the sane strains (Patriquin et al.
1983, Baldani et al. 1983). Establishment of inoculated Azospirillum
strains in roots of field grown wheat varied with strains. Soot isolates
(strains Sp 107 st and *Sp 245 sp) became dominant within roots while the soil
isolate (Sp 7) seemed less competitive (Table 5). Plant responses under field
aonditions to Azospirillun inoculation have now been reported from many plaoes
(Okon 1982, Subba Rao 1981, Vlassak & Reynders 1978) but as expected there are
large differences between strains (Freitas et al. 1982, Baldani et a^.
1983). Although such plant responses where usually accompanied by increased N
incorporation, especially into seeds, unequivocal proof of N 2 fixation has nut
been brought foreward in Azospirillum inoculation experiments. Attempts to
show N0 3" dillution in one of these experiments (DObereiner 1983) showed
higher fertilizer recovery but no sign of N- fixation. Bacterial hormones
which proportion a spronge effect on the plant roots were suggested by Okon
(1982). So far however only few bacterial strains have been tested.
In plant genotype comparisons however, N> fixation in the order of 10 -
30% of the total plant N incorporation has been shown by N balance studies
(App et al. 1980) and by l 5N 2 incorporation (De-Polli et al. 1977,, Eskew et
al. 1981) in forage grasses and rice and by the 1 5N0 3 dillution uiethod with
several forage grasses (Boddey et al. 1983a, Boddey et al. 19831) and an
additional example ÍB given in Fig. 1. There it can be seen that once the
grasses have been established, between 20 and 40% of the total N incorporation
is coming from biological fixation. If the values for one year (November to
November) are added it can be calculated that 53 kg N/ha have been fixed in
association with B^ decunbens and 23 kg with B^ hunidicola. There are also
indications of N 2 fixation in sugar cane (Ruschel * Vbae 1981). Although N2-
f ixation in association with gramineae is a very exiting field due to the
Importance of this plants for agriculture, it is improbable that complete
replacement of N fertilizers will be possible because of the more primitive
nature of these associations. Still it ramains a major challenge to soil
biologists and agronomists and prospects for new break throughs are good.
.100.
«- C - J J J t - t t r i s t l r •_: r: r . x : •
Colony type or. potato writ*• 9 " ""Jlst
lfelera.10? tc n, fornitroçenasc activity hiqh wry IOM
Assimilation of rt'j"
Cell width In». N,, « « 0 2 1-2 O.*8 1.0-1.%
Cell irrsfSíi 2-3C 2-1 2-U
Polar flaqelliaj - • •Lateral
r.-trier.*. arcarPolynrph cell* in
alcaline n d u
use of sucroceDNA base oo>f>-
G • O
_ t of siqns: • positive in rrire tkan Wi o? tne strains:7 posit ve in less than 0% of the strains; - negativefc Cells of teospirillM lippferjr anc Ájjtctacter paspali ray bear» ever, wicicrin older alcaline cultures.
TKH£ 5. Establishraent of antibiotic resistance labelled
teoBpirilltm brasilense strains in soil and
roots of field
Origin of
inoculant
strain
Control
Soil (0d)
Wheat rootsWheat roots
(Sp
(Sp
107 st)
245 st)
growni wheat
Soil
61
54
44
%
1
• 10
+ 7
1 12
*
Washed
roots
of
29
67
62
Surfacesterilized
rootsa
cultures0
• 8
• 5
_• 7
5
11 + 5
8 2 + 3
76 + 5
a Roots exposed 15 min. to It cnloramine Tb percent of cultures identified as inoculated strain.Percentage calculated from 18 cultures obtained from themaximal dillutions (MM counts) of soil or roothemogenates. Values are means of 2 harvests and 2fertilizer levels with standard deviations.
.101.
15
10
AU6 NOV F€B APR NOV JAN MAY
FIG. 1 - Seasonal variation of N accumulation by two Brachiaria spp.from nitrogen fixation and from soil . Values representmonthly means for the time intervals between the months
15,N03 dillution with a non-stated. ENF was evaluated byfixing Brachiaria as control (B^ radicans)(R. Boddey & R.Victoria, in preparation).
lhe use of the many new findings in al l fields of biological N- fixationin agricultural systems will lead to more economic but s t i l l highlyproductive farming with less risks for the environnent.
ACKNOWLEDGEMENTS: Large parts of the resu l t s reported in thispaper were obtained in experiments financed by FINEP
.102.
References
App, A.A., Natanabe, I., Alexander, M., Ventura, W., Oaez, C., Santiago, T. t
Datta, S.K. de. I960. Nonsynbiotic nitrogen fixation associated with the
rice plant in flooded soils. Soil Science 130(5): 283-289.
Baldani, V.L.D., Baldani, J.I. * Dfibereiner, J. 1983. Effects of Azospirillun
inoculation on root infection and nitrogen incorporation in wheat. Can. J.
Microbiol. 29(8): 924-929.
Barraquio, W.L., Ladha, J.K. ft Hatanabe, I. 1983. Isolation and identification
of Nj-fixing Pseudcwonas associated with wetland rice. Can. J. Microbiol.
29(8) 867-873.
Boddey, R.M., Chalk, P.M., Victoria, R. 4 Matsui, E. 1983a. lhe 15N isotope
dilution technique applied to the estimation of biological nitrogen
fixation associated with Paspalum notatum cv. batatais- in the field. Soil
Biol. Bioch. 15(1): 25-32.
Boddey, R.M., Chalk, P.M., Victoria, R.L., Matsui, E. I DObereiner, J. 1983b.
lhe use of the TI isotope dilution technique to estimate the contribution
of associated biological nitrogen fixation to the nitrogen nutrition of
Paspalum notatun cv. batatais. Can. J. Microbiol. 29(8): 1036-1045.
Bradley, R.S., Oliveira, L.A. de, Podestá, J.A. de F. 1 John, T.V.St. 1978.
Fixação de nitrogênio associado com raízes an solos diferentes na floresta
Arazonia-Central. Anais do 39 Congresso Florestal Brasileiro, Manaus.
Bradley, R.S., Oliveira, L.A. de, Podestá. J.A. de F. • John, T.V. St. 1980.
Nodulation of legumes, nitrogenase activity of roots and occurrence of
nitrogen-fixing Azosplrillun spp. in representative soils of Central
Amazonia. Agro-Ecosystems 6: 249-266.
De-Polli, H., Matsui, E., DObereiner, J. * Salati, E. 1977. Confirmation of
nitrogen fixation in two tropical grasses by N 2 incorporation. Soil Biol.
Biocnan. 9: 119-123.
Didonet, A.D., Duque, F.F. * DObereiner, J. 1982. Influência de estirpes de
R^ japonicum na eficiência e incorporação do N fixado em soja (Glycine max
(L.) Merril) em casa de vegetação e campo. XI REUR - Lima, Peru.
DObereiner, J. 1966. Azotobacter paspali sp. n., una bactéria fixadora de ni-
trogênio na rizosfera de Paspalum. Pesq. agropec. bras. 1: 357-365.
DObereiner, J. 1983. Iten years Azospirillum. In: Azospirillun II (KlingmQller,
ed.) Expsrientia Supplementun, 48: 9-23.
DObereiner, J. ft Day, J.M. 1975. Associative symbioses in tropical grasses:
Characterization of microorganisms and dinitrogen-fixing sites. In:
Nitrogen Fixation vol. 2 (W.E. Newton • C.J. Nyman, eds.) Washington State
University Press. Pullman, 518-538.
.103.
Dflbereiner, J. ft De-Polli, H. 1980. Diazotrophic rhizoooenoses. In: Nitrogen
fixation (W.D.P. Stewart ft J.R. Gallon, cds.) Academic Press, London.
303-333.
DBbereiner, J., Scotti, M.R.M.N.L., Sá, N.N.H. ft Vargas, N.A.T. 1981.
Resistance to streptomycin of Rhizobium isolates from cerrado and Amazon
soils. In: Current Perspectives in Nitrogen Fixation (A.H. Gibson « W.E.
Newton, eds.) Australian Academy of Sciences. Canberra, 434.
Eskew, D.L. ft Eaglesham, A.R.J. 1981. HfeUirotrophic 1 5N 2 fixation and
distribution of newly fixed nitrogen in a rice-flooded soil system.
Plant Physiol. 68(1): 48-52.
Faria, S.M. de, Franco, A.A., Menandro, N.S., Jesus, R.M. de, Saitello, J.
B., Aguiar, O.T. de & Dobereiner, J. 1983. Levantamento da nodulação de
leguminosas florestais na região sudeste do Brasil. Anais do Simp. Fix.
N 2 em Arvores Tropicais. Pesq. agropec. bras. Vol. esp. (no prelo).
Faria, S.H. de, Franco, A.A., Jesus, R.N. de, Henandro, N.S., Baitello, J.
B., Mucci, E.S.F., Dobereiner, J. ft Sprent, J.I. 1984. New nodulating
legume trees from South-East Brazil. New Phytologist (in press).
Freire, J.R.J. 1982. Research into the Rhizobium/Leguminosae symbiosis in
Latin America. Plant and Soil 67: 227-239.
Freitas, J.L.M. de, Roc'.ia, R.E.N. da, Pereira, P.A.A. ft DObereiner, J. 1983.
Matéria orgânica e inoculaçâo con Azospirillum na incorporação de N pelo
milho. Pesq. agropec. bras. 17(10): 1423-1432.
HUngria, N. ft Neves, M.C.P. 1984. Variação sazonal da fixação do N2: Trans-
locação do nitrogênio fixado em Phaseolus vulgar is L. XIX Congr. Bras.
Ciê. do Solo, Curitiba-Pr. (Resumo n9 42)
Lee, K.K. ft Dobereiner, J. 1982. Effect of excessive temperatures on
rhizobia growth nodulation and nitrogen fixing activity in symbiosis
with siratro. Pesq. agropec. bras. 17(2): 181-184.
Magalhães, F.M.M., Baldani, J.I., Souto, S.M., Kuykendall, J.R. ft DOberei-
ner, J. 1983. A new acid tolerant Azospirillum species. An. Acad. Bras.
Ciê. 55: 417-430.
Magalhães, F.M.M., Magalhães, L.M.S., Oliveira, L.A. de ft DObereiner, J.
1982. Ocorrência de nodulação em leguminosas florestais de terra firme
nativas da região de Manaus-An. Acta Amazônica 12(3): 509-514.
Mtms, D.N. ft Franco, A.A. 1982. Soil constraints to legume production-. In:
Biological Nitrogen Fixation "technology for Tropical Agriculture (P.H.
Graham * S.C. Harris, eds.) CIAT, Cali-Colcmbia, 133-152.
Neyra, C.A, ft DBbereiner, J. 1977. Nitrogen fixation in grasses. Adv. Agron.
29: 1-38.
Okon, Y. 1982. Azotpirilium: Physiological properties, mode of association
.104.
with roots and its application for the benefit of cereal and forage grass
crops. Israel J. Botany, 13(1-4): 214-220.
Oliveira, L.C.B. de, Franco, A.A. & DObereiner, J. 1984. Estabilidade da in-
fectividade, efetividade e capacidade de produzir pigmento de estirpes de
Rhizobium phaseoli submetidas a altas temperaturas. Pesq. agropec. bras.
(no prelo).
Patriquin, D.G., Iflbereiner, j. & Jain, D.K. 1983. Sites and processes of
association between diazotrophs and grasses. Can. J. Microbiol. 29(8):
900-915.
Peres, J.R.R. & Vidor, C. 1980. Relação entre concentração de"células no ino
culante e competição por sítios de infecção nodular entre estirpes de
Rhizobium japonicum em soja. Rev. Bras. Ciê. Solo 4: 139-143.
Ruschel, A.P. a Vose, P.B. 1981. Biological dinitrogen fixation associated
with sugarcane. In: Current Perspectives in Nitrogen Fixation (A.H.
Gibson & W.E. Newton, eds.) Australian Academy of Science, Canberra, 497.
Scotti, M.R.M.M.L., Sá, N.H.H., Vargas, M.A.T. & Dflbereiner, J. 1980.
Requirement for streptomycin resistance of Rhizobiun for the nodulation
of legumes in cerrado regions. An. Acad. Brasil. Ciê. 52(3): 650-651.
Subba-Rao, N.S. 1981. Response of crops to Azospirillum inoculation in
India. In: Associative N -fixation (P.B. Vose & A.P. Ruschel, eds.) vol.
II, CRC Press, 137-140.
Tarrand, J.J., Krieg, N.R. ft DObereiner, J\ 1978. A taxonomic study of the
Spirillum lipoferum group, with descriptions of a new genus, Azospirillum
gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov.
and Azospirillum brasilense sp. nov. Can. J. Microbiol. 24(8): 967-980.
Vargas, M.A.T. * Suhet, A.R. 1980. Efeito de níveis e tipos de inoculantes
na soja (Glycine max) cultivada on um solo de cerrados. Pesq. agropec.
bras. 15(3): 343-347.
Vasconcelos, J.I.P. ft Almeida, R.T. de. 1979/1980. Fixação biológica de ni-
trogênio em plantas de interesse econômico do Nordeste. Relatórios técni
cos I, II e III, Fundação Cearense de Pesquisa e Cultura.
Vidor, C. ft Miller, R.H. 1980. Relative saprophytic competence of Rhizobium
japonicum strains in soils as determined by the quantitative fluorescent
antibody technique (FA). Soil Biol. Biochan. 12: 483-487.
Vlassak, K. ft Reynders, L. 1978. Association of free-living nitrogen fixing
bacteria with plant roots in temperate region. In: Nicrobial Ecology
(M.W. Loutit ft J.A.R. Miles, eds.) Springer-Verlag, 307-309.
.105.
IMUNOLOGYPARASITIC DISEASES
Coordinator
Dr. DOMINGOS ALVES MEIRA
Scholl of Medicine, UNESP
Botucatu, SP
SKIN TEST WITH PURIFIED SAWADA ANTIGEN IN BRAZILIAN INDIANS
WITH OCULAR ONCHOCERCIASIS
Milton Massato HIDA
Department of Ophthalmology
Faculdade de Medicina de Botucatu
Universidade Estadual Paulista (UNESP) - Brazil
José João FERRARONI and Heitor V. DOURADO
Insti tuto de Ciências Biológicas
Universidade do Amazonas - Brazil
Kumiko SATO and Hamoru SUZUKI
Department of Parasitology
Gunma University, School of Medicine - Japan
ABSTRACT
Skin test as a method of imtunodiagnosis and study of pathogenesis of
parasitic diseases was evaluated in onchocerciasis.
The obtention of a great amount of worms of Onchooeroa volvulus to
produce antigen Is very d i f f i c u l t , therefore, purified Sawada antigen from
adult worms of Dirofilaria irmitia was used in f ive Brazilian Indians with
severe ocular lesions in consequence of onchocerciasis.
The results showed that In thirteen fractions of Sawada antigen,
positive reactions was found in the poiysaccharide antigen (fractions 1 to 4 ) .
Considering that skin test In other f i i a r i as is was observed in protein
rich fractions, this comparison suggests that 1imnuno1og1ca1 difference between
f1>arias1s and onchocerciasis by skin test Is detectable.
The value of the skin test with Sawada antigen In Yanomama Indians
infected by Onahooeraa volvulus was evidencia ted and careful Investigation Is
necessary to elucidate that positive response and the relations of ocular
changes with inmunological state of the patient.
. 1 0 6 .
INTRODUCTION
The isolation and purification of onchocrrcal antigens and development
of tests for cell-mediated immunity to Onchocerca volvulue received attention
as a method of investigation (CIFERRI e t a l . , 1965 and MUELLER et a l . , 1976).
However the d i f f icu l ty to obtain worms of Onchocerca volvulus in quantity ÕS
source of antigen and the evaluation of a specific antigen is the great problem
in this method for the imunodiagnosis and understanding 01 the pathogenesis of
the disease.
Dirofilaria imitia as antigen have been probably as the most widely
used to help in cl inical diagnosis of f i l a r ias is and several reports have des
cribed the results of skin testing with the purif ied extract of Sawada antigen
(SMITH et a l . , 1971 and SATO et a l . , 1982).
The application of this antigen in patients l iv ing in Brazil affected
with onchocerciasis presenting c l in ical ly only ocular sequels was studied and
observed the effects of testing as valuable aid to the diagnosis of this filai
r i a l infection.
MATERIALS AND METHODS
1. SUBJECTS
Five Brazilian indians from Yanomama tribe living in Roraima Territory,
four males and one woman with onchocerciasis, age between 21 to 57 years old,
characterized by severe ocular lesions was studied.
2. ANTIGENS
Dirofilaria imritie adult antigen or Sawada antigen, lyophilized and
separated in 13 fractions was supplied in ampoules containing 10 vg of antigen
and di lui ted in ] ml of 1:10000 merthiolate and 3 ml of saline.The purification
of skin test antigen FST from the Dirofilaria inmitie worms (SAWADA e t al. 1969;
SATO & SANADA, 1969 and SAWADA & SATO, 1969) 1s schematized in Figure 1.
. 1 0 7 .
FIGURE 1 . SCHEME OF THE PURIFICATION OF SKIN TEST SAWADA ANTIGEN FROM ADULT
WORMS OF Dirofilaria immitis.
IPPT
ADULT WORMS
defatted with diethyletherhomogenizedsonicated
centrifugation
Sup (F)
Treated with 10* TCA (pH 3,7)
centrifugation
PPT ( FP ) Sup ( FS Vi 1
CM-C Colum Chromatography
FSC2 FSC3,-- FSC4r , FSC5
) 0DEAE-S Column Chromatography
I 1FSCDKFST)^ FSD2 FSCD3 FSCD4
Rechronatography on DEAE Sephadex
~>SFST2/~^FST3
t) 0 1Di l tDisc electrophoresis
I
FSTS/
.108.
3 . TECHNIQUE OF SKIN TEST
The antigen solution was injected using tuberculin-type seringe dosis
of 0,02 ml of solution in the forearm producing blebs of 5 to 5,5 mm. The
lecture was performed 15 minutes after the injection and the induration was
outlined with a ballpoint pen and was transferred to a celophan tape forming
a permanent record. Two diameters of wheal met at right angles were measured.
For a positive reaction was adopted the criteria of KATAMINE (1969) conside-
ring any increase in diameter above 3 mm produced by the injection, of the
antigen should be regarded as positive.
4. MAZZOTTI TEST
The patients was submitted to Nazzotti test which consist in adminis-
tration of 100 mg of diethyicarbamazine by mouth.
5. OPHTHALMOLOGICAL EXAMINATION
The eyes of the patients was examined by slit-lamp and ophthaimoscopy.
6. SKIN SNIP EXAMINATION
In one patient a piece of skin from scapula region was excised and
examined for demonstrating microfiiariae in the skin.
7. PHOTOGRAPHIC DOCUMENTATION
The patients was photographed in parts as face, eyes, skin reactions,
and interely the body.
RESULTS
I. SKIN TEST
The skin test revealed that fractions 1 to 4 was positive in patients
with onchocerciasis and the fraction 11 presented delayed reaction in two
cases (Table 1). Two healthy controls was tested and resulted negative in all
13 fractions of Sawada antigen.
.109.
TABLE 1 . POSITIVE REACTIONS OF SKIN TEST WITH PURIFIED SAWADA ANTIGEN IN FIVE
CASES OF ONCHOCERCIASIS. DIAMETER OF INDURATION IN MILIMETERS.
^S. antigen^^fraction
patient ^ v
1
2
3
4
5
1
10x9
10x9
10x9
10x10
10x10
2
10x9
9x9
12x11
12x12
10x8
3
8,5x8
8,5x9
9x9
10x9
8x9
4
9x9
8x9
8x8
9x12
8x8,5
11
-
-
-
8,5x9,5*
8x8,5*
* delayed reactions.
II. OCULAR EXAMINATION
Patient 1: N.S. 35 years old, female, presented in both eyes trichiasis,
diffuse cornea1 opacity, complicated cataract and vision: hand
movement.
Patient 2: A.G.A. 21 years old, male, presented in right eye vision 20/50
with cornea, iris and fundus normal; in left eye cornea1 opacity,
complicated cataract, anterior chamber shallow, ocular atrophy
and vision null.
Patient 3: E.P. 28 years old, male observed in both eyes superficial diffuse
opacity of the cornea and lens opacif1cation. Vision: light pro-
jection.
Patient 4: C.6. 34 years old, male. In right eye presented vision 20/40,
opacities in perl feral cornea and normal fundus; the left eye
observed lens opacification anterior chamber shallow and cornea1
opacity diffuse, vision null.
.110.
Patient 5: E.P. 54 years old, male, in right eye, vision: hand movement, se
condary cataract and in left eye, diffuse corneal opacity, cyst in
anterior chamber, lens opacification, paralitic mydriasis and
vision null.
I I I . OTHER EXAMINATIONS
The five patients presented Mazzotti test positive, the skin snap exam^
nation revealed presence of microfilariae and subcutaneous nodules was noted in
one patient.
DISCUSSION
Considering that positive skin reaction to a parasitic antigen is a
proof of presence of this corresponding antibody, in our results the skin test
showed evident cross reactions of nivofilarta vmitis with Onchocerca volvulus.
This correlation in our five cases of onchocereiasis was specific in
f i rs t four fractions rich in polysaccharide. The fractions 5 to 13 rich in
protein, presented negative response. Two delayed reactions was observed in
fraction 11 (Table 1) .
This specificities in polysaccharide fraction to Onchoaevoa volvulus
antibody is very interesting since the reactions of other f i lar ias is , skin
test are related to protein fractions (8 and 12 fractions) separated from FST,
fraction 5 of Sawada antigen (SATO et a l . , 1982).
These results suggest that have possibility to differenciate f i lariasis
and onchocerciasis with this simple intradermical test.
The eye examination showed a marked predominance of anterior segment
lesions producing blindness by severe kerato-iridocyclitis.
CHOYCE (1972) considered that ocular complications of Central Americans
onchocerciasis are greatly heightened reactives due a low immunity compared with
africans onchocerciasis whirh present apparent immunity producing fundusocular
lesions predominantly in small percentage.
. 1 1 1 .
The investigation of «ore simple test to help in clinical diagnosis of
onchocerciasis is very important and justify the risk in the population in
prevention of ocular complications principally in Central and South America
which present high incidence of blindness in patients infected by Onchooerca
volvulus.
BIBLIOGRAPHY
1..CHOYCE, D.P. Kerato-uveal changes in leprosy and onchocerciasis: a question
of immunity. Proc. roy. Soc. Med. 65: 955-960, 1972.
2. CIFERRI, F.; KESSEL, J.F.; LEWIS, W.P.; RIEBER, S. Imnunologic studies in
onchocerciasis and bancroftian f i l ar ia s i s . I . Intracutaneous tests with
antigens extracted from Onchoaerca and Dirofilaria. Am. J. Trop. Med.Hyg.
14: 263-268, 1965.
3. KATAMINE, D. Skin test of bancroftian f i lar ias i s with purified antigen FST
prepared from canine f i lar ia , Dirofilaria irrnnti.». Trop. Med.11:1-10,1969.
4. MUELLER, J.C.; MITCHELL, D.W.; GARCIA-MONZA, G.A.; AGUILAR, F.J.; SCHOLTENS,
R.G. Evaluation of a skin test for onchocerciasis in Guatemala. Am. J.
Trop. Med. 22(3): 337-342, 1973.
5. SATO, K. 4 SAWADA, T. Studies on skin test antigen FST for immunodiagnosis
of f i lar iat i* . I I . The fractionation of the main proteins contained in
fractions FST 1,2,3 and 5 disc electrophoresis. Jap.J.Exp.Med. 39/5: 435-
440, 1969.
6. SATO, K.; SUZUKI, M,; THOMAS, V. Enzyme imnunoassay for human f i lar ias is
using purified Dirofilaria irmitie (FST). Proc. Symp. Third Japan-Brazil
Synp. Sci. Techn. 1: 288-289, 1982.
7. SMITH, D.H.; WILSON, T.; BEREZANCEC, Ju A.; LTKOV, V.; PAING, M.; CHARY,M.V.;
DAVIS, A. Evaluation of the Dirofilaria inmitie f i lar ia l skin test anti-
gen in the diagnos: f f i l ar ias i s . Bull.wld.Hlth.Org. 44: 771-782, 1971.
8. SAWADA, T.; SATO, K,; SATO, S. Studies on skin test antigen FST for immuno-
diagnosis of f i l ar ias i s . I . Electrophoretic analysis and fractionation of
antigen FST. Jap. J. Exp. Méd. 39/5: 427-433, 1969.
9. SAWADA, T. & SATO, K. Studies on skin test antigen FST for immunodiagnosis
of f i lar ias i s . I I I . Separation and characterization of FST 3-1 by isoelee
trie focusing technique. Jap. J. Exp. Med. 39/6: 533-539, 1969.
. 1 1 2 . -
CORRELATION BETWEEN MICE ACUTE TRYPANOSOMA CRUZI
INFECTION AND PLASMATIC LEVEL OF LIPID PEROXIDE
- PAULO ERNESTO SOARES PALHARES
INSTITUTO OSWALDO CRUZ - RIO DE JANEIRO
- PEDRO FONTANA JUNIOR
INSTITUTO NACIONAL DE CANCER - RIO DE JANEIRO
SUMMARY
Lipid peroxidation is a consequence of free radicals
reactions on polyunsatured fatty acids of cellular membranous structures
that occur mainly in inflammatory and degenerative process. On the other
hand, first host vertebrated response to the Trypanosoma cruzi infection
is of inflammatory nature. Based on these facts we verifyed if there is
any relation between evolution of mice acute T. cruzi infection and their
average plasmatic levels of malondialdehyde, a metabolic intermediary
of lipid peroxidation. Correlation coefficient was r=0.931 (p < 0.001).
These results show that malondialdehydemic levels are a direct function
of evolution of mice acute T. cruzi infection, and could be used pehaps
to avaliated and monitorise general host damage on this protozoosis.
INTRODUCTION
Trypanosoma cruzi, the etiologic agent on Chagas'
disease, is an intracellular parasite. Host general damage found in acute
.113.
infection is mainly caused by intracellular parasite replication and its own
metabolic toxity that occur in some cells, as well as consequence of
oxidative response that happens in others. At both process polyunsaturec
fatty acids from phosphoUpids of cellular membranous structures would
be hydroliucd and pct-oxidibud as result of nit reused free rudn:;iL.s
reactions usually present in these stress conditions (1) (2). In these
metabolic pathways oxigen free radicals are produced by partial reduction
of molecular oxygen, raising others radicals at chain reactions (1) (3) (4).
These radicals may cause extensive r Uular damage through oxidative
attack to polyunsaturtd fatty acids of cellular membranous structures,
giving rise to lipoperoxides free radicals and malondiaidehyde an
metabolic intermediary of lipid peroxidation (5) (6).
The present study was developed based on these
facts and on intensive T. cruzi replication inside cells of vertebrated
host in acute phase of infection. This phase induce a stress condition
where enhancement of free radicals reactions can occur, with
overproduction of malondialdehyde. We intended to verify if there is
any relation between «volution of mice acute T.cruzi infection and
their plasmatic levels of malondialdehyde.
MATERIALS AND METHODS
ANIMALS AND PARASITES
One hundred and twelve male albin S-W mice weighing
2l .0?l ,2g were random distributed at fourteen groups of eight animals
each, numered of zero to thirteen (0-13) respectively to the experiment
.114.
days. Animals of zero group were i. p. inoculated with 0. lml of sterile
phosphate buffered saline (PBS) pH 7.2 and blood was obtained by orbital
punch after cervical deslocation. Blood was collected in heparinized
glass tubes at 0°C and centrifugated at same temperature on 2000g for
ten minytes to obtain plasm. Groups 1-13 where i. p. infected at zero
day (0 day) with 0.1ml of bloodstream forms suspension Ü05
trypomastigotes of T. cruzi Y strain). At each day (1-13) the
respective group was killed and plasma collected by same procedure
used for zero group. Parasites were obtained from mice at the
seventh day of infection i. p. with 10* trypomastigotes of T. cruzi Y
strain, by differential centrifugation of blood and then twice washed
with sterile PBS.
- CHEMICALS
Malondialdehyde bis (dimethyl acetal) from Aldrich
Chemical Company, Inc. Wiscosin, USA; 2-thiobarbituric acid from
Merck, Darmst d, Germany; sodium sulfate anhydrous, Merck, Brasil;
sodium bicarbonicum, Ecibra, Brasil; Trichloroacetie acid and sulfuric
acid, Merck, Brasil; n-butanol, Reagen, Brasil. All are PA Chemicals.
LIPID PEROXIDATION DOSAGE
Plasmatic malondialdehyde (MDA) of all animals was
determinated by Satho's procedure (7) with modifications mainly for
thiobarbituric acid and malondialdehyde standar solutions preparations;
scaled down method; and reagent concentrations, such is described
elsewhere (8).
.115.
RESULTS AND DISCUSSION
The mice plasmatic levels of malondialdehyde and
respective everyday evolution of infection was compared and the
correlation coefficient was calculated (see table). We observed that aging
of mice along the experimental time don't induce malondialdehydemia
change (8).
Correlation coefficient was statiscaly significative
suggesting that acute phase of experimental Chagas* disease evolution is
a direct function of lipid peroxide production. Thus, these results
suggests the possibility of using malondialdehyde determination as a
procedure to avaliate and monitorise general vertebrated host damage on
this protozoosis.
ACKNOWLEDGEMENTS
The authors thant Mrs. Alcidineia Ivo for technical
assistence and Mrs. Roselia Lins de Souza for manuscript typing.
.116.
REFERENCES
1. Pryor, A.L. Fed. Proc. 32 (8): 1862, 1973.
2. McCay, P.B. Fed. Proc. 40: 173, 1981.
3. Root, R.K., and Cohen, M.S. Rev. Inf. Dis. 3 (3): 565, 1981.
4. Nathan, C .F . Fed. Proc. 41 (6): 2206, 1982.
5. Fong, K. L . , McCay, P . B . , and Poyer, V. L .J . Biol. Chem.248 (22):
7792, 1973
6. Tappel, A. L. Fed. Proc. 32 (8): 1870. 1973.
7. Satho, K.Clin.Chim.Acta. 90:37, 1978.
8. Palhares, P.E. S., and Fontana, P. Jr . 1984 (in press).
.117.
MALONDIALDEHYDEMIA (MDA-nH/ml) DOSAGE ON MICE GROUPS DURING Vklht EVOLUTION (d)
. OF ACUTE T. CRUZI INFECTION.
M D A / d 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
4.03.54.0
4.53.03.53.5
4.5
3.54.5
4.54.5
4.SS.O
4.5
4.5
4.5S.S
5.5S.SS.O5.0
4.S
4.5
4.04.0
S.O
4.5S.O5.0
6.56.0
S.O4.55.4
5.0
S.65.54.0
5.5
5.05.05.0
6.0
S.S5.0
S.S6.0
5.54.0
5.06.0
4.5 '4.55.0
S.5
5.5,S.SS.O
4.55.55.0
S.S5.0
5.06.0
4.5
4.54.55.5
S.57.0
6.4 .6.5
7.56.5
6.56.0
6.5
S.5
6.07.5
7.5
7.56.06.57.0
4.5
7.55.S
5.56.0
7.57.0
7.59.0
7.56.0
6.58.0
6.58.0
6.06.0
X
s3.81
0.53
r •
4.44
0.42
0.93
5
0
P
.00
.46
5.00
0.89
).00J
5
0
.06
.57
5.
0.
38
44
5
0
.00
.65
5.
0.
19
37
S.
0.
31
88
6.
0.
43
56
6.
1.
56
05
6
1
.94
.21
6
0
.81
.88
Mortality began at 13th day of infection.
GRANULOMATOUS FORMATION IN MICE LIVER FOR ABSORTION OF S. HANSONI
WORMS. AFTER TREATMENT WITH OXAMINIQUINE
A. MAGALHÃES and M.E. BEZERRA DE MELO
Centro de Pesquisas Aggeu Magalhães
Fundação Osvaldo Cruz
N. TELES PONTES
Department of Pathology
Universidade Federal de Pernambuco
Introdution:
The mechanism by which schistosomes evade the immune
response has been suggested by Smithers et al (1969). The reason
for the survival of adult worms in the blood vessels of their host
has been indicated by Clegg (1974) to the presence of hostlike
antigenic determinants on the surface membranes of adults
schistosomes. Sogandares-Bernal (1976), described an antibody type
attached to the integument of the worms which preclude complement
Íixation and hence prevent damage to the cell surface of the
arasite. Some others papers has demonstrated that a striking
eature of the Senistosoma mansoni tegument is that it contains
hosphohydrolase activity directed a variety of substrates, Cesa-
|ri, et al (1981).
On the other hand, Kohn et al (1979), described morpho-
logic alterations of worms in mice after treatment with oxamini-
[uine. Those lesions were found to be hyaline thikening of the
rane associated to some other alterations the parenchyma like
acuolization and wide spaces called "boble lesions".
More recently, worms also removed by perfusion after
reatment of infected mice, were examined by electron microscopy.
e drugs effects were described as swelling of the surface collapse
f the sensory bulbs and host cells attached (Voge 1980 and Kohn 1982)
.119.
In view of the above cited evidences, it was decided
to examine the damage in the tegument of the parasites using
histological serial sections of the livers, to follow the defense
mechanism of the host, that usually occurs in the process of
absorption.
Material and methods:
One hundred mice were exposed each one to 100 cercariae
obtained from snails infected in the laboratory. The infected
mice were submited to one single dose of oxaminiguine, 100 mg/kg
weight) injecting gastric intubation on the 45th day after been*
infected. 50 animals from the infected group were kept without
treatment for control.
Mice were killed by cervical dislocation at regular
intervals of 24, 48, 72, 96 hours and 5, 10, 15 and 30 days after
treatment.
Autopsy were performed and the liver were carefully
examined in fixed sections for histological routine parafine and
sections were frozen in acetone-dried-ice for cryostate microtome.
The sections were than attached to slides, bringing to laboratory
temperature and treated with anti-mouse antibody boti f luorescein-
conjugate and unlabeled. Corresponding controls for antimouse
fluorescein conjugated immunoglobulines were performed by
pretreatment with the unlabeled immunoglobulin for 30» followed
by 30m wash in PBS. All slides were mounted in buffered glycerin
at pH 7.3. All antiserum were tested for purity by reacting
against antigen. The conjugate as wellas nan labeled irtmunoglobu
Lines were obtained from P&stc-ur Lab. in Paris and diluted to
L/80.
,120.
Formalin fixed sections for routine parafine process
were stained with hematoxilin-eosine and P.A.S. (periodic-acid
Schiff)and examined under light microscope Leitz. Reflected fluo
rescence microscope equipped with an HBO 50 W. Burner, BG12+
| KV418 exiters and OG515 barrier filters were used, with an
I authomatic câmara for micro photografies.
Results;
Treatment of infected mice with a single oral dose of
oxaniniquine produced profound alterations in the surface of
worms. In the H+E and periodic-acid Schiff (P.A.S.) stained
sections, the most frequent lesion was a segmentar hyaline swel-
ling of the tegumenta1 membrane, in the worms at 24 to 48 hs
after treatment (Fig. 1 ). The frozen sections from the same
livers submited to fluorescein-conjugate antiserum showed positive
fluorescence in the tegument (Fig. 5 ). At this stage in the
correspondent portal space, cellular infiltration was present
with some irregular anorphus fluorescent material deposit (Fig.6).
Besides those lesions, the next damage, more proeminent,
48 to 96 hs after treatment, was swelling of the worm parenchyma
j under the damaged tegumenLar membrane. However the most preeminent
feature was the presence of numerous host cells, identified as
lymphocites and macrophages, attached to the outer tegument
membrane (Fig. 2 ). Those cells increases in number in the
worms with a difuse and severe parenchyma1 alterations, caracte-
rized by a cellular distention identified as vacuolar or hydropic
(degeneration (Fig. 3 ). Sections from liver of mice sacrificedi
r more advanced period of time from treatment, the tegument of
.1/1
worms was disrupted or absent and the host cells recorvering the
parasite which showed a dense cellular infiltration (Fig. 4 ).
At that time a complete granuloma was formed and the host cells
occupied the total portal structure.
Sections of mice liver killed after 10 days of treatment
showed regressive stage of the granulomas with presence of
hystiocites. A space between the central area, where the parasite
showed a great reduction in size or was reduced in fragments and
the portal vein wall, could be visualized.
At 30 days period, the host cells still present, diffuse
distributed inside of the portal area and the portal vein showed
a hystiocitic regular "ring like" distribution on the endothelium.
No worms could be found, nor fibrosis was present in the liver
froa the mice treated with oxaminiquine. On the other hand the
control group showed a large number of granulomas around eggs
and a severe fibrosis in the portal tract.
Contents:
The changes produced in schistosomas after treatment
with oxaminiguine, already described by removing the worms by
perfusion,were confirmed studing the histological aspects of
both worms and host.
The alterations previously described in the parasites
on tegujnental surface as well as in the parenchyma, were well
recognized in tissue sections from liver, at different period of
time. These are primarily hyaline swelling of the tegument mem-
srane, constriction or folding. The relationship of the parenchy
nal damage and the segmentai alteration of the membrane, suggest
that» maintenance of structural and funcional integrity of the
.122.
tegument may depend ~.r.n osmotic balance, resulting the vacuolar
infij*í t-. n of the parenchyma.
it^is also possible to relate the- danage of the worir.
surfa--t A_ the alteration on the mechanism of protection, as was
exemplified by the atachment of host cells to the damaged
surface-tegument. This could be confirmed by the presence of
host's immunoglobulines on the worm's surface, using the
fluorescent intiserum.
Following the host response one can obviosly consider
the gratmlomas around the dead parasites as a mediate cells
reaction. However, the immune response to the parasite, is first
dependnet on the alteration of the tegumental outer membrane.
This, unusual structured membrane is belived to play an important
role in protecting the worm from the host's immune response. So,
in circunstance where the worm is damaged by the drug,the surface
exposed act as target antigens for the host antibodies.
According to our findings, the host response apparently
iniciates early, just after the tegumental membrane is damaged.
Sections from liver taken 24 hs after the drug administration,
;showed that some worms could be found with segmentai swelling of
the membrane.i
Í At the sane tine the drug-induced alterations such as
swelling suggest the possibility of a modification in the osmotic
balance, responsable for the water infiltration and later the
vacuolar degeneration of the parenchyma.
Damage to the worm's surface them may expose the orgmism
to cellular response as exemplified by the attachment of host
cells. After 48-96 hs, the granulomatour formation takes place
and at this stage there is evidence that host's fagocitic
.123.
mononuclear ceils can infiltrate ttu* deyerieiate parenchyma easily.
At later period of time 30 days the most interesting
fe^-ure was the complete remotion of worm's particles and
restauration of the portal vein.
Studies with other effective drugs should contribute
to the clarification of this question, in special studies of
the biochemical effects to worms membranes.
References:
CESARI, I.M.; SIMPSON, A.J.G. & EVANS, W.H., 1981. Properties of
a series of tegumenta1 membrane-bound nhosphohydrolase activi
ties os Schistosoma mansoni. Biochem. J. London. 198: 467-473.
CLEGG, J.A., 1974. Host antigens and the immune response in schis
tosomiasis. In paraistes in the Immunized host. Ciba Foundation
Symposium 24 (New Series). Associated Scientific Publishers ,
New York, p. 161-176.
KOKN, A.; SERAPIÃO, C.J.; KATZ, N. & DIAS, E.P., 1979. Ação da
oxaminiquine sobre o schistosoma mansoni em camundongos expe-
rimentalmente infectados. Rev. Inst. Med. trop. São Paulo,São
Paulo. 21. (5): 217-227.
KDHN, A.; LOPEZ-ALVAREZ ft KATZ, N., 1982. Transmission and scan-
ning electron microscopical studies in the tegument of male
Schistosoma mansoni after oxamniquine treatment. Ann.Parasitol.
Paris, 57 (3): 285-291
3MITHERS, S.R.; TERRY, R.J. ft HOCKLEY, D.J/, 1969. Host antigens
in schistosomiasis. Proc. of the Royal Soc. Series B, 171:483-
494.
.124.
LÍUGANUAKI :;-f.KunAL, f., 1976. lmmunog Lobu If ti:i att.»<-h<"l to ami in
the integument <>i .tdult Schistosoma mansoni Sambom 1907,from
first lnfccti<in of CF mice. Jorn. Parasitol. 62 (2):222-226.
VOGK, M. a BUEDING, E., 1980. Schistosoroa mansoni: Tcqumental
surface alterations induced by subcurative doses of the
schistosomicide amoscanate. Exp. Parasitol. 50: 251-259.
11 ustration:
igures:
- S. mansoni (24 hs after treatment). The worm shows tegument
with swelling and vacuolization of the adjacent parenchyma.
2 - Male schistosoma (48 hs after treat.) Showing the host cells
attached in the tegument.
3 - Schistosoma (5 days after treat.). The granuloma complete
formed; the tegument disrupted and the host cells occupy the
total parenchymal structure.
4 - Schistosoma in advanced stage of absorption (20 days after
treatment). Presence of hystiocites arranged along the endo-
thelial cells.
5 - Fluorescence of portal vein endotheliura; segments of tegumen-
tal membrane and digestive tract. (24 hs).
6 - Fluorescence in the portal tract granulomatous reaction (15
days).
.125.
4
Fig. I
Fig. 2
Fig. 3
Alb,
Fig. 4
Fig. 5
Fig. 6
. 127 .
P r o t e c t i o n of Athyaic (Nu/Nu) BALB/c Nice Against Plasmodium
berghei by Splenocytes from Normal (Nu/+) BALB/c Mice.
José J. Ferraroni ' , Thomas G. Douglass and Clarence A. Speer
Universidade do Amazonas/CNPq, Campus Universitário, Departamento de Patologia
ICB - 69.000, Manaus, Amazonas, Brasil.
Department of Microbiology, University of Montana, Missoula, Montana, 59812
- USA.
f—
Abstract
Athynic BALB/c (Nu/Nu) mice died at 7-13 days after inoculation (UA1) of f.
erghei NK6S, whereas their heterozygous (Nu/*) littermates died at 7-8 UAI. Nu-
de (Nu/Nu) mice, reconstituted with 2 x 10 splenocytes from uninfected heterozy-
gous (Nu/+) littenaates at 20 days before parasite inoculation (OBI), died about
2 days earlier than control nude mice; nude mice reconstituted at 10 or 2 OBI
lived 2 to 4 days longer than control nudes; and nude mice reconstituted 2 UAI
lived even longer and some survived. These findings indicate that P. berghei
NK65 induces at least two T-cell dependent immune phenomena, one suppress!ve
and the other stimulatory. Reconstitution of nude mice with T-cells from BALB/<
(Nu/+) mice appeared to reduce or bypass supressive T-cell activities which all-
owed the formation of a protective immune response by some of the nude mice.
Introduction
Long lasting immunity to P. berghei NK65 has been achieved only by using para-
site inoculation and drug cure . Lymphocytes from such solidly immune animals a-
re capable only of transferring some degree of resistance to P. berghci . Acqui-
red res*stance to plasmodia has been shown to depend upon both bone marrow and2 13
thymus-derived lymphocytes . Recently, Waki & Suzuki were able to abrogate re-
sistance to P. berghei NK65 by reconstituting athymic nude mice with thymocytes
at three weeks before parasite inoculation. It is still not known how soon after
reconstitution by thymocytes that athymic mice become immunologically competent.8 9
The effects of reconstitution on both humoral and cell-mediated immunity have
been determined only for three or more weeks after thymic implantation or inje-
ction of thymocytes.
In order to determine the efficacy of mature lymphocytes upon £. berghei in-
fections in athymic nude mice, splenic lymphocytes and culture-derived spleen
cell supernatants from naive heterozygous (Nu/+) mice were adoptively transfer-
red to nude mice at various intervals before or after parasite inoculation. The
results obtained from these studies are reported herein.
Materials and MethodsAnimals, Congenie athymic nude (Nu/Nu) BALB/c mice and their hirsute (Nu/+)
littermates were raised in the Microbiology Departament animal facility at the
University of Montana. All mice were 8-weeks-oId at the beginning of each expe-
riment, except for experiment n9 3 in which the athymic mice were 5-weeks-old. .
These mice were originally obtained from the National Institute of Allergy and
Infectious Diseases, Rocky Mountain Laboratory, Hamilton, MT.
Parasite, Plasmodium berghei NK65 was originally obtained from the Universi-
ty of New Mexico, Albuquerque, NM, and had been maintained by weekly blood pas-
sages in Swiss-Webster mice for 5 yr before being used in this study. All mice
were injected intraperitoneally (IP) with 10 £. berghei-infected erythrocytes.
Splenocytes, Spleens were removed aseptically from 8 to 10 week-old donor
BALB/c (Nu/+) mice and forced through a 60-mesh stainless steel screen into
; Minimal Essential Medium (MEM, Gibco, Santa Clara, CA), pH 7.4, containing\ _ •
.129.
100 pg/ml (w/v) streptomycin sulfate and 100 D/al (w/v) potassaium penicillin G.
after allowing the tiss«e debris to settle for 10 «in, the call supernatant was
removed and washed three times with MBM. Exythrocytes were lysed with 0.15 M am-
monium chloride, after which the remaining nucleated cells were washed three ti-
mes in PBS. Nucleated cells less than 10 Urn in diameter were found to be 96X vi-
able as determined by trypan blue exclusion. Each recipient animal was injected •
IP with 0.2 ml MEM containing 2 x 107 viable splenocytes.
In order to compensate for two major variables (i.e., splenocytes and parasitt
inocula), two cell transfer experiments were performed. In the first experiment,
all animals received splenocytes from the same preparation, whereas fresh inoculi
of P_. berghei were obtained from mice at the time of each parasite inoculation.
Nine groups of 6 mice each (3 males and 3 females) were used. Croups 1-6 and 7-9
were BALB/c athymic (Nu/Nu) and BALB/c (Nu/+), respectively. Croups 1-5 received
splenocytes at 20, 10 or 2 days before parasite inoculation (DBI), at the time of
parasite inoculation (D0I), or 2 days after parasite inoculation (DAI), respecti-
vely. Splenocytes were given to group 7 two DAI and to group 8 at D0I. Groups 6
and 9 received no splenocytes and served as constrols.
Splenocyte supernatants, Three types of splenocyte supernatants (SCS 1, 2 and
3) were obtained as follows. Eight hundred million splenocytes suspended in 50ml
MEM containing 5% fetal calf serum (PCS) were inoculated into each of three 32 oz
Brockway culture flasks and than incubated at 37°C, 5% CO., 95Z air. Spleen cell
culture n9 1 was cultured 24 h in the presence of 5 ug/ml Concanavalin A (Con A,
Sigma Chemical Co., St. Louis, Mo.); after 24 h incubation, 5 yg/ml Con A were
added to culture n9 2; culture n° 3 was incubated 24 h without Con A. After incu-
bation, the supernatant was removed from each culture flask, centrifuged at 450 g
for 5 min, dialized 16 h at 4°C in 75 1 distilled water and then concentrated t-20
times by 6 h of evaporation dialysis.
Each mouse in three groups of 5 BALB/c (Nu/Nu) mice was injected IP with 0.5
ml SCS 1, 2 or 3 (approximately the amount derived from 2 x 10 splenocytes) at
two DAI of £. berghei. One group of 5 BALB/c (Nu/Nu) mice served as an untreated
control.
Results
Course of P. berghei infection, Figure 1 shows the mean daily parasitemia
(MDP) up until the time of death of BALB/c athymic mice inoculated with P.
berghei at various times relative to their receiving 2 x 10 splenocytes from the
same splenocyte preparation. The MDP rose slower and the mice survived signifi-
cantly longer in nude mice which received splenocytes at 10 DBI, 2 DBI, D0I and
2 DAI than in control mice. Splenocytes given 2 DAI caused the most significant
delay in rise of parasitemia as well as in day of death. It was in this group
only that any animals survived.
Figure 2 shows the MDP until the time of death of nude and heterozygous (Nu/+)
mice which were infected with the same inoculum of P. berghei but which received
splenocytes from different preparations at the same time of or one 2 DAI.
.130.
Heterozygous nice always succumbed tar Her with a lower parasitemia than nude
mice. The greatest beneficial effect-was observed in those mice that received
splenocytes at 2 DAI; their parasitemia remained relatively low (<SZ) for 17 DAI
and then increased to > 85Z by 27 DAI, which was also the HDD.
Figure 3 shows the MDP of nude mice that received SCS 2 DAI of 10 P. berghei
parasitized erythrocytes. No significantly differences occurred between the gro-
ups of nude mice which received SCS 1, 2, or 3 and the control group.
Discussion
Waki & Suzuki found that athymic nude (Nu/Nu) mice infected with P. berghei
NK65 survived significantly longer than their hirsute (Nu/+) littermates. Waki &
Suzuki also found that the survival time of nude mice that had been reconsti-
tuted with neonatal thymocytes from hirsute mice three weeks before parasite i-
noculation was not significantly different than that of their thymic competent
littermates. Based upon these observations, these authors suggested that nude
mice lived longer during a P. berghei NK65 infection because they lacked a T- j
cell-dependent immunopathologic response. The results obtained in the present s-j
tudy indicate that there is no immunopathologic response associated with mature :
splenic derived T-cells in mice infected with £. berghei NK65. We found that nu-
de mice reconstituted with splenocytes at 20 DBI showed no beneficial or harmful
effect and that those reconstituted at 10 or fewer DBI or at 2 DAI showed a de-
crease in onset of parasitemia and an increased survival time, and some mice e-
ven survived. Reconstitution with neonatal thymocytes probably provides a grea-
ter number of .T-cell recognition and effector functions than reconstitution with
splenocytes. Reconstitution of nude mice with thymocytes at three weeks before
inoculation may allow sufficient time for the development of suppressor T-cell
activity. Splenocytes may lack a full complement of T-cell subsets necessary to
generate suppressor T-cell activity of a magnitude of a similar to that obtain-13
ed by thymocyte reconstitution as observed by Waki & Suzuki . Thus, P. berghei ,
appears to induce at least two T-cell dependent immune phenomena, one suppressi-
ve and the other stimulatory. Reconstitution of nude mice with splenocytes from j
phenotypically normal heterozygous mice appears to reduce or bypass the suppresH
sive T-cell activities which allows the formation of a protective immune respon-*
se to P_. berghei by some of nude mice. ,
Several mechanisms are probably involved in the ability of splenocytes from ,
thymic competent mice to protect nude mice against P_. berghei NK65. Macrophages <
of nude mice have a greater capacity to phagocytose and kill certain viruses, •
bacteria and yeasts than macrophages of thymic competent mice * ' ;
However, enhanced activity by the reticuloendothelial system (RES) does j
not appear to be responsible for the increased survival time and protec-
tion against Plasmodium and Babesia by mice that had received Bacille
Calmette-Guérín (BCG) or killed Corynebacterium parvun ' .
In the present study, nude mice did not clear malarial parasites from their
peripheral blood as rapidly as thymic competent mice. Thus, tht delay in onset :
.131.
of parasitemia in nonreconstituted nude mice does not appear to be due to enhan-
ced activity or the RES..14
Recently, Waki & Suzuki reported that nude mice infected with P_. berghei
and cured by pyrimethamine primed the animals so'that a secondary antibody res-
ponse was elicited upon parasite challenge. They concluded, therefore, that at
least one parasite antigen was thymus independent and that primed B-cells were
the dominant cells generated during a primary infection in nude mice. However,12
B-cells can be primed by thymus dependent antigens in the absence of T-cells
Immune serum from mice driven to immunity by repeated cycles of P_. berghei infe-
ction and Fansidar cure (pyrimethamine + sulfadoxine) has been found to suppress
parasite development in naive mice, but only if administered on a daily basis .
Two lines of evidence indicate that antibody is probably not involved in the
antimalarial protection of nude mice reconstituted with normal splenocytes. Fir-14
; st, Wàki & Suzuki found that nude mice immunized against P. berghei were notprotected against parasite challenge even though they developed significant le-
vels of parasite-specific antibodies. Second, we found that the nude mice, which
had been reconstituted with splenocytes and survived a primary P_. berghei infec-
tion, succumbed to challenge inoculation 40 days later (about 10 days after par-
asite rlearence of the first inoculation). Thus, such reconstituted nude mice e-
vidently have no memory of their earlier infection and the factors involved in
i the elimination of the parasites in the primary infection are short-lived.
During the ontogeny of the immune system the ability to suppress a specific
immune response usually develops after the ability to respond to specific anti-
gen . In addition, soluble T-cell suppressor substances are usually not produc-
ed for about 48 h following the interaction of T-cells with specific antigen,9
, whereas T-cell helper substances are known to be produced as early as 12 h .A
significantly greater degree of protection was obtained in nude mice that recei-
ved splenocytes at or several days before parasite inoculation (present study).
This indicates that helper T-cells may play an inportant part in the resistance
of splenic reconstituted nude mice to £. berghei.
?ig. I Mean daily parasitemia of BALB/c nude mice which received splenocytes
i from different heterozygous BALB/c (Nu/+) sources after inoculation of
> 10 jP. berghei-infected erythrocytes. i|j> , received splenocytes 20 DBI;
j O,control, received no splenocytes; Q, received splenocytes 10 DBI; LJ »
received splenocytes 2 DBI; J^ , received splenocytes DOT; £ , receiv-
ed splenocytes 2 DAI.
Fig. 2 Mean daily parasitemia of BALB/c (Nu/Nu) and phenotypically normal
(Nu/+) mice inoculated with 10 P. berghei-parasitized erythrocytes and
B|.l«-nocytes from the same preparation from BALB/c (Nu/+) mice. Q , con-
ifil nude mice, received no splenocytes; & , control heterozygous
'.II/*) mice, received no splenocytes; Q » BALB/c (Nu/+) mice received
9pit norytes DOI; A • nude mice received splenocytes DOT; | , nude mice
.132.
received splenocytes 1 M I ; 0 , nude aice received splenocytes 2 DAI
Fig. 3 Mean daily parasiteua of BALB/c nude mice receiving splen cell sup-
ernatant (SCS) froa splenocytes of BALB/c (Nu/+) nice with or wkh-
out ConA .and inoculated with 10 £. berghei-parasi tized erythrocytes
2 DAI. £ .control, no SCS; O > scsi i\ > i r o" splenocytes cultu-
red in presence of ConA for 24 hr; Q , SCS from splenocytes cultur-
ed for 24 hr and then treated with ConA.
.133.
REFERENCES !í
1. Bazar-Malik, G. Increased resistance to malaria after Hycobacterium 1
tuberculosis infection. Indian J. Med Res. 61:1014, 1973. '
2. Brown, I. N. Immunologic&l aspects of malaria infection. Advan. Immunol.
^1:267, 1970.
3. Cauley, L. R. ft Murphy J. W. Response of congenitally athymic (nude) and
phenotypically normal mice to Cryptococcus neofoimans infection.
Infect. Immun. 23:644, 1979.
4. Clark, I. A.; Allison A. C. ft Cox, F. E. G. Protection of mice against
Babesia and Plasmodium with BCG. Nature 259:309, 1976.
5. Emmerling, P.; Finger, H. ft Bockemuhl J. Listeria monocytogenes infection'
in nude mice. Infect. Immun. 17:437, 1975.
6. Ferraroni, J. J. ft Speer, C. A. Fansidar prophylaxis, therapy and immune I
responses in rodent malaria (Plasmodium berghei) J. Parasitol. •
68:609, 1981.
7. Ferraroni, J. J. ft Speer C. A. Adotive transfer of resistance to |
Plasmodium berghei with spleen cells and serum from Fansidar-cured |
mice. Infect. Immun. 36:1190, 1981. j
8. Haaijman, J. J.; Teunissen, J. S,; Oudenaren, A. V; Mink, J. C. ft Benner i
R. Kinetics of recovery of serum Ig levels and of cytoplasmic Ig posi-j
tive cells in various lymphoid organs of nude mice after thymus trans-j
plantation Immunology 41:279, 1980. j
9. Lair, S. V. ft Lozzio, B. B. Thymocyte reconstituion of athymic and athym-
ic-asplenic mice: Graft rejection and antibody synthesis. Exp. Cell
Biol. 48_:439, 1980.
10. Metcalf E.; Schrater A. F. ft Klinman, N. R. Murine models of tolerance
induction in developing and mature B cells. Immunol. Rev. 43_:143,1979
11. Mongensen, S. C. ft Andersen, H. K. Role of activated macrophages in resis-
tance of congenitally athymic mude mice to hepatitis induced by herpes
simplex virus type 2. Infect. Immun. ViiVil, 1978.
12. Schrader, J. W. The role of T cells in IgC production: Thymus-dependent
antigens induce B cell memory in the absence of T cells. J. Immunol.
114:1665, 1975.
13. Waki, S. ft Suzuki, M. A study of malaria immunobiology using nude mice. In
Proceedings of the 2nd International Workshop on nude mice. University
Tokyo Press, p. 37. Stuttgart, Gustav Fisher Verlag, 1977.
14. Waki, S. ft Suzuki, N. T-independent antibody production in nude mice imm-
unized with a rodent malaria parasite (Plasmodium berghei). In M. 0.
Reed (ed.) , 1980.
.134.
Fig. -1
9 4 a
DAYS AFTER INOCULATION
Fig.-2
as M tr
DAYS AFTER INOCULATION
Fig.-3
14
1 1 1
• • rDAYS AFTER INOCULATION
.135.
MALARIA IN HUMAITA COUNTY, AMAZONAS STATE. BRAZIL. XXIX - SOM1COMPARATIVE EPIDEMIOLOGIC ASPECTS IN 1976. 1979 AND 1983.
D.\ MEIRA; P.R. CURI AND B. BARRAVIERA
Faculdade de Medicina de Botucatu - UNESP
INTRODUCTION
The factors which raise difficulties to malariacontrol program are complexes and not always well defined foreach region' . In this way malaria evolutive study in anendemic region submitted to erradication of the disease mustbe considered very important. In August 1983 the authors accomplished again a malaria survey in Humaita County, Amazonas Sta-te, in which some epidemiologic aspects were studied and thencompared to those observed by them in 1976 and 1979^ K
MATERIAL AND METHODS
In 1976, 1979 and 1983 the authors studied 409,330 and 177 individuals respectively, all of them inhabitantsof Humaita County, residing along the highways, along the banksof the Madeira river and within the urban area. The sane re-gions were studied during each of the three years. Sex and agedistribution were similar and male individuals were predominantin the roads, while the number of the female was bigger in urbanzone. All the individuals were submitted to clinical examina -tion and the epidemiologic evaluation included prior history ofprevious attacks of malaria, splenomegaly and spleen index. The
results were statistically compared through X test ( « • 0,05)(3,4,5 e 6)
RESULTS
The results are shown in Tables 1,11, III, IV and Y.
Research accomplished with support of National Council of Scien-
tific and Technological Development - CNPq (Proc. 40.3705/82)
,136,
T A B L E I_
DISTRIBUTION OF STUDIED SAMPLES IN 1976, 1979 AND 1983 ACCORDING TO ORIGIN
AND MALARIA PREVIOUS ATTACKS.
ORIGIN
Roads (R)
Madeira River (M)
Urban Zone
ABSENT
N» (\
50
90
32
(33.
(40.
(84.
)
11)
91)
21)
1976
PRESENT
N* m
101
130
6
(66.89)
(59.09)
(15.79)
1979
ABSENT
N» (I)
47
1*2 7
49
(57
(69
(77
4 3)
10)
08)
PRESENT
N* (*)
35
57
15
(42.57)
(30.90)
(22.92)
1983
ABSENT
N» (t)
25
62
64
(59
(81
(81
.52)
.58)
.01)
PRESENT
N* (I)
17
14
15
(40
(18
(18
48)
42)
99)
Comparison of previous attacks related to the year:
21976:
1979: x
X" = 32.71* (p < 0.001) - R > M > U
2
1983: x'
6.50* (0.02 < p < 0,05) - R > U
8.738*(0.01 < p < 0.02) - R > (M = U)
Comparison of previous attacks related to origin
R: x2 = 4.29 (0.10 < p < 0.20) n.s. in the 3 years
M: X = 53.21* - The fal] in the previous attack:;
proportion is quadratic, and it was more pro-
nounced from 1976 to 1979 than from 1979
1983 (Pig. 1)
U: x2 = 0.79 (p > 0.50) n.s. in the 3 years .
to
T A B L E I I
DISTRIBUTION OF CHILDREN ON AGE GROUP 2 TO 9 STUDIED IN 1976 , 1979 AND 1983 ACCORDING
TO ORIGIN AND PRESENCE OF ENLARGED SPLEEN (SPLEEN INDEX).
ORIGIN ^ ^ ^ ^
Roads (R)
Madeira River (M)
Urban Zone (U)
1976
ABSENT
N* (I)
33
57
16
(63
(79
(94
46)
.17)
12)
PRESENT
N« m19
15
1
(36.54)
(20.83)
( 5.88)
1979
ABSENT
N' (I)
22
So
26
(68
(91
(96
75)
80)
.30)
PRESENT
10
5
1
(31
( 8
( 3
25)
20)
70)
1983
ABSENT
N' {%)
6
27
23
( 46
( 96
(100
.15)
.42)
.00)
PRESENT
N« (t)
7
1
0
(53.85)
( 3.58)
( 0.00)
Comparison of enlarged spleen according to the year:
1976: x2 = 7.70* (0.02 < p < 0.05) - R > U
1979: x2 =12.43* (p < 0.001) - R > (M = U)
1983: R x M: G = 3.53* (p < 0.05)
R x U: G = 3.89* (p < 0.05)
M x U: G = 1.02 * (p > 0.10)
Compar i son of enlarged spleen according to the o r ig in :
R: x 2 - 2 .04 ( 0 . 3 0 < p < 0 . 5 0 ) - n . s . in the 3 years
M: x • 7 . 3 3 * ( 0 . 0 2 < p < 0 . 0 5 ) - E n l a r g e m e n t o f
spleen was less frequent in children on agegroup 2 to 9, and i t was more pronounced from1976 t o 1979 ( F i g . 2)
U:x = 1-25 (p > 0.50) - n.s. in the 3 years.
T A B L E iu_
DISTRIBUTION OF INDIVIDUALS OLDER THAN 15 YEARS STUDIED IN 1976, 1979 AND 1983
ACCORDING TO ORIGIN AND ENLARGED SPLEEN.
ORIGIN ^ ^ ^ f r
Roads (R)
Madeira River (M)
Urban Zone (U)
1976
ABSENT
N«
8
15
1
(*)
(12
(16
(20
.31)
.13)
00)
PRESENT
N*
58
80
4
(t)
(87.69)
(83.87)
(80.00)
ABSENT
N«
26
63
7
(%
(50.
(80.
(87.
1979
)
98)
77)
50)
PRESENT
N'
25
15
1
('
(49
(19
(12
.02)
23)
50)
ABSENT
N»
6
26
. 20
(\
(37.
(92.
(66.
1983
)
50)
86)
66)
PRESENT
N»
10
2
10
m(62.50)
( 7.14)
(33.34)
Comparison of enlarged spleen according to the year:
1976: n.s.
1979: xZ - 14,28* (p < 0.001) - R > (M = U)
1983: x 2 = IS.23* (p < 0.001) - (R = U) > M
Comparison of enlarged spleen according to the
origin:
R: x2 = 22.79* (p < 0.001) - There was a fall in
the period from 1976 to 1979, with little in-
crement from 1979 to 1983.
M: x = 68.64* (p < 0.001) - There was a pronounc-
ed f a l l in 1983 when compared to 1976 and 1979.
U: x2 " 9.62* (p < 0.01) - There was a b igger
p ropo r t i on in 1983.
3O 2
1 1 5
isis
! 5 V
I** O O
o -• o =
I 3
Kl O - s e -
I 3 8 3 ! =
i l l
s s sS r • f
O Q. C_
I =
- s x =
^ - .9.
Í ? z z
I « 9 S2 d. S o
5 ?. s f,
I V ".' 75 x i i
5 -» »• *« 1. 7
. 1 4 0 .
II';. J - M-.M.P'.T.L» ;-)•«>H.i'l .".:• )•'• :
J'J«).yjO«J-*, /-.TlACKb I ROM J'J- '
HIVI.P J'. TAB H A N T S .
PI
Ü.GÜ
0,4!
0.30
0,15
< ! . J ••-.
1970 1979 1983 YI:AR
M C . 2 - OBSERVED PROPORTION OF ENLARGED SPLEEN ] CHILlf-:
ON AGE GHO'iP 2 TO 9 LIVING IN MADE I PA PI VER V] LU-
GES, FROM 1976 TO 1983.
PI
0,20
0,10
1976 1979 1983 YEAR
. 1 4 1 .
DISCUSSION
In 1976 a history of prior malaria was most fre -quent among inhabitants of Madeira River. In 1979, the greatestfrequence was amongst roadside residents when compared to thoseliving in urban area. In'1983, prior malaria history was mostfrequent in roadside inhabitants than in those living alongsidethe river and in urban area. However, both of the latter hadthe sane frequence. There also was variation in frequence ofprior malaria attack among individuals living alongside the roadsin the three years studied.
The incidence of splenomegaly and average size ofthe spleen was greater in highway residents than in those livingalong the river or in the urban zone during each of the threeyears.
These results show that malaria was mesoendemicalong the river in 1976, but became hypoendemic in 1979 and 1983.Along the higways it was mesoendemic in 1976 and 1979, but beca-me hyperendemic in 1983. In the urban zone, malaria was alwayshypoendemic.
.142.
REFERENCES
1. ALVARADO, C.A. - Malária in: Doenças Infecciosas e Parasita -rias. Editado por Ricardo Veronesi. 6a. Edição. Rio de Ja-neiro, Guanabara Koogan, 1976, pag. 660-682.
2. BOLETIN DE LA OFICINA SANITARIA PANAMERICANA - III Reunion deDirectores de los Servicios Nacionales de Erradicacion dela Malaria en las Americas. 87: 172-176, 1979.
3. CURI, P.R. 5 MORAES, R.V. - Associação, homogeneidade e con -trastes entre proporções em tabelas contendo distribuiçõesmultinomiais. Ciência e Cultura. 3^(5): 712-722, 1981.
4. FLEISS, J.L. - Statistical methods for rates ans proportion.New York, John Wiley and Sons, 1973.
5. FREEMAN, G.H. (, HALTON, J.H. - A note on exact treatment ofcontingency goodness of fit and other problems of signifi -cance. Biometrika, 38: 141-149, 1951.
6. HOLLANDER, M. & WOLFE, D.A. - Nonparametric statistical methods.New York, John Wiley and Sons, 1973.
7. MEIRA, D.A.; CORRÊA, F.M.A.; SOGAYAR, R.; BARRAVIERA, B.; COSTARDI, A.C.; RUI,P.; SALATA, E. Ç PIROLA, J.A.G. - Maláriano município de Humaitá. Estado do Amazonas. II - Alguns as>pectos epidemiológicos comparativos entre 1976 e 1979. Rev.Inst. Med. trop. São Paulo, 23 (Supl. 5) (5): 5 - 11, 1981.
8. RUSSEL.P.F.; WEST, L.S. & MANWELL, R.G. - Malaria Surveys. In:Practical malariology. Edited by Paul F. Russel, Luther S.West 5 Reginald D. Manwell. Philadelphia, W.B. Saunders Company, 1946, pag. 378-403.
.143.
MALARIA IN HUMAITA COUNTY. AMAZONAS STATE. BRAZIL. XXX - AN INQUIRYINTO COMPARATIVE PARASITOLOGY DURING 1976, 1979 AND 1983.
D.A. MEIRA; S.M. DI SANTI; B. BARRAVIERA AND P.R. CURIFaculdade de Medicina de Botucatu - UNESP.
P. RUISUCAM de Humaitá - Ministério da jde
INTRODUCTION
Blood parasitology has been used as the only methodfor malaria diagnosis in endemic regions. However not always is the
real number of cases expressed by this procedure, what is in con-trast with clinical diagnosis for example.
The evolutive study in a same region based onparasitologic survey could concur to confirm these observations ,mainly when compared to other procedures.
In August 1983 the authors made a parasitologic survey in Humaita County, Amazonas State based on thick smears thatwere subsequently compared with those made by them in 1976 and 1979.
MATERIAL AND METHODS
In 1976, 1979 and 1983 respectively 409, 293 and182 individuals were studied in Humaita. All were inhabitants ofvarious regions of the County, living either along the highways, inlocalities in the banks of Madeira River or in an urban districtAll patients were submitted to finger punction and thick smearswere made and stained by Giemsa method for parasitology examinationof 100 microscop fields examined under oil immersion. In 1976 and1983, only one smear was collected from each individual while in1979 samples were obtained on four cpnsecutive days.
RESULTS
The results are shown in Tables I and II.
Research accomplished with support of National Council of Scienti -fie and Technological Development - CNPq (Proc. 40.3705/82).
.144.
T A B L E I
DISTRIBUTION OF STUDIED SAMPLES IN 1976 AND 1983 ACCORDING TO ORIGIN AND
POSITIVITY IN PARASITOLOGICAL EXAM DONE ONLY ONCE IN EACH INDIVIDUAL.
O R I G I N ^ ^
Road
Madeira River
Urban Zone
TOTALS
N»
12
0
0
12
(I)
(8.0)
(0.0)
(0.0)
(2.9)
1976
N»
139
220
38
397
m
( 92.0)
(100.0)
(100.0)
( 97.1)
N*
0
0
0
0
(t)
(0.0)
(0.0)
(0.0)
(0.0)
1983
N'
39
64
79
182
— 4
m
(100.0)
(100.0)
(100.0)
(100.0)
T A B L E U_
DISTRIBUTION OF STUDIED SAMPLES IN 1979 ACCORDING TO ORIGIN, NUMBER OF PARASITOLOGICAL EXAMS
IN EACH INDIVIDUAL AND POSITIVITY OF PARASITOLOGICAL EXAMS.
EXAM- ^ < í ^
1»
2»
3 '
4»
TOTALS
0
0
1
2
3
ROAD
(t)
(0.00)
(0.00)
(5.88)
(6.90)
(4.17)
N*
9
17
16
27
69
(100.00)
(100.00)
( 94.12)
( 93.10)
( 95.83)
N»
0
2
0
1
3
MADEIRA
(0.00)
(8.70)
(0.C0)
(2.78)
(2.86)
RIVERi—i
N»
22
21
24
35
102
(»)
(100.00)
( 91.30)
(100.00)
( 97.22)
( 97.14)
N»
0
0
0
0
0
URBAN
CD
(0.00)
(0.00)
(0.00)
(0.00)
(0.00)
ZONE
N»
23
27
33
33
116
(t)
(100.00)
(100.00)
(100.00)
(100.00)
(100.00)
N»
0
2
1
3
6
SUB
( «
(0.00)
(2.02)
(1.36)
(3.07)
(1.07)
TOTALS
N»
54
65
73
95
287
w(100.00)
( 97.98)
( 98.64)
( 96.93)
( 98.93)
TOTALS
N»
54
67
74
98
293
(I)
(100.00)
(100.00)
(100.00)
(100.00)
(100.00)
REFERENCES
1. MARQUES, A.C. - Situação atual do controle das grandes ende -•ias. Exposição feita no XV Congresso da Sociedade Brasi -leira de Medicina Tropical, realizado em Campinas, São Pau-lo, de 4 a 8 de fevereiro de 1982.
2. MEIRA, D.A.; PITA. H.J.; BARRAVIERA, B.; SPERANDIO. L.; LI -MA.J.R.; CORRÊA,F.M.A.; SOGAYAR, R.; SALATA, E.; BRASIL, M.A.M.; MENDES, R.P. { CAMPOS, E.P. - Malária no município deHumaitá, Estado do Amazonas. I - Alguns aspectos epidemiológicos e clínicos. Rev. Inst. Med. trop. São Paulo, 22:124-134, 1980.
3. SOGAYAR, R.; CORRÊA, F.M.A.; SALATA, E.; MEIRA, D.A.; MENDES ,R.P.; CAMPOS, E.P.; BARRAVIERA, B.; PITA, H.J.; BRASIL.M.A.M. & SPERANDIO, L. - Malária no «unicípio de Humaitá, Esta-do do Amazonas. XI - Aspectos paTasitologicos. Rev. Inst.Med. tTop. São Paulo, £(Supl. 5): 65-71. 1981.
4. SOGAYAR, R.; BARRAVIERA, B.; MEIRA, D.A.; MENEGUIM, J.M.; DlSANTI, S.M.; PIROLA, J.A.G.; VADILETI, C. 5 BARBOZA, A.F. -Malária no município de Humaitá, Estado do Amazonas. XV -Inquérito parasitolôgico em dias consecutivos nos habitan -tes da região. Rev. Inst. Med. trop. São Paulo, 24_ (Supl.-6): 29-31, 1982.
,147.
MALARIA IN HUMAITA COUNTY. AMAZONAS STATE. BRAZIL. XXXV - INQUIRYINTO THE EPIDEMIOLOGY OF THE GENERAL POPULATION USING PASSIVE HE-MAGGLUTINATION.
O.A. METRA; J. MARCONDES AND R.P. MENDESFaculdade de Medicina de Botucatu - UNESP
A.B. EL-KHOURY AND V. CURYHospital do Servidor Publico Estadual
P. RUISUCAM DE HUMAITA - MINISTÉRIO DA SA0DE
INTRODUCTION
Malaria parasitological diagnosis is a very simpleand specific one. Neverthless, in endemic regions there are somesituations which make Balaria diagnosis impossible to be nade. asit happens in under-microscopic parasitenia. However in such these regions malaria control through patient treatment depends onparasitological diagnosis made by means of active and passivesearching. In this way, the adoption of new and more sensibletechinics for diagnosis, that also could be easily put to usecould contribute in a more efficient way for breaking the epide -miological chain.
The objective of this study was to evaluate passivehemagglutination behaviour in a sero-epidemiological survey in Hu-maita County, Amazonas State inhabitants.
MATERIAL AND METHODS
In August 1983 the authors studied 13S individualsall inhabitants of some regions in Humaita County. Fifty six ofthem came from Madeira River Villages; twenty-one were inhabitantsof Transamazonia Highway; forty-one were living in the urban zoneand seventeen were soldiers from the 54 Batallion of the JungleInfantry Service. The soldiers were the control group and therefore
Research accomplished with support of National Council of Scienti^
fie and Technological Development - CNPq (Pr>c. 40.3705/82).
.148.
were,selected by the Batallion Health office from volunteers bornin the State of Amazonas. They had never had previous malariaattacks, had no spleen enlargement nor parasites detected in bloodfilm at the time of examination.
All studied individuals were submitted to clinicalexamination including past history of malaria attacks, evaluationof spleen enlargement and blood smear for parasite counting. Bloodwas collected by venopuncture for antibody determination by passi^ve hemagglutination method using antigens obtained from Plasmodiumfaleiparum merozoite* '.from dillution 1:16 on.
RESULTS
The results are expressed in Table I.
DISCUSSION
The results showed four (7.It) positive tests awngthe River inhabitants, two (9.51) among the Highway inhabitants
and 10 (24.4t) in the urban zone inhabitants, making a total of16 (13.51) positive tests among the 135 individuals studied. Allthe results of the control group were negative.
There was no correlation between history of previousattacks of malaria (four patients), spleen enlargement* ' (fivepatients) and positive hemagglutination.' There was also no conco-mitance of passive hemagglutination and blood smear since parasite
f 21counting was negative in all individuals studied* .
The analysis of the results suggests that passivehemagglutination is more accurate than blood smear for the diagno-sis of malaria in endemic regions.
.149.
T A B L E I
PASSIVE HEMAGGLUTINATION FOR ANTIBODY DETERMINATION USING Plasmodium falaiparum
AS ANTIGEN, IN INHABITANTS OF HUMAITA COUNTY, AMAZONAS STATE.
inO
ORIGIN
ROADS
MADEIRA RIVER
URBAN ZONE
SOLDIERS*
TOTALS
1/16
2
2
8
0
12
1/32
0
0
2
0
2
PASSIVE
1/64
0
1
0
0
1
HEMAGLUTINATION
1/128 1/256
0
0
0
0
0
0
0
0
0
0
1/10 24
0
1
0
0
r
N»
2
4
10
0
16
SUB - TOTALS
(%) N»
( 9.5)
( 7.1)
(24.4)
( 0.0)
(11.8)
19
52
31
17
119
(I)
( 90.5)
( 92.9)
( 75.6)
(100.0)
( 88.2)
N»
21
56
41
17
135
TOTALS
(100.0)
(100.0)
(100.0)
(100.0)
(100.0)
* Soldiers from the 54 Batallion of the Jungle Infantry Service.
REFERENCES
1. MEIRA, D.A.; CURI, P.R. $ BARRAVIERA, B. - Malária nc municí-
pio de Humaitã, Estado do Amazonas. XXIX - Alguns aspectos
epidemiologicos comparativos entre 1976, 1979 e 1983. Apre-
sentado no XX Congresso da Sociedade Brasileira de Medicina
Tropical e I Congresso da Sociedade Latino-Americana de Me-
dicina Tropical, Salvador, Bahia, fevereiro de 1984.
2. MEIRA, D.A.; Dl SANTI, S.M.; BARRAVIERA, B.; CURI, P.R. 6 RUI,
P. - Malária no município de Humaitá, Estado do Amazonas
XXX - Inquérito parasitologico comparativo entre 1976, 1979
e 1983. Apresentado no XX Congresso da Sociedade Brasileira
de Medicina Tropical e I Congresso da Sociedade Latino-Ameri_
cana de Medicina Tropical, Salvador, Bahia, fevereiro de
1984.
3. OLIVEIRA, R.M. - Personal comunication.
.15],
MALARIA IN HUMAITA COUNTY, AMAZONAS STATE, BRAZIL.XXXIV - IMMUNERESPONSE OF PATIENTS WITH Plasmodium faleiparum according to ga-metocytes.
D.A. MEIRA; P.R. CURI AND J. MARCONDESFaculdade de Medicina de Botucatu - UNESP
E.S. MATSUOKA; M.A. FAVRIN AND A.B. EL KHOURYHospital do Servidor Público Estadual
N.G.S. MOTTAInstituto Básico de Biologia Médica e Agrícola da UNESP
INTRODUCTION
The association between the amount of circulatinglymphocyte and the presence of gametocytes in Plasmodium falcipa^rum malaria patients* ' ' has indicated the need to investigatethe eventual relations among such factors and the immune responseof the patients.
The purpose of this paper was to demonstrate thebehaviour of leukocytes, total lymphocytes and T and B lymphocytesin Plaemoâium falciparum malaria patients before and after thetreatment, according to gametocyte presence.
MATERIAL AND METHODS
In August 1983 the authors studied 36 patientswith Plasmodium faloiparum malaria and 14 normal individuals ,boin in Humaita Region who never had malaria, with no spleen en-largement and with negative blood parasite counting as wellas passive hemagglutination. All the studied individuals weresubmitted to complete clinical observation and to hematologicalexamination, blood parasite counting and lymphocyte typing. Thelymphocyte were separated and then frozen in liquid nitrogen ^for later typing by rosette formation.
Research accomplished with support of National Council of Scientifie and Technological Development - CNPq (Proc. 40.3705/82).
152.
The patients were treated with clindamycin, 20mg/Kg/'day alone or in association with chloroquine for 3 to 5days. None of then received primaquine.
Blood parasite counting was repeated daily duringthe treatment while the hematological examination and lymphocytetyping only afteT the end of it.
The patients were divided in two groups accordingto the presence (13 patients) or absence (23 patients) of gametocytes before treatment. Severe malaria was predominant in thegroup without gametocytes.
The results were analysed by "F" statistical me-thod and paired and unpaired "t" test.
RESULTS
The results are expressed in Table i, II, II-I,IV, V and VI and in Figure 1.
DISCUSSION
The results showed that the amount of leukocyteand total lymphocyte was similar in normal individuals and inthe patients with gametocytes before and after treatment. Leuco-penia and lyjnphopenia were observed in the patients without gametocytes before treatment, with tendency to normalization after it. The amountsof T lymphocyte were very low in both the patient groups beforetreatment, with tendency to increase in the convalescence period.The B lymphocyte béhaviouT was different in the two patient group:patients with gametocytes had normal amounts and those with' atgametocytes had very low amounts of it.
The results suggest that in accute malaria thereis a depression of T cells and that the immune response is rela-ted to the presence of gametocytes and B lymphocytes.
.153.
T A B L E I
COMPARISON OF LEUKOCYTE AND LYMPHOCYTE AVERAGE BETWEEN NORMAL INDIVIDUALS AND Plaamodium falciparum MALARIAPATIENTS BEFORE THE TREATMENT ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE THE TREATMENT.
GROUPS
6 1
6 2
G3
N* OF
INDIVIDUALS
14
13
21
LEUCOCYTES
AVERAGE
/inn 3
5679
5100
3671
STATISTICAL
ANALYSIS
F = 1 3 . 2 1 *
SIGNIFICANCE
LEVEL
p < 0 . 0 0 1
COMMENTS
(Gj • GO > G j
lYMTOCYTES
AVFMGE
/m?
2095
1708
1101
STATISTICAL
ANALYSIS
F - 1 4 . 1 7 *
SIGNIFICANCE
LEVEL
p < 0 . 0 0 1
COMMENTS
(Gj • GO > G.
G, = Normal individuals G2 = Patients with gametocytes before the treatment
G, = Patients without gametocytes before the treatment.
T A B L E 12
COMPARISON OF LEUKOCYTE AND LYMPHOCYTE AVERAGES BETWEEN NORMAL INDIVIDUALS AND Plaemodium falei-pavum
MALARIA PATIENTS AFTER TREATMENT ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE TREATMENT.
GROUPS
H
G3
N» OF
INDIVIDUALS
14
11
23
LEUCOCYTES
AVERAGE
A m
5679
4873
4352
STATISTICAL
ANALYSIS
F = 4.79*
SIGNIFICANCE
LEVEL
0.01 <p< O.OS
COKWENTS
G 1 > G 3
Ç>2 i s i n t e rmed ia te
LYMPH0C1TES
AVERAGE
/mm3
2095
2013
1836
STATISTICAL
ANALYSIS
F = 0 .59
SIGNIFICANCE
LEVEL
p > 0 .50
O&MENTS
The groupsdon't differ
G, * Normal individuals G2 = Patients with gametocytes before the treatment
G-, = Patients without gametocytes before the treatment.
T A B L E III
COMPARISON OF LYMPHOCYTE T AND B AVERAGES BETWEEN NORMAL INDIVIDUALS AND Plaamodium falciparum MALARIA
PATIENTS BEFORE TREATMENT, ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE TREATMENT.
GROUPS
G2
G3
N« OF
INVIDIDUALS
14
13
20
T LYMPHOCYTES
AVERAGE
/ran3
1064
529
357
STATISTICAL
ANALYSIS
F = 14.88*
SIGNIFICANCE
LEVEL
p < 0.001
COMMENTSB LYMPHOCYTES
AVERAGE
/mm3
346
284
220
STATISTICAL
ANALYSIS
F = 2.58
SIGNIFICANCE
LEVEL
P>0.05
COMMENTS
((G -G ) >.G3'Trend"
Normal individuals G2 = Patients with gametocytes before the treatment
Patients without gametocytes before the treatment.
T A B L E IV
COMPARISON OF LYMPHOCYTE T AND B AVERAGES BETWEEN NORMAL INDIVIDUALS AND Plaamodium faloiparum MALARIAPATIENTS AFTER THE TREATMENT, ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE THE TREATMENT.
GROUP
G3
N* OFINDIVIDUALS
14
10
20
T LL4PHOCYTESAVERAGE/mn3
1064
786
869
STATISTICALANALYSIS
F - 1.51
SIGNIFICANCELEVEL
p > 0.10
COMMENTS
The groupsdon't differ
B LYMPHOCYTESAVERAGE/mn3
346
506
332
STATISTICALANALYSIS
F - 0.10
SIGNIFICANCELEVEL
p>0.80
GOhMIOTS
The groupsdon't diffei
G, = Normal individuals Patients with gametocytes before the treatment
G, * Patients without gametocytes before treatment.
T A B L E V
COMPARISON OF LEUKOCYTE AND LYMPHOCYTE AVERAGE OF Plaamodium faloipurum MALARIA PATIENTS BEFORE AND AFTERTREATMENT, ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE THE TREATMENT.
GROUPS N» OFINDIVIDUALS
LEUCOCYTESAVEARGE/mn3
BEFORE AFTER
STATISITCAL SIGNIFICANCEANALYSIS LEVEL
COMMENTSLEUCOCYTES
AVERAGE/mm ANALYSISBEFORE AFTER
STATISTICAL SIGNIFICANCELEVEL
COMMENTS
11
23
5136 4873 t - 0.44 p > 0.50 Before • After
3578 4365 t = 1.75 0.05<p<0.10 Before i After
1724 2013 t - 1.03 p > 0.30 Before • After
1015 1836 t - 6.30* p < 0.001 Before < After
G2 = Pat ients with gametocytes before the treatment
G3 " Pat ients without gametocytes before the treatment.
T A B L E V I
COMPARISON OF T AND B LYMPHOCYTE AVERAGES OF Plaamodium /aZetparumMALARIA PATIENTS BEFORE AND AFTERTHE TREATMENT, ACCORDING TO THE PRESENCE OF GAMFTOCYTES BEFORE THE TREATMENT.
GROUPSN» OF
INDIVIDUALS
T LYMPHOCYTES
AVERAGE/nm3
BEFORE AFTER
STATISTICAL SIGNIFICANCE
ANALYSIS LEVELCOMMENTS
B LYMPHOCITES
AVERAGE/mm3
BEFORE AFTER
STATISTICAL SIGNIFICANCE
ANALYSIS LEVEL COMMENTS
10
17
490 786 t = 2.41* 0.02 <p< 0.05 Before < After
357 812 t • 5.34*. p < 0.01 Before<After
299 306 t = 0.52 p 0.50 Before-After
203 325 t - 2.27* 0.02<p<0.05 Before < After
G2 * Pat ients with gametocytes before the treatment
G, a Patients without gametocytes before treatment
FIG. 1 - LEUKOCYTE, TOTAL LYMPHOCYTE AND T AND B LYMPHOCYTE FOLLOW-UP IN NORMALINDIVIDUALS AND IN Plaamodium falciparum MALARIA PATIENTS, BEFORE ANDAFTER TREATMENT, ACCORDING TO THE PRESENCE OF GAMETOCYTES BEFORE THETREATMENT.
5900/
4700
3500 .
LEUCOCYTES
2500
900
TOTALS LYMPHOCYTES
1000 <
600
200
LYMPHOCYTES
360 '.
2 8°200
360'.
-*-"/ 280.
200.
BEROFE AFTER BEFORE AFTER BEFORE AFTER BEFORE AFTER
NORMAL INDIVIDUALS
PATIENTS WITH GAMETOCYTES
PATIENTS WITHOUT GAMETOCYTES
REFERENCES
1. HORS, J.; PREUDM1OMME. J.L.; TOULZE-ZAPATERIA. M.T.; GUILLET-BIGOT, J.; ROY. J.P. 5 DAUSSET, J. - A simplified methodfor freezing lymphocytes in nitrogen vapors. Transplant. ,15: 417 - 419, 1973.
2. MEIRA, D.A.; MARCONDES, J.; BARRAVIERA, B.; PEREIRA, P.CM. ;RUI, P. 6 CURI, P.R. - Malaria at Humaita County, AmazonasState, Brazil. XVI - Gametocytes and lymphocytes studied inpatients with Plaamodium falciparum. Rev. Inst. Med. trop.São Paulo, 2±(Supl. 6) , (6) : 32-39, 1982.
3. MEIRA, D.A.; MARCONDES, J.; CURI,P.R.; GUIMARÃES, F.R. G DISANTI, S.M. - Malária no município de Humaitã, Estado doAmazonas. XXXIII - Comportamento dos gametócitos e linfóci-tos em doentes com Plaemodiun faloiparum. Apresentado noXX Congresso da Sociedade Brasileira de Medicina Tropical eI Congresso da Sociedade Latino-Americana de Medicina Tropi^cal. Salvador. Bahia, fevereiro de 1984.
4. SNEDCOR, G.W. § COCHRAN, W.G. - Statistical methods. Ames.Iowa. The Iowa State University, 7a. ed., 1980, 505 p.
.161. .
MALARIA IN HUMAITA COUNTY, AMAZONAS STATE. BRAZIL. XXXII - F R E -QUENCE OF THE HLA ANTIGEN IN THE GENERAL POPULATION AND PATIENTS.
D.A. MEIRA; J: PELLEGRINO-JUNIOR AND J. MARCONDESFaculdade de Medicina de Botucatu - UNESP.
K. TSUJITokai University
E.S. MATSUOKA; E.E. HAIDA AND A.B. EL-KHOURY
Hospital do Servidor Público Estadual
INTRODUCTION
The importance of natural factors of resistanceto malaria in individuals from the general population in HumaitaCounty, Amazonas State was shown by the authors in early study^.In the same way the importance of gametocytes.in patients withPlasmodrum falciparum malaria as well as the relations of thosegametocytes with prognosis and immune response were suggested bythe authors in precedent papers^ ' ' . All of these studies showthat eventual relations between the normal factors of resistanceand predisposition or resistance of inhabitants of endemic regionto malaria should be investigated.
The purpose of this study was to investigate theeventual associations of HLA - A, B, C and DR antigens in inhatútants of Humaita County and in patients as well as the eventualconnections of these antigens with predisposition or resistanceto malaria.
MATERIAL AND METHODS
In August 1983 inhabitants of different localitiesof the country and urban zone in Humaita County as well as 39patients with Plaemodium falaiparum malaria were studied. Boththe individuals and the patients were submitted to clinical observation
Research accomplished with support of National Council of Scien-
tific and Technological Development - CNPq (Proc. 40,3705/82).
.162.
including previous history of malaria attacks, avaliation of
spleen enlargement, parasitoloi;)' of the blood and passive heinag
glutination for detection of malaria antibodies.
Three groups were constituted :
I - CONTROL - 2S individuals born in Amazon
Region with no previous history of malaria
attacks, without spleen enlargement and
blood exams for parasites and antibodies ne
gatives.
II - AMAZONIAN PATIENTS - 38 individuals born in
Amazon Region with history of previous mala
ria attacks and/or spleen enlargement, posi
tive blood exams for parasites and positive
passive hemagglutination.
Ill - NON-AMAZONIAN PATIENTS - 22 individuals all
of them immigrants from other parts of the
Country, with the same characteristics seen
in group II patients.
Blood was collected from all individuals of the
thTce groups for lymphocyte obtention. The lymphocytes were then
frozen in liquid nitrogen for posterior determination of HLA -
A, B, C and DR antigens.
The results were analysed by x test (ot = 0.05).
RESULTS
Results are expressed in the Tables I, II, III ,
IV and V.
DISCUSSION
First of all, it should be stressed the high fre-
quency of blanks among normal individuals born in Amazon Region.
JOBIN 6 Cols/ \ in typing HLA - antigens of Tucuna indians, ex
plained it as being due to the high incidence of hemozygous cells
for certain antigens or to the existence of still unknown antigens.
.163.
T A I I. E I
NUMBER OF SELECTED CYTOTOXIC SERA.USED TO TYPE HUNAITA INHABITANTS AND MALARIA PATIENTS AND TlllilR
HLA SPELIF ITUS
HLA N»
LOCUS A
I I A - A ,
MA - Aj
IIA - Aj
M A - A ,
* * * *»(w24)
* * - *io«A-A, ,MA-A,,
«* - \S ( H 29)
" * * *«(«»)
IIA - Aj j - j j j
IIA - * , . , , , , . ,
ItA - Al9,wJJ)
MA-A,,
-OF SERA
USED
J
4
I
2
1
1
4
3
J
4
4
4
I
1
J
HLA
LOCUS »
I IA - Hs
MA-1WIIA - «^j.
MA-9,
MA-i,
" * - • «
MA - » l 3
H L A - B , ,
MA - t , 5
' " " *MIIA - l>|7
IIA - bJt
' " * " *» • ;
HA - »M,2
* * " »22|wS.
'«A - B ^ " *
H1A - H^JJ
« A - « 4 0
« A - h, | ) (w(i,
N« OF SERA
USED
4
3
.•
7
4
5
S
3
3
1
1
3
3
S
ill 2
11
HI A
1.OCOS CM
I IA - «;,
IIA-Ctó
" A - l«A
ItA - l^,,
MA-C^
IIA-C^
« A - Cv7
* * " Cw«
N> OF SERA
USED
3
1
5
2
-
1
1
1
II LA
I.OCUS OR
I IU - UK,
I I U - URj
IDA - I H ,
HI .A - tV)
I H - DRS
IkA - !«„
ILA - DR7
HLA - DRj
H U - DH,
N» OF SERA
usi-.n
*
0
I
4
4
6
: A B i. L
PHENOTYPE AND CENE FREQUENCY I \ HLA ANTlfiiNS OF .'5 NORMAL AMAZONIAN INDIVUDUALS
u . i u m r l i
UUft AANT
l«.\ -
HLA -
HLA -
HLA -
HLA -
* A -
I I A -
U A -
HLA -
II v -
MA -
It» -
1 1 A -
ULANk
\
*3
*9(«23)
N(nM)
*10
* U
Al!)
*I»(«J1
A_,s
N» IK
CAStS
2
12
s0
«
3
S
4
. 3
0
a
1
2
s
I1KHÍ1.
«tQ.
s.o
4).O
20.0
-
32.0
12.0
20.0
16.0
12.0
-
-
4.0
D.O
-
UJ«.
FMQ.
O.lMO
0.27a
O.IK
-
0.17S
0.0»!
II. IDS
COM
0.061
-
-
0.020
0.041)
0.C10
UJLTJb h
ANT1CZN
..JHLA
HLA
HLA
HLA
HLA
IDA
IIIA
I1A
II \
I L \
HU
IIA
HU
HU
IRA
"-V"" •«SI
- » - 5 ,
- » 7
••l2- « „- » 1 4* * 1 S
• "l..- »,7
- » u
" " 2 !
" B22cw56
- B , .
S' HI
CASLS
t l - ' M
1
4
1
f
1
0
u
mi MII .
32.0
4.0
16.0
4.0
24,0 .
•).()
H.I)
24.Ü
H . l l
V2.0
-
(INI.
Ü.175
0.020
0.OS3
0.020
O.US
n. ii2(i
U.IUII
O.l.'S
n.fti»
O.UCl
-
HIA
!0A
HA
IDA
ISA
II.A
" ' «1
" C w4
" ^
" C w 7
- i:w8
N» 01
CASIS
(I-.'S
3
3
IS
1
3
II
4 . 0
4 .
;n.
4.
12.
0
n
u
I)
0
0
ft
u
II
.020
.020
.105
. (Mi l
IMl.MVI
20.11 U.IOS
1 2 . 0 U.iXil
12.0 O.Ool
60.0 0.307
4.0 0.020
12.Ü «.Obi
UUK1IKMUXH-.
IW1).
H i
IBA -
HLA -
HU -
HU -
VIA -
IDA •
IIA -
IIU -
M.A'iK
i 1
I..,
PN,
l*t}
W 4
0Rs
"V.mi.
I«V,,
I I I
:.
2
1
4
1
1
1
1
2
1
1
IS .4
18.1
9.0
36.3
9.0
9.0
Ü.U
D.O
U . I
_
I I
U
0
0
0
0
0
u
a
. ;iii
.an
.04b
.202
.046
,04b
.(Mii
.(M(i
. « «
PKtNOIYPt AND ULNb PRMJ1II.NCV IN III.A - ANTICUNS OI 38 AMAZONIAN MALARIA PAT1KNTS
(CROUP I I )
LOCUS A
IIA -
«A -
11A -
HtA -
U A -
HLA-
HLA -
H U -
HLA -
ILA -
H \ -
• A -
BLAfft
Aj
A,
AJ
N-24)A,o
A j ,
A19f«29)
A19(w31)
A,8
N» OF
CASES
(1-38J
3
13
l»
}
17»
S
1
a
4
3
2
3
6
PHEM3T.
IRKJ.
1
?.;.
3 4 . :
i s .a
7 . 9
44.7
13.1
2.6
21.0
10.5
7.y
5.3
7.9
-
IKIJQ.
0.U40
0.1S»
U.US-!
O.Ú40
0.2S(J
O.ObB
0.013
0.114
0.0S4
u.inu
0.020
rt.o.'n
0.040
0.082
LOCUS B
IILA -
H A -
IUA -
HLA -
111 A
HLA -
H L A -
H1A -
HU -
IIIA -
IILA -
lll.\ -
IUA '-
IILA -
nLA -
HA -
HU -
HA -
WAV.
"5
"*M
" * s :
»7
».
B12
B u
B14
B.S
hiB : : i k54 i
"27
B M35
B 4 0 , * 0 )
B4O|«<,1)
III
11
1
4
I
\
4
3
4
1.
I.
1
i
1
1
0
u
1
i
1
IttfCT.tWi).
-
.\V7Ul.S
10.S
i.t,
7.8
10.5
7.8
10.5
11.7
IS.7
10.5
.'.6
-
1U.S
10.5
GfcNK
IK11).
-
0.121,
(J.0S4
0.054
0.013
0.040
0.OS4
0.040
0.054
0.082
n.nii
O.U54
0.013
0.UI3
-
l>.205
0.054
0.054
0.1154
LOCUS Cw
W l IIJ N
IILA - l^j
HJ\ - l ^ ,
IUA - l.M ,
I < W " Lw4
IIIA - l ^
HU - ^
I I U - C g
N« orCASI:S
(i-38)
0
0
3
5
Ü
6
0
PHEMUI
IlilQ.
t
-
-
13.1
-
15.7
-
1 Kl i l -
,. 1MII
U.LKtS
-
U.Ü8J
AMI'111
IILA -
IDA -
IIIA -
HLA -
IIIA -
IIU -
HU -
HU -
HLA -
UlwXNk
m
UK,
!«,.
l«(,
1*4
s
DR7
wu
li-10)
1
2
1
8 "
1
2
1 °3
1
IK
10
20
1U
80
10
10
20
30
1
.0
.0
. 0
.0
.0
.0
.0
.0
awinn}.
O.DSl
11.105
«Mil
0.552
0.1151
0.051
o.io:
-
0.16S
ll.UM
* mg/100 ml of culture filtrate.
i 3i
O & f. 43 ^ 3
t*\ C. •» S » irt
e S o = —
u » _J _ J _^
a = s
r i O ^
o o 33 O d
irt -O O
. 3 —• O
o oo o
1131 A IS
fM M
S 2 5 : 5 2 2 5 S 2 í 2 2 5
r>j -^ r ^ 4 ^ ^ r v * r 4
i c o o3 íí3 d
• a 2r. o rj o oT T. r- O% Oi
° % R
5: 3 fc. PT 5 â S 5? R** '' i i i i
<" -f ^ <- <- -r -r <- J* <-
.167.
T A B L E V
COMPARISON BETWEEN THE STUDIED GROUPS (I, II AND III) ACCORDING TO THE PHENOTYPE AND GENE
FREQUENCY IN HLA - ANTIGENS
COMPARISON
BETWEEN
GROUPS
I x II
I x III
II x III
LOCUS A, B, C
HIGH FREQUENCY
ANTIGEN
-
-
H L A " A9(w24)
STATISTICAL
ANALYSIS
N.S.
N.S.
X2 = 4.138*
0
0
0
P
.05
.05
.05
COMMENTS
I =
I =
II
II
> III
> III
LOCUS DR
HIGH FREQUENCY
ANTIGEN
HLA - DR4
-
HLA - DR4
STATISTICAL
ANALYSIS
4.072*
N.S.
8.824*
0
0
0
.05
.05
.005
COMMENTS
I •
I >
II
s II
• III
> III
I - Normal amazonian individuals (25)
II - Amazonian patients with malaria (38)
III - Non-amazonian patients with malaria (22)
N.S.- Not significant
Otherwise, COLAUTO & Cols. , showed a high frequence of 0 - Rh
positive blood group in Amazon Region inhabitants and only this
blood group in indians of this region. These observations may
suggest that the high incidence of homozygous cell could be
the responsible factor of the frequence of blanks found in this
study.
The results obtained in this research showed
differences between the behaviour of group I, II and III. When lo
cus A, B, and C were considered, they revealed a high frequence ,
of AQfW24i antigen *n Group II when compared with Group III. There
was no differences in genie distribution when Group II was compa-
red with Group I. Locus DR showed a high frequence of DR. anti -
gen in Group II when compared with Groups I and II.
Although the size of the studied population was
small, these observations seem to suggest a relashionship between
malaria, origin of the patient and HLA - antigens. These results
also show the need of further studies on this theme.
.169.
I -1
REFERENCES
1. COLAUTO, E.M.R.; MEIRA, D.A.; MENDES, R.P.; SILVA, E.A.; BARBOZA, A.F.; COLAUTO, R. 5 GOMES, M.C.G. - Malária no muni-cípio de Humaitá, Estado do Amazonas. IX - Freqüência desistemas de grupamentos sangüíneos em habitantes da regiãoe em doentes. Rev. Inst. Med. trop. São Paulo, ^3 (Supl.-5) (5): 54-60, 1981.
2. COLAUTO, E.M.R.- BARRAVIERA, B.; MEIRA, D.A.; MATSUBARA, L.S.PALLEGRINO-JÚNIOR, J.; MACHADO, P.E.A.; SOGAYAR, R.; BARBOZA, A.F.; SILVA, E.A.; COLAUTO, R.; PIROLA, J.A.G. 5 MEN -DES, R.P. - Malária no município de Humaitã, Estado do Amazonas. XXI - Freqüência de fatores de resistência eritroci^tária na população geral e em doentes: Hemoglobina S e Si£tema Duffy. Rev. Inst. Med. trop. São Paulo, 5 (Supl. 5):72 - 78, 1981.
4. DANILOVS, J.; TERASAKI, P.I.; PARK, M.S. 5 AYOUB, G. - B lymphocite isolation by thrombinnylon wool. 8 Internatio -nal Histocompatibility Workoshop and Conference. Newalletter,6, December, 1978.
5. HORS, J.; PREUD'HOMME, J.L.; TOULZE-ZAPATERIA, M.T.; GUILLET-BIGOT, J.; ROY. J.P.; DAUSSET, J. - A simplified methodthe freezing lymphocytes in nitrogen vapors. Transplant.,xS: 417 - 419, 1973.
6. MEIRA, D.A.; MARCONDES, J. ; BARRAVIERA, B.; PEREIRA, P.CM. ;
RUI, P. 5 CURI, P.R. - Malaria at Humaita County, AmazonasState, Brazil. Gametocytes and lymphocytes studied in pa-tients with Plaemo£ium falciparum. Rev. Inst. Med. trop.São Paulo, 24(Supl. 6) (6): 32 - 39, 1982.
7. MEIRA, D.A.; MARCONDES, J.; CURI, P.R.; GUIMARÃES, F.R.:§ DI
SANTI, S.M. - Malaria no município de Humaitá, Estado doAmazonas. XXXIII - Comportamento dos gametócitos em doentes
.170.
com Plasmodium falciparum. Apresentado no XX Congresso daSociedade Brasileira de Medicina Tropical e I Congresso daSociedade Latino-Americana de Medicina Tropical. SalvadorBahia, fevereiro de 1984.
8. MEIRA, D.A.; MATSUOKA, E.S.; FAVRIM, M.A.; CURI, P.R.; MAR -CONDES, J.; MOTTA, N.G.S. Ç EL KHOURY, A.B. - Malária nomunicípio de Humaitá, Estado do Amazonas. XXXIV - Comportamento da imunidade em doentes com Flasmodium falaiparum emrelação aos gametócitos. Apresentado no XX Congresso daSociedade Brasileira de Medicina Tropical e I Congresso daSociedade Latino-Americana de Medicina Tropical, Salvador-Bahia, fevereiro de 1984.
9. RAY. J.Q.; HARE, D.B.; PEDERSEU, P.D. & MULLALLY. D.I. - Ma-nual of tissue typing techniques dhew publication n9 (NIH),74 - 545, Bethesda, 1976, National Institutes of Health.
.171.
; MALARIA IN HUMAITA COUNTY, AMAZONAS STATE, BRAZIL. XXXVIII - DE-
TERMINATION OF HLA ANTIGENS IN THE FAMILY OF PATIENTS PRONE TO
\ CEREBRAL MALARIA.k.
I D.A. MEIRA; J.PELLEGRINO-JÜNIOR AND J. MARCONDES
I Faculdade de Medicina de Botucatu - UNESP
' K. TSUJI
j; Tokai University
E.S. MATSUOKA; A.B. EL-KHOURY AND E. HAIDA
Hospital do Servidor Público Estadual
INTRODUCTION
The authors ' observed severe malária in two fa-
milies with cases of cerebral form of the disease.
Many mechanisms are involved in the pathogenesis
of cerebral Plasmodium faleiparum malaria and one of them is
immunecomplex deposition in the capillary vessel sistem' '
Familiar occurrence in more than one member of the same family
and the probability of a mechanism with immuropathogenic basis
in this form of the disease denote that eventual associations of
HLA antigens in the families presenting cerebral malaria must be
studied.
This was the purpose of this study.iI
\ MATERIAL AND METHODSi
IIn August 1983 a family from Humaita County con -
sisting of mother and five children was studied. The principal
characteristics of the family are shown in Table I. All its
members had Plasmodium falciparum malaria seeing that two of
' them had severe forms of the disease and the remaining were mild
cases. One of the two children with severe disease developped
cerebral malaria. Blood was collected from all of the six
Research accomplished with support of National Council of Scien-
tific and Technological Development - CNPq (Proc. 40.3705/82
.172.
individuals for lymphocyte obtention which were then frozen in
itrogi(4,5)
liquid nitrogen1 ' for !ater determination of HLA - A, B and DR
antigens
RESULTS
The results are expressed in the Table I and Figu
re 1.
DISCUSSION
The results showed that the two children with se-vere malaria shared HLA - A*Q antigen which probably was inheri -ted from their father. This antigen was not observed in themother nor in her other three children. It must be stressed thatthese children had identical HLA antigens.
These results are not sufficient to propose mutualdependence of severe malaria and HLA - antigens; however theysuggest the need of further familiar observations under thesesame conditions.
.173.
T A B L E I_
CARACTERIZATION OF THE MEMBERS OF A SAME FAMILY ACCORDING TO MALARIA CLINICAL
FORM, ENLARGED SPLEEN DEGREE AND HLA ANTIGENS,
INDIVIDUAL RELATIONSHIP AGE MALARIA ENLARGED SPLEEN HLA
N» DEGREE (YEARS) CLINICAL FORM CLASS (LOCUS A AND B)
Mother 36 Benign 0 A2.w29 Bw51.18
Daughter 16 Benign ,11 Bw51.18
Daughter 11 Benign V2.11 -vM.18
Son 10 Cerebral A2.m V51... •
Son Severe A2.10 Bw51.w21
Son Benign A2.11 Bw51 .18
FIG. 1 - MALARIA IN AN AMAZONIAN FAMILY ACCORDING TO HLA - A, B ANTIGENS:
raQ&l Unknown form
Cerebral form
V .\ Severe form
O Benignform
A2. Bw51
\l. B18
A2. Bw51
B18
uw51
A 2 . B18
A2. Bw51
A10. Bw2]
Not done
O Male and
female
O Age
36
A 2 . Bw51
.18
REFERENCES
1. DANILOVS, J.; TERASAKI, P.I.; PARK, M.S. f, AYOUB, G. - B lym-
phocyte isolation by thrombim-nylon woll. 8 Internatio -
nal Histocompatibility Workshop and Conference New Sletter,
6, December, 1978.
2. HORS.J.; PREUD'HOMME, J.L.; TOUZA-ZAPATERIA, M.T.; GUILLET -
BIGOT, J.; ROY, J.P. (, DAUSSET, J. - A simplified method for
freezing lymphocytes in nitrogen vapors. Transplant. , 1_5 :
417 - 419, 1975.
3. MEIRA, D.A.; MARCONDES, J.; BARRAVIERA, B.; PEREIRA, P.CM. ;
RUI, P. & CUR1 , P.R. - Malaria at Ilumaita County. Amazonas
State, Brazil. XVI - Gametocytes and lymphocytes studied
in patients with Plasmodium falciparum. Rev. Inst. Med.trop.
São Paulo, _2_4 (Supl. 6) (6): 32 - 39, 1982.
4. PAYFAIR, J.H.L. - Immunity to malaria. Br. Med. Bull . , 38^(2):
153 - 159, 1982.
5. RAY, J.G.; HARE, D.B.; PEDERSEU, P.D. $ MULLALLY. D.I. -MANUAL
of tissue typing techniques. Dhew Publication no (NIH),74-
545, Bethesda, 1976, National Institutes of Health.
.176
LIMNOLOGY
Coordinator
Dr. JOSÊ GALIZIA TUNDISI
Federal University of S. Carlos
São Carlos, SP
JAPAN-BRAZIL COOPERATIVE STUDY ON BIOLOGICAL PRODUCTIVITY OF RIO DOCE
VALLEY LAKE SYSTEM
Y. SAIJO
Water Research Institute, Nagoya University
J. G. TUNDISI
Departamento de Ciências BiológicasUniversidade Federal de Sio Carlos
ABSTRACT
Japan-Brazil cooperative limnological study was realized from June
to July 1983. The sain research lake system of this project was Rio Doce
Valley lakes, Minas•Gerais. The Pantanal lake system, Mato Grosso was
also included for the comparative study. The main subject of project is
the biological productivity of lake systems including the study of species
diversity, the characteristics of biological population, the food chain
mechanism, the productivity of each trophic level, the matter cycle, etc.
These are summarized under the following 4 headings.
1. Clarification of the mechanism of the structure and functionof the biological population and the matter cycle which main-tains the high productivity of the tropical lake systems.
2. Evaluation of the significance of the biological productivityand the natural resources of lakes in Rio Coce Valley and ofthe largest wet and submerged areas in the world.
3. Knowledge of the succession process of tht tropical lakesthrough study of the relationship between the depth andthe ecological characteristics.
4. Elucidation of the impact of human activity on the lake systemthrough comparison of lakes in natural and artificial forests.
.177.
In Rio Doce Valley lakes, the research was carried out fro» 23 June
to 20 July 1983 according to the following steps.
1) Comparative detail study on 4 lakes of L. D. Helvécio,L. Carioca, L. Jacaré and L. Belgo Mineira.
2) An intensive study in L. Carioca.
In Pantanal lakes, Mato Grosso, a preliminary survey was undertaken
from 25 to July 1983.
This paper report the outline of this project and some of the
results chained.
If
\ .178.
JAPAN-BRAZIL COOPERATIVE STUDY ON BIOLOGICAL PRODUCTIVITY OF RIO DOCE
VALLEY LAKE SYSTEM
Y. SAIJO
Mater Research Institute, Nagoya UniversityChikusa-ku, Nagoya 464, Japan
J. G. TUNDISI
Departamento de Ciências BiológicasUniversidade Federal de São Carlos
13560, São Carlos, SP, Brazil
INTRODUCTION
When one considers the biological production of freshwater bodies in
the world, the contribution of tropical lakes is very great.
In temperate lakes the seasonal changes of water temperature are
remarkable, and there is also a vast difference in water temperature
between the surface and bottom of the lake when the lake is stratified
from early summer to early autumn. Influenced by the physical condition,
generally the biological productivity in a temperate lake is high in
shallow lakes and low in deep lakes. On the other hand, in tropical lakes,
the seasonal changes of water temperature are minor and the range of
diurnal change often exceeds that of seasonal change.
Supported by the high water temperature throughout the water column
and enough light, in tropical lakes, the photosynthesis and other biolog-
ical activities are far more active than in temperate lakes. Consequently,
the biological productivity is very high even in deep lakes. However, our
knowledge of the ecological and biochemical mechanisms whi ;h maintain the
high productivity of tropical lakes is still very limited.
.179.
HISTORICAL BACKGROUND
The Aatazon is the largest Mater system in the world. Extensive limnolog-
ical studies were initiated by a German group after World Mar, and the close
relationship between the river and lakes has been clarified fro» the aspect of
biological productivity [1, 2]. Now, active research is being continued by
lianologists of Brazil and other countries mostly connected with INPA (Insti-
tuto National de Pesquisas de Amazonia/CNPq). Compared with the Amazon regior
the limnological knowledge of the lake systems in central Brazil had been very
limited. About ten years ago, J. G. Tundisi and his group started their
limnological study from the reservoii at the suburb of São Carlos City, S. P.
[3, 4]. Then their study was extended to the Rio Doce Valley lake systea in
Minas Gerais [5, 6, 7, 8]. On the other hand, a big scale cooperative study
on the biological productivity in reservoirs was carried out in Sid Paulo State
[9, 10]. Pantanal is another huge water system in Brazil, but there has been
almost no lianological research in this region except for one report on some
lakes near Cuiabá [11].
As a basis for close cooperative study between Japan and Brazil, there
has been a rather long history in the exchange of limnologists. The first
visit to Brazil was made by N. Nakamoto of Shinshu University, in 1974.
He visited to Brazil again the Laboratory of Limnology, Department of Biolog-
ical Sciences, Sao Carlos Federal University (UFSCar) in 1976 relating to the
study of phytoplankton [12]. In 1977, T. Sunaga of Kagawa University visited
UFSCar to study fish.
In 1978, J. G, Tundisi and T. Matsumura-Tundisi of UFSCar visited Japan
thanks to an exchange system between the Japan Society for the Promotion of
Science (JSPS) and the Brazilian Academy of Science*(BAS). They visited
several universities and institute and some lakes in Japan. During this visit
the possibility of a cooperative research was discussed for the first time.
In 1979, Y. Saijo of Nagoya University visited UFSCar and other universities
.180.
on the JSPS/BAS exchange and encountered the aajor lake systeas in Brazil.
The first draft of a project Mas coapleted in October 1979 during the visit of
Tundisi to Japan to participate in the first Brazil/Japan Syaposiia» on Science
and Technology. In 1980, K. Hino of UFSCar visited Japan to study phytoplank-
ton in lakes in Japan by JSPS/BAS, while J. G. Tundisi and T. Matsumtira-Tundisi
visited Japan to participate in the International Lianological Congress in
Kyoto. In 1981, Ikushiaa of Chiba University visited UFSCar to study the
aacrophyte [13]. In 1982, H. Fukuhara of Niigata University visited UFSCar to
study the benthos in lakes. Both on JSPS/BAS exchange systea. J. G. Tundisi,
K. Hino and R. Henry (UNESP) visited Japan to participate in the third Japan-
Brazil Syaposiua, and the present cooperative project was discussed in detail
with all Japanese participants.
STUDIES ON RIO DOCE VALLEY LAKES
The sain research lake system of this project is the Rio Doce Valley
Lakes, Ninas Gerais (Fig. 1). A series of extensive lianological study was
carried out since 1976 in these lakes by the group of J. G. Tundisi aainly
relating to the priaary production, phytoplankton and zooplankton. The aia
of this project is to extend their research to all trophic levels, and also
to clarify the biogeocheaical cycle of aatters. These are suaaarized under
the following 4 headings.
1) Clarification of the aechanism of the structure and function of
the biological population and the aatter cycle which aaintains
the high productivity of the tTopical lake systeas.
2) Evaluation of the significance of the biological productivity
and the natural resources of lakes.
3) Knowledge of the succession process of the tropical lakes through
the study of the relationship between the depth and the ecological
characteristics.
.181.
4) Elucidation of the impact of human activity on the lake systems
through comparison of lakes in natural and artificial forests.
This project is also planned in relation to the Japanese MAB (Man and
Biosphere) project organized by UNESCO.
ORGANIZATION AND CONTENT OF THE RESEARCH
In 1983, the research was carried out from 23 June - 20 July to clarify
the feature of dry seasons. The field work was done by dividing the scientists
into the following four groups, i.e., 1) physicochenical environment and
plankton, 2) macrophyte, 3) benthic animals and 4) fish. The list of
participants and the contents of field research were shown in Table 1.
Description of the studied area
The lakes in Rio Doce Valley distribute in and around the "Parque Florestal
de Rio Doce" (19*S0(S lat and 42*35* and 42*40'* long). In the basins of
Pleistocene, there are about 150 lakes formed by damming of the drainage rivers
of the River Doce watershed. The depth of the lakes ranged from about 30 m to
the shallow swamps.
In the park, the forest and lakes are protected very well, though a part
of the forest was burned by a forest fire about 10 years ago. Here, we chose
two lakes for our study, one was a Lake Don Helvécio,another Lake Carioca.
Their general morphometric properties (Fig. 2) are as follows.
Uke
Lake
Don Helvécio
Carioca
6
0
area
km2
.87 K"
.135
max
32
11
depth
.5"
.8
mean depth
12-1 *
5.28
i
| On the other hand, outside the park, from 1954 the original tropical
forest was replaced by the artificial forest of eucalyptus as the material for
\ charcoal which is used in the iron industry instead of coal. Me chose two l?Ves,
\
Table 1. Organization of Japan-Brazil Cooperative Study in Rio Doce
Valley Lakes (June 23 - July 20, 1983)
Coordinator
Meteorological observations
Physics, chemistry ( primary production
1. Temperature, light, conductivity 6 pH
2. Dissolved gases (02, CO2)
3. Major inorganic elements % trace metals
4. Nutrients
5. Chlorophyll f organic matters
6. Photosynthesis t respiration
Nutrient limitation
1. MBOD bioassay
2. Enrichment bioassay.
Phytoplankton
Zooplankton1. Distribution in four lakes2. Vertical migration in L. Carioca
Aquatic macrophytes
1. Ecology of at,uatic macrophyte*
2. Decomposition of aquatic macrophytes
3. Oxygen budget at a littoral region
Benthos
1. Community structure
2. Migration ecology of Chaoborus
3. Feeding ecology of Chaoborus
4. Taxonoaical studies on benthos
Y. Saijo(Nagoya)
N. Nakamoto(Shinshu)
Y. Saijo0. Mit.amura
(Osaka)
N. Nakamoto
N. Nakamoto
I. Ikushima(Chiba)
H. Fukuhara(Niigata)
Brazi1
J. C. Tundisi(S3o Carlos)
J. G. Tundisi
J. G. TundisiK. Hino(SSo Carlos)
R. Henry(Botucatu)
K. Hino
T. Matsumura-Tundisi(SSo Carlos)
J. G. Gentil(São Carlos)
G. Torres(Belo HoTizonte)
S, Maria Clare(São Carlos)
.183.
Fish
1. Species composition of fish
2. Diurnal rythm of fish movement
3. Diurnal change in feeding activitiesof juvenile fishes
4. Feeding experiments of juvenilesAstyanax spp.
Sediments (Core sample)
1. Eh 6 nutrient
2. Nutrients in interstitial water
T. Sunaga(Kagawa)
0. Nitamura
J. R. Verani(São Carlos)
T. Matsumura-Tundisi
F. A. Barbosa(Belo Horizonte)
Universities
Japan
Y. Saijo: Nagoya University
I. Ikushima: Chiba University
T. Sunaga: Kagawa University
N. Sakamoto: Shinshu University
0. Nitamura: Osaka Kyoiku University
H. Fukuhara: Niigata University
Brazil
Universidade Federal de SãoCarlos (UFSCar)
J. G. Tundisi
T. Matstaura-Tundisi
K. Hino
J. G. Gentil
S. Maria Claro
J. R. Verani
Universidade Federal de MinasGerais (Belo Horizonte)
F. A. Barbosa
G. Torres
Universidade Estadual Juliode Mesquita (Unesp)[Botucatu]
R. Henry
.184.
Lake Jacaré and Lake Belfo Mineira, as examples of lakes which are receiving
the influence oi active human impact. In Lake Jacaré, the eucalyptus was
first planted in 1962. First and second cuts were in 1970 and 1978 respec-
tively. Concerning the latter, the first plant was in 1965, and the first
and second cuts were in 1973 and 1983, respectively.
Morphcaetric properties of Lake Jacaré (Fig. 3) is as follows.
area M X depth aean depth
Lake Jacaré 1.03*" 9.8s 3.65a
Though there is still no bathymetric map of Lake Belgo Mineira, this
lake is very shallow and one third of the water surface is covered with float-
ing leaves of Nyphaeceae. In this region, before cutting the trees, the
forest is treated with pesticide and then burned.
Results of interest
Though the processing and analysis of the saaple and data are still in
progress, soae interesting features obtained in Rio Doce Valley Lakes,
especially coapared with teaperate lakes in Japan, are as follows.
1) Although a general feature of the tropical lake, the concentrations of
dissolved oxygen were generally undersaturated even at the lake surface. The
concentrations of ammonium in the water were very high coapared with Japanese
lakes, and the concentrations of nitrate were generally very low. These facts
seem to suggest a high decoaposition rate of organic aatter and a low nitrifi-
cation activity in these lakes.
2) Results of the studies on the nutrient limitation in lakes by the MBOD
assay technique of Japan as well as the enrichment technique of Brazil showed
the limitation by phosphorus. Nitrogen also seemed to be poor compared with
the eutrophic lakes in Japan.
3) We succeeded in obtaining the core samples of bottom sediments in four
.185.
lakes. The length of the core ranged fro» 60 cm to 90 ca. Compared with the
sediments in lakes in Japan, these samples showed extreaely soft character,
even 50 ca or aore below the bottoa surface.
4) The species diversity of aquatic aacrophytes was low in four lakes
which were closely distributed in the Rio Doce Valley. Fro» the dominant
species and the floristic composition of aquatic aacrophytes, it nay be under-
stood that four lakes does not stand on the serial stage of successional
sequence.
The rate of decomposition of aquatic aacrophytes was variable in relation
to species. In situ percentage loss of dry organic aatter and nitrogen in the
short period was determined. The greatest losses, about 80 \ of the initial
dry weight and nitrogen of floating leaves in Nyaphaea sp., occurred during
the first 8 days. In lakes in Japan, it takes about one month to obtain
a siailar percentage of decomposition.
5) Regarding the benthos, compared with lake* in Japan, a characteristic
feature of four lakes was dominance of Chaoborus froa shallow lakes to the
deepest one.
6) Comparison of fish fauna of each lake, revealed very few common
species among the community of the lakes. Similarity value was very low, even
though these lakes were located in the same river systea of Rio Doce and in
short distance. This fact accords with the distribution of aacrophyte flora
described above.
FTOB the general concept of the lakes in Japan, the standing crop of fish
in studied lakes seeaed to be very high compared with their low standing crop
of phytoplankton. It was also interesting to note so aany species of fishes
which have a carnivorous character. In Japan, carnivorous fishes are rare.
PRELIMINARY STUDY OF PANTANAL
Pantanal is a huge wetland extending over the boundaries of Brazil(Fig. 1),
.186.
Bolivia and Paraguay and has an area of about 300,000 km2, i.e., comparable to
Japan itself. The mean altitude is only 120 m. The yearly mean temperature
is 24- 27 C, and the yearly mean precipitation is 1200- 1400 mm. The change
of dry season (April to September) and rainy season (November to March) are
definite. In the rainy season, though the change of water level is 2 m or so,
most of Pantanal is submerged and appears like an inland sea, and the isolated
hills remain like islands. During July and August, the water is confined to
the large rivers or to the low parts of the plain, and in some regions numer-
ous individual lakes are.formed. Pantanal seems to be comparable to the Amazon
water system in its importance for limnology. However, so far as we know,
there is no published limnological work concerning this region except a paper
by Silva [11].
A preliminary limnological research in Pantanal regions was carried out
from 25 to 30 July 1982, using the research vessel of Mato Grosso State.
Ne went down the Paraguai River from Corumbá and went up Miranda River up to
Passo da Lontra (Fig. 4).
Mater temperature, conductivity, pH, and other chemistry of water,
chlorophyll and plankton, bottom sediment and benthos, macrophyte and fish
were investigated. Our study was mostly limited to rivers, because it was
difficult to enter the lakes which locate along the rivers. The level of
water was still high and the water temperature ranged 19-20 C at most sta-
tions. The river waters were rather tranrparent. It was interesting to note
that the dissolved oxygen contents were undersaturated even in the Paraguai
River at 71 - 81 % (72-73 \, 87-91 \, 49 - 57 * in Taquari R., Aboral R. and
Miranda R., respectively). Another point of interest was the difference in
conductivity of river waters, in spite of their similar physiognomy.
The highest was Miranda River (130 uS.cm2) and the lowest was the Taquari
River (25 uS/cm2) ana about 40 uS/cm2 in other rivers. All of these values
are considerably low from our general Japanese view of conductivity ifl rivers.
.187.
FUTURE PERSPECTIVE
Japan-Brazil cooperative lianological study in Rio Doce Valley Lakes was
first realized fro» June to July 1983 after a Ions preparation period. Though
our research was Halted to the dry season, the knowledge hitherto obtained by
Tundisi and his group on this lakes was extended to the whole ecosystem, and
•any interesting features of the lake system are being clarified. Especially,
the comparison with the temperate lakes in Japan, the distinct differences
between the tropical and teaperate lakes in ecological and biogeocheaical
characteristics were suggested. In our future research, if one consider the
iaportance of active decomposition in Batter cycle in lakes, the studies on
microbiological process would seen indispensable. Now we are planning our
next research in the wet season, and we look forward to intensive studies on
the whole ecosystem for two or three years. The knowledge obtained through
this series of research will contribute not only- to the understanding of
tropical lakes but to the general lianological principle of global lakes.
In Pantanal, only a short preliminary research was carried out during
the past several years, Pantanal seems to be a very coaplicated water system
composed of many types of wetland. To understand the ecological and biogeo-
chemical mechanism of the Pantanal water system as a basis for the protection
of nature and the future development of natural resource in this region,
an organization of Pantanal research comparable to INPA in Amazon might be
required.
Acknowledgements
This research was financially supported by the grant of the Ministry of
Education , Science and Culture (Overseas Research Project; No. 58041035) in
Japan, and by the CNPq (The National Research Council), FAPESP and the Organi-
zation of American States (Project: Limnological, ecological studies on
Brazilian lakes and reservoirs) in Brazil.
188.
1 Ne wish to express our thanks to the Japan Society for the Promotion of
I Science and to the Brazilian Academy of Science for its kind support in
I realizing this project.
í Thanks are also due to the authorities of Ninas Gerais State and South
I Mato Grosso State for their generous permission to undertake research ands' sake use of their facilities.
REFERENCES
1) Schiadt, G. W., Lago do. Castanho, Amazonas, Brazil (1973).
2) Sioli, H., Coupling of lanJ and water systems. A. D. Hasler (Ed.)(1975) 199.
3) Matsumura-Tundisi, T. and Tundisi, J. G., Oecologia (Berl.) 25 (1976) 265.
4) Tundisi, J. G., DsC Thesis. Dep. of Biology of Fac. Fil. Cienc. e Letras
de Ribeirão Preto, USP. (1977) 409pp.
5) Tundisi, J. G., Matsuaura-Tundisi, T., Barbosa, F. A., Gentil, J. G.,
Rugani, C , Forte Pontes, N. C , Aleixo, R, C , Okano, W. Y. üid
Santos, L. C , UFSCar, DCB, São Carlos (1978) 146pp.
6) Barbosa, F. A. and Tundisi, J. G., Arch. Hydrobiol. 90 (1980) 139.
7) Tundisi, J. G., Matsuaura-Tundisi, T., Forte Pontes, N. C. and Gentil, J. G.,
Revta brasil. Bot. £ (1981) 5.
8) Barbosa, F. A., Esteves, E. A. and Tundisi, J. G., Tropical Ecology 23_
; (1982) 155.
i 9) Tundisi, J. G., Verb. Internal. Verein. Lianol. ÍU (1981) 1031.
l 10) Matsimura-Tundisi, T., Hino, K., Claro, S. M., Verh. Internet. Verein.
| Linmol. 21. (1981) 1040.
l 11) Silva, V. P., Programa de Posgraduacao eu Ecologia e Recursos Naturais,
UFSCar. (1980).
12) Nakaaoto, T., Marins, M. A., Tundisi, J. G., Oecologia (Berl.) 23 (1976) 179.
13) Ikushina, I,, Hino, K., Tundisi, J; G., Jap. J. Liimol. 44 (1983) 304.
.189.
,Cuiabá / Brasilia o /
Coruaba
\ MINAS GERAIS
Rio Doce Valley Lakes
Belo Horizonte
BOLIVIA
O Slo Carlos /
O y Rio de JaneiroSão Paulo
ATLANTIC OCEAN
Fig. 1. Location of Rio Doce Valley Lakes and Pantanal Lakes.
.190.
Fig. 2. Bathyaetric aaps of Lake Don Helvécio (upper)and Lake Carioca (lower).
.191.
N
18»
Fig. 3 . Bathyaetric sap of Lake Jacaré.
MIRANDA
Fig. 4. Studied area in Pantanal.
DESERTIFICATION AND DEFORESTATION
Jiro KONDO Dr. Eng.,
Director
The National Institute forEnvironmental Studies
Professor Emeritus
University of Tokyo
ABSTRACT
The enlargement of desert and the reduction of forest in the present
•orld vill change the global environment in the future. There is an evidence
of increasing the concentration of carbon dioxide in the atmosphere «hich «ill
change the clisate and the agriculture on the earth. However, it is possible
to prevent the enlargement of desert and the climate change by increasing
forest. The concentration of carbon dioxide can be reduced by increasing the
photosynthesis of plants. The experience of Japan for saving energy vill be
useful to other countries.
Studies of Infurenaces of Atmospheric Pollution on Plants and Plants
Cossunity
The first-period Phytotron (Phytotron I) in the National Institute for
Environmental Studies(NIES) «as built in Deceamber, 1975 and the second-period
Phytotron(Phytotron II) «as completed in August, 1981 in order to obtain basic
data for planning the environmental policy. These facilities belong to the
big and modern Phytotron in the «orld. The Phytotron I has controlled green
houses and grovth cabinets for the air pollutant exposure and the phytotron II
includes simulators to analyze the plant-environment system. Overall views of
the Phytotron I »nd II shown in Photo 1.
.193.
(a) Pbytotron I. (b) Phyiotron II.
Photo 1 Phytotron (NIES)
lhe toxic effects of S0 a , NO» and 0 3 on plants have been extensively
studied at the Institute by conductinc a special research program since 1976.
The results of the first three years prograa «ere published in the Research
Report fa.11(1981) entitled * Studies on the Effects of Air Pollutants on
Plants and Mechanises of Phytotoxicity ".
In the first Procraa aost studies «ere concerned on the effects of the
air pollutant of single toxic gas. However plants are usually exposed to the
•ixc* air pollutants in the urban area and few results have been reported on
the effects ofaixed air pollutants on plants. For clear understanding of the
effects of theaixed pollutants the second three years research prograa ' Stud-
ies on Effects of Air Pollutant Mixtures on Plants " have been conducted froa
1979 to 19S2.
Nixed gee shoved either additive synergistic or antagonistic effect of
the single gases. The sensitivity of plants to aixed pollutants «as changed
by species and by combination of the pollutants. The aechanisa of phytotoxici
ty «as studied froa physiological biocheeical and aicroaeteorol^gicaJ stand-
points. These rsults are collected in the Research Report No.66' Studies on
Effects of Air Pollutant Mixtures on Plants. ' The detailed description of the
facilities in «hich the experiments are conducted is also included.
The «intensive studies should be continued to reach the coaplete understanding
of I tie acchanise of phytotoxicity.
.194.
The function of plants for the filtering process of purification of the
polluted atsosphere should be emphasized At the National Institute for Envi-
ronmental Studies, therefore,studies on the ameliorating- function of plants,
the effects of air pollution on plants, and image instrumentation systems for
their evaluations have been curried out (1,2).
Plants and the surrouding atmosphere exchange C 0 a and «ater vapor through
the stomata of the leaf, which are needed for photosynthesis, respiration and
transpiration. In atmospheric po'luted areas, toxic air pollutants also enter
the plants through the slomata. Photo.2 is a picture of the stoma of sunflower
plant. The photograph is taken by the electric microscope at NIES.
( Inhingnr l ( n e e r r m l N )
Photo 2 Stosa of Sun Rover
At the Institute, a light Microscope system by which one can observe con-
tinuously the opening and closing of stomata of an intact leaf under growing
conditions has been developed(3),
.195.
then this systea is used for continuous observation of the stoaatal move-
•ent of growing intact plant. Me obtained microphotographs like those in
Photo 3, which show the response of an intact stoaa of the adaxial epidermis
of bro.idbe.in plant to illumination change. The illumination is changed from
30 to 2 klx at 0 ain(a) and fro» 2 to 20 kin at 20 ain(e). The loveient of
the central pore of the stoaa can be continuously observed.
(d) 15 min (e) 20 min (f) 40 min
(i) 200 min
Photo 3 Hicrophotoraphs of an intact stoaa of an adaxial epidermis
of a broad bean plant to illumination change. The time after the
first illumination change is shown under the photographs.
The illumination «a* changed froa 30 to 2a klux at 0 ain (a) anu
froi 2 to 20 lkux at 20 min (e).
.196.
Th* absorption of CO» and air pollutants by plants is affected by light
intensity, wind, air temperature, humidity and gas concentration in the plant
coaaunity. It is also influenced by temperalure and «ater content in the soil
and physiological activity of the plants. At HIES, Plant Community Environment
Simulator has been built to study the relationship between the gas absorption
and the plant's environment. The size of the test section is 2.4 • X 2.4 a,
and the length is 3a as being shown in Fig.l.
1:2;J:4:
5;<v.7;8;9;
10:I I :12:13:14.
Killing chamberKreenhoneycombcontraction conehumidity profile unittemperature profile unitvelocity profile unitlamp home air condilioncrsolar simulatorgrowth roomsoil environment cnntorl unit
doordust filtercorner
IS:16:
17:IX:19:20;21:22;2.1;24:25:2ft;27:
main blowerelectric motordiffusermain air conditionercooling coilhealing coilhumidifiercorner vanefrnh air fillerventilation blowerventilation control valveexhaust air nitergases supply system
Fig. 1 Plant Community Environment Simulator (NIES)
.197.
Fig.l shows a configuration of the simulator. This simulator is a kind
of circuit type «ind tunnel of the loa «ind velocity. The sain air condi-
tioner is installed in the lover tunnel and the profile units for «ind veloci-
ty, air temperature and hunidity are equipped in the upper tunnel. The growth
rooa is also installed in the upper tunnel. On its upper and side surfaces»
the solar simulator, which can automatically regulate light spectrua and
intensity, are wounted- The soil environment control unit is installed on the
lower surface of the room. Viewing the air stream within the equipment» the
air circulated from the growth room comes to the main blower through the cont-
raction cone. The fresh air is mixed with the circulated air in the cone and
with toxic gases in the corner.
The light spectrum and the intensity in the growth room are regulated by
the solar simulator, which consists of various fluorescent lamps (110 V X
224 lamps ) with the SCR for power control. Photo 4 shows a general view of
the inside of the growth room. The control system of fluorenscent lamps can
divide into 6 seres for the upper surface and 3 series for the side in the
growth room. Fluorescent lamps coated with various luminous paints are made
to order.
Photo 4 Growth roos in the Simulator
Finally, the soil environment control unit is described. Six portable
units ( 0.6 V X 0.9 D X 1.2 H nf ) with carriers are provided in the growth
room. This equipment has a brine tank installed around the soil container.
The soil temperature can be regulated by the control of brine temperature.
Also, an automatic sprinkling unit is furnished to adjust the water content in
the soil.
.198.
Fig.2 shows an experiaental evidence on the function of plants for the
purification of polluted ataosphere. Introducing 200 young poplar trees in
the growth rooa of the siaulator we exaained the absorption of 0 3 and N0 s by
the plant coaaunity. Air flow froa section A to section B was set at a verti
cal profile as shown in Fig.2. Air teaperature and huaidity were Maintained
at 25' C and 60 % RH. The vertical distributions of the gas concentration at
each section were coapared. A reaarkable difference of the gas concentration
between A and B under lighting conditions(40klx) was noticed. The decrease of
the gas concentration is resulted froa the gas absorption by the plant coaauni
ty.Experimental layout
Gas sampling point (B)
Gas sampling point (A)•
0.0-O.5 1.0 1.5
Distance (m)
60s concentration (dork) 60s conceniroiion
002 0.04 OOC
60s conctnrronon (ppm)
• » • 0 , 1*1—O—0, III—*—N0 fUI 1
• - ^á 6
\ 1
k 1
\ i
i002 004 00»
60s concentration (ppm)
Fig. 2 Atsospheric Purification
.199.
The Senario, Increase of Carbon Dioxide and Cliaate Chance.
The senario is shown in Fig.3 in the fora of PDPC (Process Decision Pro-
graa Chart).
The energy deaand, caused by the increase of po'jlation in the vorld «ill
increase the consumption of firewood and of charcoal in developing countries
and the combustion of fossil fuel in developed countries. These «ill increase
the concentration of C0 a in the atmosphere. Due to the green house effect
the increase of C0 a causes the rise of global temperature.
The ocean tide, agriculture in the «orld «ill be changed and the sea lev-
el «ill be increased due to the melting of ice at poles be melted.
The increase of firewood consumption means the loss of forest which again
causes the increase of C0 a in air due to the decrease of plant photosynthesis.
The climate change, the rise of temperature «ill increase the vapori-
zation of C0 s from sea. This results also the increase of CO» in the atmos-
phere.
The influence on ecosystem, caused by the accumulation of radioactive
materials in environment is also included in the figure.
tn«r|jr
fir»woodcharcoal
foai i l
nucttur
vaporltation otCO2 fro* ata
lncctmof C02 I chan|t
M 01
- * - •
Man tide
— K agricultura food
1 • aaa lavtl
global carbonbnlmtca
rmltoactlvtiiatf
~H plliliototyntlitalairowch ofplant»
r«.H• • U n i t
tcoiyittm
Fig. 3 Senario, Increase of Carbon Dioxide and Climate Change
.200.
On* can vrita May aea*ri*a. Fit.3 ekoaa a» «xaapl*. b***v*r, tb*
probability of tha final m t t ia mat high eiace tba probability la tka pr*d-
uct of tha tranaiaat probability of aach *v*at.
There ara aciantiata *ho iaeiet that tka earth ia currently geiag- through
an lea Age. Tb* traperatur* «ill certainly iner*aa* due to tb* gr*m bo***
eff*ct aa th* concentration of C0« ia th* •tsoepkere increaeas, bo**v*r,
thia fact do** not nacaaaarily predict that th* t*ap*r*tffr* of tka *arth «ill
be increased in tbe future.
Increase of Photosynthetic Activity of Crop Plant
Fig.4 illustlats the aaount of photoproducta of plant under varioua
concentration circuastancea of CO» .
MOMMNplMlfcCO,
Fig. 4 Photophroducta in tarai of CO» concentration
C« plants like asize, sugar cane and sorghus exhibit a reaarkable photo-
synthetic activity about two tiaes aa larga as that of C» plants like wheat,
rice and soybean.
Tha aechanisa of carbon dioxide fixation in a plant is particular to
species and genua. Aaong the C» crop plants^ tkara baa net yet bean discover
ed varieties exhibiting C« type photosynthesis.
.201.
It aay be possible to introduce C« type photosynthesis system into C 3
plants by interspecies crossinc. According to preliminary experiments of
hybridizaton between C« and C 3 species using Aatriples, soae hybrids rised
whose leaf structure was siailar to that of C« plants.
However, it was also fund that theier physiological function «ere inter-
mediate between C» type and C« type, or more close to C a and there was none
that had the function of typical C« photosynthesis. Therefore, if further
studies are aade on physiological function of CO» fixation system and inher-
itance, there aay be a possibility of plant improvement by changing C* plants
into C . or CAN plants like pineapple and cactus which i<as a strong photosyn-
thesis capability.
The objective of the project is to increase the total energy output
without increasing input of fossil energy by increase in material production
capability of plants. However, the increase of photosynthesis of crop results
not only the increase of primary food production but also the decrease of
CO a since crop is the extensively cultivated in larg area.
.202.
A Study on Forestry Control
A Mathematical model is introduced in order to simulate the change in
biosphere. Fie5 shows the preliainaly study on the process of increasing
trees froa the are land. Nonte Carlo method is introduced by assuaaing a
Narkovian process. Trials to simulate the long rane change in the future.
The results are compared vith the observation. The industrial policy of
controling forest, like the deforestation or the production of tiaber can be
examined by using this model. The utilization of forest in order to aaintaine
the green area can also be studied by the model.
I! II II I] H II II 17 II II• i i
Fig. 5 Formation of forest on land ( Simulation )
The transition probabity is expressed in a form of a transition matrix as
tree
veed
land
ttree
0.9
0.3
0.1
veed
0.1
0.6
0.6
land
0.0
0.1
0.3
«here the probabity to change tree from weed is 0.3.
The matrix should be corrected according the regional condition.
.203.
Forest and Desert
In wood, tall trees shut out the solar light soil contains water, fallen trees
are decomposed by micro organise and turns out to nourishment for plants. Large
amount of CO» is abosrbed by trees and photosynthesis take place inside the leaf.
The production rate of bioaass is large in a tropical rain forest. Nany species of
insects, birds and animals can be observed in a forest.
Vhen trees are removed from forest for timber producrton or for pulp industries,
soil become dry and easily be removed.
Plants can not flow under a severe condition in a hat area «hen humidity is
decreased.
Every living thing can not essist as forest disappears. The ecosystem will be
destroyed.
It vill take a long time for recoverring forest. If the veather is dry, bare 1
and turns out to desert and forest vill never be formed again.
Obviously, it is not an esy task to convert desert to forest. In the area of
desert the climate is very dry, humidity is very low. The water vapor can not
exsist in the atmosphere due to the strong radiation from the white sand on the
surface of the desert. However if the earth be covered by green plants, albedo the
radiation from the earth will be decreased. The 30 S of atmospheric humidity is due
to the vaporization of water from the leaf of plants.
The climate will be changed, the rain will turn to fall on forests if desert
changed to green land. The plant species which can stand for dry weather and salted
soil should be selected.
According the autorotation of the earth, the dry climate expands toward east,
thus the area of desert is enlarging as much as 6 X 10* ha. in every year, on the
earth.This can be observed by an artificial satellite, like Landsat.
.204.
Cnefb) Polity in Japan
l;ig.6 .shows the energy d e u n d in terns of oil V>>!IIBC. Although the total
deaand is inrrp.ising over the ye a r s , the ;>».junt of imported oil is de c l i n i n g .
This is due Io lhe saving of energy and the utilization of new energy s o u r c e s ,
such as nuclear and gcothcrsai e n e r g y .
9.73
n. i:
A.\7
Kl
s n v l n g
import
1977 i a or, I'!!)(
Fig. 6 Energy Deaand - Oil Usage
Fig.7 shows the plan of the usage of various energy sources in the future.
The use of nuclear energy, geotheraal energy and other new energy sources will
increase by the year 2000 while the use of fossil fuel will decrease in Japan.
40Owind power, wave energy,
tolar battery, geothennal energy
19771980 !99O 2000 2010 year
¥i$. 7 Transition to self-sufficiency of energy in Japan
.205
lit.S shows the aaount of crude oil imported to Japan in this decade.
.ia?.noase industries have tried to reduce the consumption of energy and at the
saac tiae, industri.il construction within Japan has been chanced. Heavy indus-
tries have chanced their production systcas and adopted every innovation to
save cnercy. The elasticity of oil to the Gross National Product «as 2.4 in
cjr'y 1970 and fell to zero in recent years. In other words, it «as necessary
Jo increase the consumption of oil by 2.4 t in order to increase the GNP by 1
*, however, in 1983 the GNP «ill be raised without an increase in the oil con
suaption.
PRICE OF OIL ( dollars/Barrel)26.00 34.00
1572 73 74 75 76' 77
10 7 si
78 79 SO SI 32 33
OIL (
rmmmmmm1970 71 11 73 74 75 76 77 78 79 80 81 82 83
Fig. 8 Price of Oil and Aaount of Oil Imported to Japan
.206.
Th« innovations and experiences of Japan can be transferred to other coun-
tries in order to reduce the burning of fossil fuel. The technology of ne«
energy sources should also be transferred to developing countries «here the
burning of firewood and charcoal is increasing. In this *my, the increase of
energy demand «ill not necessary result in an increase of CO» in the ataos-
phere.
The Ipplicate of Shevhart Control Chart
On the otherhand, «e have a clear evidence that the backgroud level of
CO, is rising. Ve should carefully analyze the daIa in order to determine On-
instance when preventive action Bust be taken.
Fig.9 is one example «here the difference of the the concentration of
fO s of the sane nonth of two consective years is plotted on the Shevhart
.ontrol chart, used for statistical quality control in industries. The data
outside of the upper control liait Beans that there is sonc possibility of in-
creasing the concentration extraordinary. The confidence level of this statis
lioal test is 2.5 t.
Global environmental nonitoring as veil as theoretical investigation of
the rcosystes should be strengthened in the future.
Conclusion
The biotechnology which is developed in recent years should be introduced
to intensify the function of botanical photosynthesis.
The iic-rion lo prevent the expansion of dry area on tiie earth should be
taken up immediately. Deforestation caused by the need for firewood or char-
coal should be stopped by providing energy to the people in developing coun-
tries.
The quality of atmosphere should examined continuously, the concentration
of carbon dioxide should be monitored carefully.
The author «old like to his sincere appreciation to Dr. I. Aiga and Dr. K.
Omasa for their help to arrange the materials and to Dr. A, Amemia for pro-
viding the information on the Green Energy Program.
.207.
mi7
6
5
32
I
0
-1
-2i
Maun a
_„„. „_ „ •«..—•• . .
. • •
i—
t -
LoaU k | . . . t . l > . . i . | i
• • .• • • • . • • • •
. • •
*
• " • ' • I ' " 1 '
••„••'.. "•v '"'."v
—-
N- 68
MEW- 1.409
SIGHÍ1- 0.595
3trriti34S
3*0
335
330
335
330
1973 1974 1975 1976 1977 1970 1979 1980
1973 1974 197S 1976 1977 1970 1979 1900
l ' ig. 9 Shcvharl control chart and CO a. concentration til launa Loa
.-208.
References
1. The National Institute for Environmental Studies: Studies on the Effects
of Air Pollutants on Plants and Mechanises of Phytotoxicity, Res. Rep. XIES
No. 11. PP. 1 - 165. NIES, Tsukuba ( 1980 ).
2. Omasa, K., and I. Aiga: Imafe instrumentation for evluating the effects
of envieonmentla pollution on plants. In * Encyclopedia of Systems and
Control * Editor-in-Chief H. Singh. Pergaaon Press, Oxford, in press.
3. Omasa et al.: Observation of stomatal movements of intact plants using an
image instrumentation systea with a light Microscope. Plant t Cell
Physiol. 24:281-288 ( 1983 ) .
4. Oaasa et al.: Iaage instrumentation for avaluation the effects of air
pollutants on plants. In ' Acta IHEKO 1982 Vol. 3 " . Akadeaiai kiado,
Budapest ( 1983 ).
5. Tsuchiya H.: Civilization depending on crop cultivation by energy use,
Toyokeizai Shimpo-sha, 158-195 ( 1980 )
image instrumentation systea with a light microscope.
6. Green Energy Program, Agriculture, Forestry and Fisheries Research
Council Seretriat, Ministry of Agriculture, Forestry and Fisheries,
Tokyo Japan. March 1981.
.209.
SPATIAL DISTRIBUTION OF A BIVALVE POPULATION (Dlplodon delo-
dontus expansus) IN A SMALL TROPICAL RESERVOIR WITH B1PHASIS
ON DISTRIBUTION NEAR THE BASES OF TREES.
R. HENRY and C. A. SIMXO
Departament of ZoologyInstituto Básico de Biologia Médica e AgrícolaUniversidade Estadual Paulista (UNESP)Botucatu, SP, Brazil.
INTRODUCTION
Few studies on the benthic fauna have been con-
ducted in brazilian reservoirs so far, except for some stu-
dies on spatial distribution and abundance of Chironomidae
and Chaoboridae (STRIXINO ft STRIXINO, 1980, 1982) and Oligo
chaeta (CÕ, 1979). Recently, density and biomass of a mol-
lusk population was estimated in a small reservoir (HENRY a
SIMXO, 1984a). The bivalve distribution in function of
depth in thet reservoir and the population distribution pat-
terns were also described (HENRY ft SIP1Ã0, 1984b) .
The abundance and biomass of mollusks in Brazi-
lian lacustrine ecosystems were evaluated in lakes located
in the Amazon (FITTKAU et al, 1975) and Rio de Janeiro (AL-
VARENGA et al, 1979). However, studies on benthic density ,
biomass, growth and productivity have received more empha -
sis in Africa than in Brazil (BUROI& et al, 1973; DARLIN-
GTON, 1977; DEJOUX et al, 1971; LÊVÊQUE, 1971, 1972, 1973 ;
LÊVEQUE et al, 1983; McLACHLAN ft McLACHLAN, 1969, 1971).
In December, 1982, the dam of the Reservoir of
,210.
Rio Pardo cracked and consequently the whole reservoir was
.emptied. An opportunity was supplied to conduct intensive stu
dies on a bivalve population living in this environment (HENRY
ft SIMÃO, 1984 a and b).
During the samplings, a great abundance of mollusks
was observed in the sediment near the bases of trees which re-
mained in the reservoir after the innundation. The results of
these observations are presented in this paper.
.211.
MATERIAL AND METHODS
The aolluska were collected near the edge of the Re-
servoir of the Rio Pardo, Botucatu, State of São Paulo (Pig. 1).
The reservoir is located at 22° 59' S and 48° 25' W and its
morphonetric characteristics are: maximum lenght: 600 m; maxi-
mum width: 525 ra; maxiipum depth: 4.0 m; average depth: 2.5; sur
face: 15,28 ha and volume: 4.06 x 105m3 (HENRY, 1981). The gra-
nulometric composition of the sediment in the sampling area con
sists of silt (41%) and clay (44%).
The bivalves were collected manually at nineteen sta
tions (area: 0.25 m ) and the distance between the stations was
approximately 50 to 70 cm. All sampling stations were situated
in transects connecting the bases of three trees (Fig. 2).
The size structure of the specimen was studied at
each sampling station. The bivalves were measured with a pachy-
meter. Eleven size classes were determined and the specimen we-
re classified according to lenght. Biomass (alcohol weight,
shell dry weight and body dry weight) was obtained through the
following equations according to HENRY & SlrtÃO (1984a):
alcohol weight: -12.13 + 4.14 x lenght
shell dry weight: -8.21 + 2.75 x lenght
body dry weight: -0.38 + 0.13 x lenght
RESULTS
The population density of the bivalves at the 19 st£
tions is presented in Table 1. Greater abundance of bivalves was
found at the stations 1, 7, 12 and 19 near trees A, B, C and A,
respectively. The two stations at the extrenities of each tran-
sect and near the trees presented more than 50* of the whole
.212.
density (Table 1). In the transects C to A, an increase of the
bivalve occurred near station 16. This sampling station is lo-
cated near tree D (Fig. 2).
Fig. 3 shows the size structure of the bivalves in
each of the three transects. Size class 8 (lenght from 5.10 to
5.65 cm) was dominant at almost all stations and contributed
to approximately 50% of the size frequence. However, at the sta_
tions near the trees» the bivalves presented a clearer size va
riation than the specimen collected in the squares located in
the middle of the transects.
Fig. 4 shows the fluctuations in bionass of the bi-
valves at each station. The alcohol weight, shell and body dry
weight at the sampling stations presented the sane variation
pattern as that of density. Higher biomass (alcohol and dry wei
ghts) was recorded at the stations located near the trees.Shell
and body dry weights corresponded approximately to 65 and 3%
of the bivalves alcohol weight, reflectively. The body dry
weight of the mollusks was approximately 4.5% to the shell dry
weight.
DISCUSSION
Previous studies (HENRY, 19S1; HENRY S CURI, 1981 a
and b; 1983; HENRY ft SOUZA, 1984) have shown that the Reservo-
ir of the Rio Pardo is well oxygenated during the year and in
the whole water column and presents low conductivity i< 25 uS.
an ) and retention time (annual mean: 6.3 days). The water
flux from the Rio Pardo and Córrego Água da Madalena is very
fast (0.3 m.s ) ,a11owing a high renewal of water in the reser
volr. The granulometric composition of the sediment is domina*
.213.
ted by silt. Correlations between granulometry and abundance
of D. delodontus expansus showed no significant relationship
(HENRY ft SIMAO, 1984a). The depth caused no limiting effect
on the distribution of D. delodontus expansus in the reservoir
(HENRY ft SIMAO, 1984b).
Apparently, there exists no limiting factor on the
abundance and spatial distribution of the mollusks. In fact,a
high population density and biomass of the bivalves was recox
ded in the reservoir. In December 1982, the population was
estimated at approximately 5.74 x 10 specimen. Alcohol, dry
shell and body weights were, respectively, 55, 35.6 and 1.7
tons. Despite this high density in the reservoir, the popula-
tion can increase much more in other areas such as for exam-
ple near the bases of trees preserved in the reservoir after
the innundation. Comparison of the data obtained in the whole
reservoir (98 sampling stations) with the mean density recor-
ded at 19 stations in the transects (Table 2), showed that
the density and biomass near trees was approximately two or
three times higher than in the whole reservoir.
According to BONETTO (1959), the larvae or glochi-
deas of the species o£ Diplodon complex consists of two types:
parasitic or no-parasitic forms. If the larval development con
sist of a free life form, the glochideas could stick to the
substrata such a» rushes, trees, etc, near the water surface.
When reaching the adult stages, they would get free from the
substrata and go to bottom of the reservoir (MANSUR, pstrsonal
information). The life cycle of D. delodontus expansus in the
reservoir of the Rio Pardo is unknown. Assuming that the lar-
val development is not parasitic, the high densities of bivajl
ves found in the sediment near the bases of trees can be ex-
.214.
plained by larvae adherence to trees near the water surface fol
lowed by release and down fell of the adult species. This expla-
nation may be reasonable since the size structure study on mol
lusks showed a great variation in lenght of the specimen at the
sampling stations near the bases of the trees.
The locomotion power of the bivalves in aquatic
ronments can not be neglected. HEBLINC t PENTEADO (1974) repor-
ted a displacement of 46.6 cra.h~ for Djplodon rotundus gratu»
when the level of the reservoir decreased due to opening of
the floodgates. When the dan of the Reservoir of the Rio Pardo
cracked, the bivalves probably moved with the decrease of the
water level in the reservoir. When bivalves meet an obstacle du
ring their displacement, they are unable to turn around it
(MANSUR, personal information). This can be another explanation
for the high density of roollusks in the sediment near the bases
of trees.
The bivalves are filterers and can contribute to a
decrease of the suspended matter in the water. Although we do
not know the filtration rate of D. delodontus expansus, the ro-
le of raollusks in the Beservoir of the Rio Pardo is certainly
very important. Some papers (LEWANDOWSKI * STANCZKOWSKA, 1975 ;
ELLIS, 1978) reported that Unionidae could filtrate approximate
ly 0.3 1. h"1 to 3.6 1. h"1. If the filtration rate of D. Delo-
dontus expansus were within this variation range, the whole vo-
lume of the reservoir would be filtered by the bivalve populati
on In approximately one to ten days.
Other studies on the biology and ecology of D. delo"
dontus expansus will be necessary to clarify the observations
presented.
.215.
SUMMARY
The spatial distribution of a bivalve population in the
sediment near the bases of trees was studied in a snail tropical
reservoir (Reservoir of Rio Pardo, Botucatu, São Paulo, Brazil).
The abundance and biomass of mollusks were evaluated in three
transects. The size structure was also examined.
The results showed that: 1) the density increased signifi-
cantly near the bases of trees when compared with the abundance
of bivalves located in the middle of the transects; 2) the increa
se and decrease of biomass (alcohol and dry weights) of D. delo-
dontus expansus were correlated with the density changes in the
density changes in the transects; 3) near the trees, the bival-
ves presented a clearer size variation than the specimen collee
ted in the stations located in the middle of transects.
Biological and ethological hypotheses were presented to
explain the occurrence of bivalves in the sediment near the
bases of trees. The role of D. delodontus expansus in the eco-
logy of reservoir is discuted.
RESUMO
A distribuição espacial de uma população de bivalve no se
dimento próximo ã base de árvores foi estudada em um pequeno re
servatõrio tropical (Represa do Rio Pardo, Botucatu, São Paulo,
Brasil). A abundância e a biomassa dos moluscos foram estimados
em três transectos. A estrutura em tamanho foi tanbém examinada.
Os resultados mostraram que: 1) a densidade aumentou sig-
nificativamente oróximo a base das árvores quando comparada con
a abundância dos bivalves coletados â meia distância nos transec
.216.
tos; 2) o aumento e a diminuição da bionassa (pesos alcoólico e
seco) de D. delodontus expansus fo-an correlacionados con as nu
danças de densidade nos transectos; 3) prõxino âs árvores, os M
valves apresentaran una variação em tacanho nais evidente que os
indivíduos coletados na netade dos transectos.
Hipóteses de natureza biológica e conportaraental slo apre-
sentados para explicar a ocorrência dos bivalves no sedinento
próxino ã base das árvores. 0 papel de D. delodontus er.nansus na
ecoloqia do reservatório ê discutido.
.217.
We are grateful to Dr. C. Lever*no (O".STO't, Paris ,
France) for this encouragement and suggestions at the start
of our study; to CNPq (Proc. 40.3022/33) and IDBMA/FAPESP for
their financial support; to Dr. L.C.F. Alvarenga for identify
cation of the bivalve; to Dra. n.C.D. Mansur for confirmation
of the identification of the aninal and for the suggestions;
to Dr. P.R. Cufi for the statistical assistance; to Dra. V.A.
Carvalho for facilitating the granulonetric analyses by
putting at our disposal laboratory equipment; to H. Carneiro,
E'.M. Pellegrini-Caranaschi, U. Caramaschi, J.''. Pavan and S.
V. Basso, for their contribution to field research; to T'rs. 0.
Henry for the english version of the manuscript and M./\. Ilunes
de Oliveira, for typing the manuscript and drawing the figures.
Miss C.A.S. received financial support fron the Fi'IIDAP. Speci-
mens were given to the National Museum of Rio de Janeiro (Bra-
zil) and the Museuin of Natural Sciences of the Zoobot3nic Ins-
titute of Rio Grande do Sul (Drazil) (deposited in the "alaco-
logy Section under number 5218).
REFERENCES
ALVARENGA, L.C. de; COELHO, A.C. dos S.; C.N. RICCT; COMES, L.
A.L. S H.M. BAP.ROS. 1. Resultados preliminares dos trabalhos
ecológicos realizados na Lagoa de Juturnaibn, Município de
Araruama, Estado do Rio de Janeiro, criadouro natural dos
bivalves Diplodon becheanus (Dunker, 1849) (Onionoidea, Hy-
riidae) e Anodontites trapesialls (Lanarck, 1819) (Muteloi-
dea, Mycepodidae). In: Anais do V Encontro de Malacologis -
tas brasileiros, Mossoró, RN, U a 17/07/1977. n. 73-89,1979.
.218.
BONETTO, A.A. Contribucion al conociniento Oe las glochitlias del
gênero Diplodon y su aplicaciôn a los studios sistenãticos
Priner Congresso Sudanericano de "oologia. n. 43-59, 1959.
BURGIS, it.J. Bionass and distribution of organisms in Lake Geor-
ge, Uganda. Proc. r. Soc. London B, 184:271-298, 1973.
Co, L.M. Distribuição de Oligochaeta na Represa do Lobo (Estado
de São Paulo» Brasil). São Carlos, 1979. 169 fls. {Disserta-
ção - Mestrado - Universidade Federal de São Carlos)•
DARLINGTON, J.P.E. Temporal and spatial variation in the benthic
invertebrate fauna of Lake George, Uganda. J. "ool., 331; 95-
111, 1977.
DEJOUX, C ; LAUZAIJNE, L. and LÊVÊQUE, C. Nature des fonds et r£
partition des organisnes benthiques dans la region de Bol
(archipel est du lac Tchad). Cah. O^STOM Ser. Hydroblol., J5:
213-223, 1971.
ELLIS, A.E. British freshwater bivalve nollusca. Academic Press,
London, 1973. 109p.
FITTKAU, E.J. IRMLER, U.; JUNK, W.J., REISS, F. and SCiriOT, G.
VI. Productivity, bionass and population dynamics in A'lazoni-
an water bodies. In: F.B. GOLLEY and E. flEDIKA (Eds.) Tropi-
cal Ecological Systems, p. 239-311, 1975.
HEBLIIJG, H.J. & PENTEADO, A.M.C. Anatomia funcional de Diplodon
rotundus gratus Wagner, 1827 (Mollusca, Bivalvia). Rev. Bra-
sil. Biol., _3_i'67-80, 1974.
HENRY, R. Estudos ecológicos na Represa do Rio Pardo (Botucatu,
SP, Brasil). I. 0 ambiente e variações diurnas de alguns fa-
tores ambientais. Rev. Brasil. Blol., jU:153-161, 1981.
HENRY, R. & CURI, P.R. Influências de parâmetros climatológicos
sobre alguns fatores físico-qulmicos da água na Represa do
Rio Pardo (Botucatu, SP). Rev. Brasil. Biol., £1:299-306.1981a.
.219.
r, R. • CURI, P.R. Analise de parâmetros hidrolõgicos da Re
presa do Rio Pardo (Botucatu, SP). Rev. Brasil. Blol., êl:
321-326, 1981b.
HENRY, R. ft CURI, P.R. Estudos ecológicos na Represa do Rio Par
do (Botucatu, SP, Brasil). II. Distribuição horizontal e va-
riação anual do material em suspensão. Rev. Brasil. Bjol.,43
-.311-316, 1983.
HENRY, R. ft SIMXo, C A . Evaluation of density and biomass of a
bivalve population (Diplodon delodontus expansus) (Kuster,
1856) in a small tropical reservoir, (submited to publicati-
on) 1984a.
HENRY, R. ft SIM&O, C.A. Spatial distribution of a bivalve popu-
lation (Diplodon delodontus expansus) (Kuster, 1856) in a
small tropical reservoir, (submited to publication). 1984a.
HENRY, R. ft SOUZA, A.P. Represa do Rio Pardo, Botucatu, SP. Rio
ou Lago? (submited to publication) 1984.
LÉVÊQUE, C. Equation de von Bertalanffy et croissance des mol-
lusques benthiques du lac Tchad. Cah. ORSTOM. Sir. Hidroblol.
5:263-283, 1971.
LÉVÊQUE, C. Mollusques benthiques du lac Tchad: écologie, etude
des peuplements et estimation des biomasses. Cah. ORSTOM.
Ser. Hydroblol., 6_:3-45, 1972.
LÊVÊQUE, C. Dynamique des peuplements, biologle et estimation
de la production des mollusques benthiques du lac Tchad.Cah.
ORSTOM Sir. Hydroblol., Tilll-W.
LÊVÊQUE, C ; DEJOUX C. ft LAUZANNE, L. 8. The benthic fauna: e-
cology, biomass and communities. In: J-P Carmouze et al (eels)
Lake Chad. Dr. W. Junk Publishers, The Hague p. 233-271,1933.
LEWANDOWSKI, K. ft STANCZYKOWSKA, A. The occurrence and role of
.220.
Bivalves of the family Unionidae in Mlkolajskie Lake. Ekol.
Pol., 22:317-334, 1975.
STRIXINO, C. ft STRIXINO, s.T. Macroinvertebrados do fundo da
Represa do Lobo (Estado de São Paulo - Brasil). I. Distri-
buição e abundância de Chiromoraidae e Chaoboridae (Diptera).
Tropical Ecology, JU:16-23, 1980.
STRIXINO, G. * STRIXINO, S.T. Macrobentos da Represa do Monjo-
linho (São Carlos, SP). Rev. Brasil. Biol., 42:165-170,1982.
.221.
Table 1. Density and frequence variation patterns of Diplodon delodontus expanses in each one
of three transects.
T
R
A
N
S
r>
T
S
From
A
to
B
From
B
to
C
From
C
to
A
DENSITY
(Specimen
0.25 m'2)
FREQUENCE
(%)
DENSITY(Specimen0.25 m"2)
FREQUENCE(%)
DENSITY(Specimen0.25 r\~*)
FREQUENCE(%)
STATION
1 2
78 31
46.4 18.5
STATION
7 8
58 9
43.6 6.8
STATION
13 14
24 7
15.3 4.4
j
18
10.7
9
6
4.5
15
7
4.4
4
6
3.6
10
6
4.5
16
26
16.6
NUMBER
5
8
4.8
NUMBER
11
12
9.0
NUMBER
17
8
5.1
6
27
16.0
12
42
31.6
18 19
30 55
19.1 35.1
TOTAL
168
100.0
TOTAL
133
100.0
TOTAL
157
100.0
Table 2. Comparison in the means (x) and standard deviations (s) in alcohol weight,
shells and bodies dry weight and density of Diplodon delodontus expansus
in the whole reservoir and in the sampling area near the trees.
LOCAL VARIABLE STATISTICS
In the whole
reservoir (n=98) Alcohol weight (g.m" )
Shells dry weight (g.m )
Bodies dry weight (g.m
Density (specimen .m )
-2,
347.27
225.00
10.92
•Í6.62
854.32
554.13
26.96
96.42
293.97
191.07
9.30
32.31
742.25
481.56
23.39
83.91
Sampling area
near the trees
(n-19)
Alcohol weight (g.m~ )
Shells dry weight (g.m" )
Bodies dry weight (g.m~ )
Density (Specimen .m~ )
100m
FIG. I. POSITION OF THE SAMPLING ÁREA (• ) OF
PIPLOOOM DELODONTUS EXPANSUS IN THE
RESERVOIR OF RIO PARDO. BOTUCATU,
S i0 PAULO. BRAZIL.
SI9
FIG.2. SAMPLING SCHEDULE IN THREE TRANSECTS.SHOWING THE POSITION OF EACH ONE OF
19 STATIONS.
. 2 2 4 .
4*Mlêt*
4*9t*•1*
Z 4»O »•
p M
s "
' M:
nil
JL
UI * •
ã »Uiu. ••
4*
3n.18
n
7n;3;24
9»:03
n
JL
10n:06
nnr
15n.0?
16«26
Lft:i2
12
17: 06
10ft. 90
ruüTllk.I»i4*fflf»*m
RG.3. FREOUENCY-SIZE DISTRIBUTION IN EACH ONE OF THE
STATIONS IN THE THREE TRANSECTS.
LENOHT CLASSES:
I* < 1.60 cm2« 1.80 i-3> 2 J 6 » . 2.90cm4« 2401-3.49em
6» 440»-449cm
7> 4.9 S«-9.10 cm8* 9.10 »- 9.65cm9" 645 H- 6,20cm10* 6 2 0 , . 6.75 cmll> 6,76»__ 730cm12» 7.50 H-7.86cm
. 2 2 5 .
• 0 0
700
§too
400
«ft«5 soo
2 *ooj
100
O
BIOMASS
D ALCOHOL «««NT
B SHCLLS DRY «CMNT• «OMCS DRY «««MT
1 2 3 4 S • 7 • 9 10 II 12 13 M IS 16 17 » 19A B C 0 A
FIG.4 BIOMASS VARIATION PATTERNS OF DIPLODON DELOOQNTÜS EXPAMSUS , N
EACH ONE OF THREE TRANSECTS.
THE ECOSYSTEM LAGOA CARIOCA : MAJOR CONSEQUENCES OF ITS THERMALBEHAVIOR DURING THE WINTER AND SUMMER .
F.A.R. Barbosa - Department of General Biology - ICB/UFNG.J.G.Tundisi - Department of Biological Sciences - UFSCar.
ABSTRACT
In order to obtain more information for the comprehension ofthe global dynamics of Lake Carioca and taking into considerationthe data recorded during the period 1978/1981, it is proposed asimpiified -diagramm of the seasonal differences(winter/summer)between the major physico-chemical and biological variables a£ afunction of the recorded thermal structure from a systemic pointof view. The diagramm shows the major physico-chemical and biolo-gical consequences of the presence or absence of thermal stratifycation of the water column and relate them to climatological va -riables namely the lower energy input during winter and the higherone during the summer and the allocthonous contribution mainlyfrom plant origin. The data analysis, especially that one relatedto the diurnal cycles allowed us to get a general idea of the dy-namic functioning of Lake Carioca. The significative seasonal di£ferences between some of the analysed parameters show the need ofmore detailed seasonal studies to confirm or to test if we canapply to Lake Carioca the previously proposed hypothesis aboutthe functioning of tropical lacustrine ecosystems. By the otherhand taking into consideration the relative homogeneity of theseasonal variations for the great number of the parameters, itbecomes evident the importance that the studied parameters be measured at shorter intervals either in time and in space as it wasdone before for the diurnal studies, with which it becomes possi-ble to detect phenomena with such short duration and irregular r"riodicity that by the other hand would not be detectable withinthe seasonal variations as a whole.
.227.
FILTER-FEEDING RATES AND SELECTIVITIES OF TWO CIChLIDb ON
THE ZOOPLANKTON OF BROA RESERVO:?. (SAC CARLOS, SF, JRAZIL).
X. LA2ZAR0
Laboratory of Limnology
UFSCar
(O.R.S.T.O.M., CNPq)
ABSTRACT
Geophagus brasiliensis and Tiiapia rendalli are
two planktivorous cichlids abundant in most of the reser
voirs of the São Paulo State where little is known or. the
interaction fish-plankton. Fish between 2C and lOrrsr. of stand
ard length (SL) use both particulate. and filter feeding.Fish
smaller are obligate particulate feeders, while larger ones
are obligate filter feeders. In aquariums, G. brasiliensis
and T. rendalli from 36 to 16 8mm SL are observed to feel on
zooplankton as pump-filter feeders. They capture prey by
rhythmic buccal suctions not directed at individual organ_
isms. Buccal volumes, determined from silicone casts, in
crease as a power function of fish standard length. Fumping
rates, determined with chronometer, decrease as a linear
function of fish standard length. Absolute filtering rates,
computed by the product of buccal volume and pumping rate,
increase as a power function of fish standard length. Filter
ing rates per gram of fish decrease as a power function of
fish standard length. Populations of G.brasiliensis and T.
rendalli can filter rather large volumes of water per hour.
During one hour feeding trials, G.brasiliensis 30-42mm SL
and T.rendalli 29-42mm SL use a combination of erratic par
ticulate feeding (only the smallest fish and mainly at the
beggining of the feeding period) and lengthy pump-filter fe
eding (most fish and most of the time) to capture zooplankton.Fish have a highest feeding electivity for the large-bodiedand easily captured Moina minuta.
.228.
Abstract
Fish-zooplankton interactions in Americana Reservoir,
São Paulo, and the influence on the water quality.
Marlene S.Arcifa, Thomas G. Northcote, and Otávio
Froehlich.
This project consisted of two phases. At the first
phase, of one year duration, it was carried out a survey on the fish
community composition, a study of its horizontal and vertical
distribution, and its diet. Most fishes were concentrated in the
littoral and sublittoral zones and only three species explored the
limnetic zone. From these three latter, two are pianktivorous,
although not exclusive - Astyanax fasciatus and A. bimaculatus -
and their vertical distribution in the reservoir coincided with
that of the zooplanktcn.
The insects (mainlt Chironomidae l a r v a e ) , micro-
crustaceans, detrits and sediments were found to play an important
role in the diet of the community. The adults of the planktivorous
fishes were found to be selective feeders, choosing the more
conspicuous d a d o c e r a n s like Daphnia gessneri, Pi aphanosoma neotro-
pi cum and Moina sp.
The second phase was carried out in two stages, one
in 1982 and another in 1983. Experiments were done in four plastic
enclosures coupled with floating structures. Two series of experiments
were carried out, each one with a planktivorous fish species. In
each series, two enclosures were populated with fishes and the
other two were fish-free, for a period of approxinately one month.
The fishes affected the zooplankton composition and
density,' the ph /topi ank ton biomass and light penetration. At the
.21'.).
end of the experiments there was a clear difference between the fish
and the fish-free enclosures. The aspect of the fish enclosure was
of a more eutrophicated environment, with lower transparency.higher
biomass of planktonic organisms, when compared to the fish-free
enclosures. Certainly, the planktivorous fishes affect, in more than
a way, the eutrophication of Americana Reservoir.
.230.
EUTROPHICATION IN SAO PAULO STATE RESERVOIRS.
J.G. TUNDISI and T. MATSUMURA-TUNDISI
Laboratory of Limnology
Universidade Federal de São Carlos
(FAPESP, CNPq, Organization of American States)
ABSTRACT
In Southern Brasil, and particularly in São Paulo
State, several reservoirs are being subjected to increasing
eutrophication, mainly in the last ten years. The introdu£
tion of excess nutrients in these artificial ecosystems is
due to increased urbanization, and sewage disposal, produ£
tion and use of fertilizers in agriculture and industrial
waters. A comparative overview of the eutrophication prob_
lems and processes is given, based upon research developed
in the last six years in a wide geographic area of São Paulo
State. Levels of primary production, the vertical profiles
of volumetric primary productivity, nutrient distribution arri
light pene\ration are compared. The ionic composition shows
strong correlation of SO* with the oligotrophic - eutrophic
situation of the reservoirs. A case study of progressive eu
trophication in Barra Bonita reservoir in the center of São
Paulo State is presented. Discussion is directed on the fre_
quenay and consequences of algal blooms, as a result of eu
trophication, in this particular reservoir. Results presented
show the relationship between nutrient loading, blooms of
Myaroaistie sp and Anabaena sp zooplankton composition, resi^
dence time, the circulatory, and exchange processes in the
reservoirs, and the operation of these systems from the hydro
electric and hydraulic point of view. Parameters necessary
for the eutrophication model (in preparation) are discussed.
Possible corrective and preventive measures to minimize the
eutrophication processes are also presented.
.231.
LIMNOLOGY AND ECOLOGY OF FURNAS RESERVOIR.
J.G. TUNDISI*, KOZO HINO*, RAOUL HENRY**, J.G. GENTIL* and
DIRCEU MARZULLO RIBEIRO***.
* Laboratory of Limnology, UFSCar.
** UNESP, Botucatu.
***Furnas Hydroelectric Company.
(FAPESP, CNPq, Organization of American States, FURNAS).
ABSTRACT
Furnas reservoir is one of the largest man made
lakes in South America, with an area of l.UUO km2 and a
maximum volume of 22.9 50 km*. During April, 1982 a series
of intensive limnological surveys was carried out at seven
stations selected ar representative points in order to ob
tain comparative information on basic parameters. TWJ main
rivers are forming the reservoir, Rio Sapucai and Rio Gran
de. The two rivers are in different conditions of eutro
phication the former being more eutrophic them latter.This
reflected the values obtained for several parameters. Rio
Grande showed a volumetric production of phytoplankton of
20mgC x m~3 x day"1 at Pmax, while Rio Sapucai, showed a
value of 80mgC x m~J x day"1 also at Pmax. In both, the
fraction < 50y was predominant as primary producer. At the
intermediate station, receiving waters from the two rivers,
the value for primary production at Pmax was 60mg C x m~3 x
day"1. The hypolimnion was characterized by severe oxygen
deficit, high concentration of ammonia and total dissolved
phosphate. Comparison with other reservoirs in São Paulo
State, is made; some measures to prevent eutrophication are
recomended. The eutrophicati^n at Sapucai river was mainly
due to agricultural activities. The hydraulic operation of
the reservoir and its morphometric features, were the main
causes of a chemical-physical and biological stratification.
.232.
ZOOPLANKTON COMPOSITION OF FURNAS RESERVOIR (MINAS GERAIS)
T. MATSUMURA-TUNDISI and 0. ROCHA
Laboratory of Limnology
Universidade Federal de São Carlos
ABSTRACT
During April 1982 limnolbgioal surveys were made
up in Furnas reservoir located in Minas Gerais State in
order to know the trophic state of that water body. The res_
ervoirs is constructed by the function of two main rivers:
Rio Grande and Rio Sapucai. Although the chemical analyses
of water made in the several stations located in the the
Rio Grande and in the Rio Sapucai have not showed a sharp
difference in the nutrient concentrations (NOs, NO2, POi,
and NHi,) the plankton production was more higher in the
Rio Sapucai than in the Rio Grande. The diversity of zoo
plankton community although was similar in the series of
sampling stations the dominating species was quite different.
In the Rio Sapucai the larger forms like Thermoayalope ne_
gleatUB deoipiens (Cyclopoida), Diaphanoeoma braohyurum and
Moina minuta (Cladocera) and Ploeeoma truncatum (Rotifera)
dominated the zooplankton community while smaller forms like
Thevmooyalopa minutus (Cyclopoida), Boemina ep and Bosmi_
no-peia deiterei (Cladocera) and Keratella aoahlearie (Roti
fera) dominated in the sampling stations of Rio Grande.
.233.
THE LIFE CYCLE AND PRODUCTION OF Vapknia ge.66ni*i
0. ROCHA and T. MATSUMURA-TUNDISI
Laboratory of Limnology
Universidade Federal de São Carlos
ABSTRACT
The life cycle of Daphnia gessneri was studied at
the laboratory under constant conditions of temperature
and food concentration. The duration of embryonic and
post-embryonic development was determined at 18°C. Growth
rate, reproductive output and life-span at surplus food
conditions were monitored and the data used for calculat^
ing this species production at Barra Bonita reservoir. The
results are compared with those found in the literature
for tropical species.
.234.
GEOMORPHOLOGICAL AND LIMNOLOGICAL PROCESSES AS A BASIS FOR
LAKE TYPOLOGY - MIDDLE RIO DOCE VALLEY.
J.G. TUNDISI
Laboratory of Limnology
UFSCar
M. REGINA M. de MEIS
Instituto de Geociências - UFRJ(FAPESP, CNPq, Organization of American States,Acad.Brasil.de Ciências).
ABSTRACT
The characteristic pattern of morphometric features
of the Middle Rio Doce Valley lakes is related to the region
al geomorphic evolution during the Upper Quaternary. Litho,
allo and morphostratigraphic evidences indicate that the
activity of the erosional and depositional processes varied
strongly in space with time. As a consequence at the end of
the Pleistocene, part of the drainage net became non func
tional, changing into lakes. The limnological features re
suiting from the morphometry are basically processes of cir
culation, nutrient recycling, thermal, chemical and biology
cal stratification. Wind action affects very little the cir
culation pattern in the lakes. A strong stratification de
velops from September to March; thus, the thermal regime de
pends mainly on surface heating and cooling. The resulting
biological stratification is mainly related to differential
vertical migration of zooplanktonic organisms, and accumula-
tion of cyanophyta in the lower hypolimnion of the lakes dur
ing the stratification period. Based on the geomorphic se£
ting, Upper Quaternary evolution and limnological processes,
six main types of lakes and wetlands are proposed. Dom Hel
vecio, Lagoa da Barra and Carioca model lake types - and in
nundated depressions meandering creek floadplains, and re
generated "wetlands". The mechanisms of evolution of the
system are discussed from both geomorphological and ecology
cal points of view.Possible effects of human activities are disoEsad,
.235.
ECOLOGICAL MODELS AS A WAY FOR ORGANISING
CONDENSING AND TRANSMITTING KNOWLEDGE
Dr. Maurício Vieira Kritz
Laboratório de Computação Cientifica
Rua Lauro M ü l l e r , 455
22290 - Rio de J a n e i r o , RJ - Brasil
Mathematical models have several a s p e c t s and utilities such as
allowing q u a n t i f i c a t i o n , numerical s i m u l a t i o n , prediction and
numerical e x p e r i m e n t a t i o n . If is our intend to stress other
important aspects of such models which can not classified as
q u a n t i t a t i v e . These are the organizational and compactification
aspects of the Mathematical L a n g u a g e and its ability to act as
a vehycle for transmiting knowledge about relationships
among constituents (state v a r i a b l e s ) of a system and about the
structure of the system itself once the meaning of the
variables present is stablished.
Starting by recalling functions as a compactification of
punctual (measured) data relationships we shall emphizise that
if there is a function which explains the data that can be
obtained as a solution from a mathematical relation or equation
one has a "law" for the i n t e r d e p e n d e n c e of the two constituents
related by the function. This "law" is represented by the
•niethentatical relation or equation a n d , since the way a function
"explains" collected data is never uniquely defined, a great
deal of intuition and knowledge about the "real" phenomena is
involved in "accepting" such a r e l a t i o n or equation as a "law".
From the principle that people that built a model know a lot
about the phenomena described by it , even if they may have
commited some errors or their knowledge is not complete, from
the significance of the mathematical relationships and from
the mecaning of v a r i a b l e s , it is p o s s i b l e to go the way back
and learn a lot about the system being modelled. Thisreversed
path is as much as difficult as the original one and sometimes
implies some guess work to figure out what are the underlying
.236.
h y p o t h e s i s . It needs a l s o access to the b a s i c c o n c e p t s and
v o c a b u l a r y of the s c i e n t i f i c a r e a s r e l a t e d to the p r o b l e m and
i n f o r m a t i o n about m e a s u r e m e n t t e c h i n i q u e s and s t r a t e g i e s of
data acqui sition .
The e n o r m o u s c o m p l e x i t y of e c o l o g i c a l s y s t e m s forces a lot of
s i m p l i f i c a t i o n s when d e s i g n i n g a m o d e l , to rend it w o r t h y . This
in t u r n , makes the use of such m o d e l s as c o m u n i c a t i o n media
more d i f f i c u l t .demanding an i n t e r d i s c i p l i n a r y b a c k g r o u n d to
p r o p e r l y a c c o m p l i s h i t . As e c o l o g i c a l s y s t e m s include so m a n y
areas of natural r e s e a r c h , such a task m u s t be attempted by several
p e r s o n s with d i s t i n c t b a c k g r o u n d s , w h i c h imposes other and
m a g n i f y some of the normal d i f f i c u l t i e s . F i n a l y , we shall
d e s c r i b e more e x p l i c i t l y our e x p e r i e n c e w i t h an interdisciplinary
g r o u p , in formation at L C C , that has being w o r k i n g in this
d i r e c t i o n withi i the last y e a r , e x p e r i e n c e that n e v e r t h e l e s s
s u p p o r t s all the d i s c u s s i o n s in this w o r k .
. i .1 /.
THE EgJUOGICAL DISTRIBUTION OF FISH AND THEIR POOP IN Tt£ ESTUARIKE RESIGN
OF OHTA RIVER IN HIROSHIMA PREFECTUR \PAN.
Tetsuo SUNAGA
Biological Institute
Faculty of Education
Kagawa University
ABSTRACT
The estuarine region of Ohta River(main river length 103 km,
drainage area 1,690 km ) was chosen as the research site that
was one of the most typical one exposed to a great variety of
environmental impacts. Sait wedge are performed in tidal rivers
during ordinary weather, while tidal rise and faHs, exceeding
3 m in spring tide, fluctuate the environmental conditions of
roganisms inhabiting around the estuary.
Twenty nine species of fish, including freshwater, estuarine
and marine species, were collected in the research site, species
compositions of fish collected at sampling stations were differ-
ent from one another reflecting difference of salinity of water.
Stomach contents of dominant species of fish were examined and
occurrence of food items were compared among specimen collected
from different sampling stations. In the upper part of the es-
tuary, these fish fed on oligochaets and larvae of chironomids,
but in the lower part they fed mainly on polychaets.
From the result of the comparison, we concluded that the fish
selected food organism corresponding to the abundance of the
organism in their habitat.
.238.
CITRUS PESTS
Coordinator
Dr. OCTÂVIO NAKANO
School of Agriculture "Luiz de Queiroz"
Piracicaba, SP
PARASITISM OF PUPAE OF Anastrepha spp. (Dip.: Tephritidae) BY Doryctobracon
areolatus (Szépligeti, 1911) (Kym.: Braconidae) IN CITRUS AND TROPICAL FRUITS
A.S. NASCIMENTO & A.L.M. MESQUITA
Centro Nacional de Pesquisa de Mandioca e Fruticultura/EMBRAPA
R.A. ZUCCHI
Department of Entomology - ESALQ/USP
ABSTRACT
A study was made of parasitism in fruit flies of the genus Anastrepha
(Schiner, 1868), one of the main pests of fruit culture in Brazil and in the
world, the aim being to provide information for an integrated plan for
dealing with fruit flies.
8,805 pupae of Anastrepha spp. were obtained, of which 1,221 were parasited
by Doryctobracon areolatus. Pupae derived from the hosts grumichama ( Eugenia
brasiliensis), cabeludinha (E. tormentosa), pitanga (E. uniflora] and uvaia
(E. uvalha) presented the highest indices of parasitism: 30.37%, 28.80%, 20.93%
and 14.42%, respectively. The lowest indices were found in mango (Mangifera indica),
guava (Psidium guajava), citrus (Citrus sinensis) and carambola (Averrhoa carambola)
The present work discusses and proposes the use of some of these fruit trees as
trap plants for fruit flies and their parasites, in an integrated programme for
handling this pest.
INTRODUCTION
Fruit flies stand out as one of the most important insect pests of fruit
culture. The use of poisoned baits, the traditional method of control, has
proven efficient. However, besides the additional cost to the producer, it
reduces the natural enemy population which ti>- poisoned bait also attracts.
According to PAVAN (1976 - personal coircnunication), in the medium term the
efficient control of fruit flies by exclusively chemical methods will no
longer be possible, hence the need to intensify the bioecological study of
this pest and its natural enemies.
.239.
Various authors have mentioned the importance of natural enemies in reducing
the fruit fly population, especially those of the family Braconidae. GONZALEZ -
HERNANDEZ & TEJADA (1979) refer to Doryctobracon crawfordii (Vier.) as the most
important natural enemy of Anastrepha ludens in Mexico. WHAFTON & HARSH (1978)
report on various species of the Braconidae parasitising the Tephritidae and
include species introduced and established in the New World; among them the
autnours cite Doryctobracon areolatus as a parasite of- seven species of the
genus Anastrepha.
EMBRAPA's Centro Nacional de Pesquisa de Mandioca e Fruticultura is currently
developing a project in this line of research, with the object of affording
information that will enable the recommendation of alternative methods of control
(NASCIMENTO et alii 1981).
The present work aimed to determine, under field conditions, the index of
parasitism of pupae of Anastrepha spp. in citrus and tropical fruits, with a view
to the possible utilisation of the latter as plant bait for fruit flies and to
promote the population growth of their parasites.
MATERIALS AND METHODS
Field
Collections were made of oranges (Citrus sinensis), grumichama (Eugenia bra-
siliensis), cabeludinha (E. tomentosa), pitanga (E^ uniflora), uvaia (E. uvalha),
mango (Mangifera indica), guava (Psidium guajava) and carambola (Averrhoa carambola),
the plants being at' the Experiment Station of Santo Antonio de Jesus, situated in
the Banian recôncavo at latitude 12958' south and longitude 35915' west, and at an
altitude of 215 m.
The collections were made weekly during the period from 3 rd October, 1980 to
25th August, 1981. All the citrus fruits collected snowed symptoms of pest attack
while the other fruits were picked indiscriminately, whether showing symptoms of
infestation or not. After reaping the fruits were transported to the laboratory
where the pupae and adult insects were obtained.
Laboratory
The fruits from each delivery were kept on sawdust dampened with water in
plastic boxes covered with a fine mesh. After an interval of fifteen to twenty
days the sawdust was strained and the pupae collected. These were then placed
.240.
under damp sawdust in small plastic cups, each enclosed in another larger one,
bottomless and with the opening covered with fine mesh. Thus, the adult insects
remained confined together as they emerged. Afterwards they were separated by
sexes, counted and placed in bottles containing 70% alcohol. To calculate the
pupal viability (Tables 1 and 2) the following formula was used:
P.V.% = N9 of Anagtrepha «nerged x 100
Total pupae - N9 of braconids emerged
RESULTS AND DISCUSSION
Table 1 shows the levels of parasitism of Anastrepha spp. by the braconid
Doryctobracon areolatus in fruits derived from different tropical plants grown
at the Experiment Station of Santo Antonio de Jesus. Of the 8,805 pupae obtair-
from the different hosts, a mean of 14.08% were found to be parasitised, with
range from 1.32 to 30.38% of parasitism.
Similar results were obtained by other authors. HEMPEL (1906) refers to
parasites of the family Braconidae in fruit flies, in the state of São Paulo, on
plants of peach, pitanga and jaboticaba. The percentage of parasitism varied from
3% to 50%, with the highest indices in A. fraterculus. However, it is worth pointing
out that the real maximum index of parasitism was about 30%, taking into account
an overestimation in the results. Again in São Paulo FERNANDES (1983) found in caj'a-
mirim a rate of 27.86% natural parasitism of Anastrepha by D. areolatus. Apart from
this natural enemy, FONSECA (1938), in the town of Bonfim, Bahia, observed in
fruits of caja-umbu large quantities of larvae of A. fraterculus parasitised by
waspsof the genus Opius.
The important benefit of these parasites, as natural agents in the control of
fruit fly populations, can be found in the observations of SUPLICY et alii (1978).
In a study of populational fluctuations of Anastrepha spp. and Ceratitis capitata
in the state of São Paulo, they observed a sharp reduction of the pest after two
periods of populational abundance• This was attributed to the interference of
natural enemies and to other predisposing factors.
Therefore there is sufficient evidence of the occurrence of parasitism in pupae
of fruit flies by braconids in different regions of the country, notably in tropical
fruits. The hosts.grumichama, cabeludinha and pitanga showed an index of parasitism
of the fruit fly by the natural enemy of 30.38%, 28.80% and 20.94%, respectively
(Table 1), values considered relatively high.
.241.
Based on these findings, and considering the great preference of the genus
Anastrepha for tropical hosts (MALAVASI & HCRGANTE, 1990 and NASCIKEMrO et alii,
1982), a research objective is suggested to be the feasibility of these bests as
an attraction for fruit flies in comnercial orchards of citrus, peaches, nectarines
and others. FONSECA (1938) reports on the introduction from Hawaii to Brazil in
1937 of the hymenoptera Tetrastichus giffardii, airring to parasitise £. capitata.
He conments that coffee plantations lend themselves to intense reproduction of the
parasite during the period of maturation of the berries. Prom there they pass to
adjacent orchards, parasitising tue flies which were forced to migrate in search
of other fruits for oviposition.
Following this reasoning it is believed possible to increase the parasitism cf
fruit flies in the conditions of commercial orchards of citrus, peaches, nectarines
and others, using hosts such as grumichama, cabeludinha, pitanga and perhaps the
coffee tree itself as attractions or trap plants for the flies. According to
MENEZES (1983 - personal comnunication) this great preference which the braconids
show for the larvae of the Tephitidae in tropical fruits is due to the wealth and
diversity of vitamins existing in these fruits.
An interesting observation is that the number of citrus fruits picked with
symptoms of fly attack is very high in relation to the number of pupae obtaired,
and higher still in relation to the number of adults derived (Table 2). NASCIMENTO
(1980) attributed the lesser occurrence of Anastrepha adults in citrus orchards cf
the Bahian recôncavo to the greater acidity of citrus fruits. Table ? confirms this
finding in that the number of adults obtained from the citrus varieties decreases
with the level of acidity (3, 1 and 0 for the oranges Hamlin, Pera and Natal,
respectively). The low index of pupal viability for citrus of 1.88% (Table 1)
shows that flies of the genus Anastrepha have difficulty in completing their life
cycle in this host. This fact explains partly the low index of parasitism of pupae
of Anastrepha in citrus, found in this work (Tables 1 and 2), as well as the value
of 0.20% found by WHAPTON et alii (1981) in Citrus paradisi in Costa Rica.
CONCLUSION
Some tropical fruit plants as hosts of fruit flies nerkedly favour the
multiplication of natural enemies. These plants, such as grumichama Eugenia
brasiliensis), cabeludinha (E. tomentosa), pitanga (E. uniflora) and uvaia (E.
uvalha), could be used in an integrated programe for controlling fruit flies,
.242.
with the object of increasing the index of parasitism of Anastrepha spp. by
Doryctobracon areolatus.
ACKNOWLEDGEMENTS
The authors express their thanks to Dr. Almir Pinto da Cunha Sobrinho for the
facilities granted at the Experiment Station of Santo Antonio de Jesus, and to
Prof. Luiz De Santis for identification of the parasite.
REFERENCES
FERNANDES, O.A. Estudos òioecolõgicos de moscas-das-frutas de gênero Anastrepha
(Diptera, Tephritidae) em Jaboticabal - SP. Jaboticabal, FCAV/UNESP. 67 p.
(Dissertação de graduação). 1983.
FONSECA, J.P. 0 combate biológico âs moscas-das-frutas. 0 Biológico, 4_: 221 -
225, 1938.
GONZALEZ-HERNANDEZ, A. & TEJADA, L.O. Flutuation de Ia problacion de Anastrepha
ludens (Loew) y de sus enemigos naturales en Sargentia greggii s. watts. Folia
EntomalÓRica Mexicana 41: 49-60, 1979.
HEMPEL, A. 0 bicho dos frutos e seus parasitas. Boi. Agricultura, 2: 206-214,
. 1906.
MALAVASI, A, & MCRGANTE, J.5. Biologia de "moDcas-das-frutas" (Diptera-Tephritidae.
II. índices de infestação em diferentes hospedeiros e localidades. R. Bras. Piol.
Rio de Janeiro, 40 (1): 17-24, 1930.
MENEZES, E.B. Obser/ações sobre paras it ismo de moscas-das-frutas. Rio de Janeiro.
1983 (Informação pessoal).
NASCIMENTO, A.S. Dinâmica populacional de moscas-das-frutas (Diptera - Tevhritidae
no Recôncavo Baiano. Piracicaba, ESALQ/USP, 1980. 110 p. Dissertação de
Mestrado.
NASCIMENTO, A.S.; ZUCCHI, R.A.; MORGANTE, J.S.; MALAVASI, A.; MACEDO, M.M.G. &
SILVA, L.M.S. Bioecologia das iiescas-das-frutas Anastrepha sp. Diptera: Teph-
litidae. Cruz das Almas, EMBRAPA/CNFMF, 1981. 4 p. (CNIWF. Pesquisa em
Andamento, 1/81).
.243.
NASCIMENTO, A.S.; ZUCCHI, R.A.; MCRGAOTE, J.S. & MALAVASI, A. Dinâmica popula-
cional das noscás-das-frutas do gênero Anastrepha (Dip., Tephritidae) no Re-
côncavo Baiano II - flutuação populacional. Pesq. Agropec. Bras.t .Brasilia
17 (7): 969 - 980. 1982.
PAVAN, C. Comunicação pessoal. Cruz das Almas, BA. 1976.
SUPLICY FllflO, N.; SAMPAIO, A.S. & MYAZAKI, I. Flutuação populacional das "mos
cas-das-frutas" Anastrepha spp. e Ceratitis capitata (Vied., 1824) em ei -
tros na fazenda Guanabara, Barretes, SP. 0 Biológico, São Paulo 64: 279-84,
1978.
WHAFTON, R.A. & MARSH, P.H. New world Opiinae (Hymenoptera: Braconidae) parasitic
on Tephritidae (Diptera) J. WASH. ACAD., 68 (4): 147-49, 1978.
WHARTON, R.A.; CILSTRAP, F.E., RHODE, R.H.; FISCHEL-M, M. & HART, W.G. Hymenopterous
egg-pupal and larval-pupal parasitoids of Ceratitis capitata and Anastrepha spp.
(Dip.: Tephritidae) in Costa Rica. Entomophaga 26 (6): 285-90, 1981.
.244.
TABLE 1 - Occurrence, percentage emergence, pupal viability and parasitism of
Anastrepha spp. (Dip.: Tephritidae) by Doryctobracon areolatus (Hym.:
Braconidae) in citrus and tropical fruits - Santo Antonio de Jesus, Ba-
hia, 1980/81.
Host* Pupae
obtained
Adult Anastrepha
N9 % Emergence
% Pupal
viability
D, areolatus
Adults % Parasitisir
Grumicham
Cabeludiriha
Pitanga
Uvaia
Manga
Goiaba
Citros
Carambola
1.172
861
683
319
2.790
2.688
216
76
466
276
275
174
1.157
1.794
4
49
39,76
32,06
40,26
54,55
41,47
66,74
1,85
64,47
57,10
45,02
50,92
63,74
45,71
71,10
1,88
65,33
356
248
1.43
46
259
165
3
1
30,38
28,80
20,94
14,42
9,28
6,14
1,39
1,32
TOTAL 8.805 4.195 1.221
MEAN 42,64 50,10 14,08 14,08
The diverse hosts have the following specific names:
Grumichama - Eugenia brasiliensis MangoCabeludinha - E. tomentosa GoiabaPitanga - E. uniflora" CitrusUvaia - H. uvalha Carambola
- Mangifera indica- Psidium guajava- Dif. varieties- Averrhoa carambola
.245.
TABLE 2 - Occurrence, percentage emergence, pupal viability and parasitism of Anastrepha
spp. (Dip.: Tephritidae) by Doryctobracon areolatus (Hym.: Braconidae) in ci -
trus and tropical fruits - Santo Antonio de Jesus, Bahia, 1980/81
v . t Fruits Pupae Pupae/ Adult Anastrepha % Pupal D. areolatus
collected obtained fruit ~ % mergence viability Adults % Parasitism
Hamlm
Pera
Natal
173
141
131
106
53
47
0,61
0,38
0,36
3
1
0
3,77
3,77
0
2,86
1,92
0
1
1
u
u,84
1,99
0
TOTAL 455 206 1,35 4 7,54 - 2 2,83
MEAN - - 0,45 1,33 2,51 - 0,66 0,94
.246.
EFFECT OF STERILANTS ON Ceratltis capitate (WIEDEMANN, 1824)ÍOirTEKA: TEPHRITIDAE), ITS SYMBIOTES AND THE PREDATORChrysoperla externa (HAGEN, 1861) (NEUROPTERA: CHRYSOPIDAE)
C. ALBERTO-PEREZDepartament of EntomologyEmpresa Capixaba de Pesquisa Agropecuária -EMCAPA
0. NAKANODepartament of EntomologyEscola Sup. de Agricultura "Luiz de Queiroz" ESALQ/USP
ABSTRACT
The affect of sterilant in increasing dosages on vhe
pest Ceratitis capitata (Wied.) and the predator Chrysoperla
externa (Hagen) as well as the presence, of bacterial symbio-
te in the digestive tract of C. capitata on the degradation
of these substances was investigated.
Diflubenzuron, tested at dosages from 25 to 2000 ppm,
did not show any effect either viability or fecundity of C_
CEoitata, maybe due to t*e activity of some symbiotes
present in the intestinal flora.
Methoprene and PH-6044 were not efficient to cause
sterility of C. capitata.
Copper oxychloride at 0,1% even though it sterilized
females of C. capitata was not long lasting; copper
oxychloride at 0,12% showed injury on the stalk of eggs and
decreased eggs number per female.
Avermectin at 31 and 35 ppm gave the best results,
since allowed greater longevity to adults of Ç. capitata, an
important characteristic of sterilant, and females did not
oviposit during their life; on C_ externa, Avermectin at
35 ppm caused fragility of corion, decreased the stalk and
caused abnormality of the eggs.
.247.
STUDY OF CONTINUOUS FERMENTATION FOR OBTAINING BACTERIAL
INSECTICIDE TO CONTROL AGRICULTURAL PESTS
D.M.F. Capalbo and I.O. Moraes*
Summary
The continuous culture technique with Bacillus thuringiensis
was studied to produce bacterial insecticide. Investigations on
the dilution rate were done to stablish the maximum growth and spo
rulation rate of the bacillus. The results obtained confirmed that
the sporulation rate increased when the dilution rate decreased;
the best range of dilution rate was from 0,05-0,10 h~ , for the
operational conditions used. The continuous fermentation developed
run well when it was done in multiple stages and the last one run
as a descontinuous fermentor.
Introduction
Under current agricultural practice, it has been estimated that
more than 401 losses of crops in Latin America are due to pests .
In spite of the heavy and extensive use of chemical pesticides ,
these losses are occuring annually.
It is now generally recognized that chemical pesticides,parti^
* State University of Campinas- Faculty of Food and Agricultu
ral Engineering- Dept. of Food Engineering
P.O. Box 6121
13 100- Campinas-SP-Brasil
.248.
cularly chemical insecticides, are toxic to non target species
(including beneficial insects), domestic animals and man and they
have been steadily losing effectiveness due to the development of
resistance among target species.
It is important to replace the chemical pesticides by biora
tional agents that would be specific in their action.
Bacillus thuringiensis products have attributes that are es
sential for a successfull microbial pesticide: specificity, poten
cy and efficacy against several insects, in addition to the econo
mic viability of production and safety to man, animals and benefi
cial insects. By fermentation the bacillus produces ;. proteinacious
Crystal along with one toxic active spore and this parasporal bo
dy is also a lethal toxin for certain insects.
Bacillus thuringiensis can be grown and sporulate in submerged
culture in conventional equipment. The operational conditions(pH,
aeration and agitation, temperature) are controled to obtain the
maximum growth and sporulation rate.
For study of cultures undergoing both growth and sporulation,
continuous cultivation is however more suitable than batch cultu
re,particularly for examining the effects of the growth rate or
its changes on sporulation.
Material and Methods
The continuous production of the microbial insecticide with
Bacillus thuringiensis, sorotype 1, isolated from the comercial '
product Bactospeine (Rhône-Poulenc), was studied.
Corn steep liquor (60g/l) and cane sugar molasses (1Sg/l) we
.249.
re chosen as the nutrient medium. The parameters studied during
cultivation included the changes in content of carbohydrates ( ex
pressed as glucose), pH, concentration of nicrobial cells ( as
dryed cellular mass MS),optical density (DO) and sporulaticn rate
(microscopic examination). The methodology is described by Capal
bo (1982) and Moraes (1973).
The study of growth of B. thuringiensis on batch culture was
carried out on Minifermcnter M-1000 from Fermentation Design Inc.
The best conditions were selectionated for continuous culture.
Continuous cultivation was stablished in a multiple stage
apparatus. The first fermentor operates at 500 rpm and 1 vvm ( vo
lume of air by volume of medium by minute). The second and others
fermentors operated at 380 rpm of agitation and 0,3 vvm of aeration
rate. The dilution rate varied as follows: 0,05-0,10 h"1; 0,10 -
0,15 h and 0,15-0,30 h . These dilution r;ites were controlled
by means of a periostaltic pump for the first fermentor .md the
culture medium passed from this to the others through a syphon.
The last fermentor operates as a static fermentor, d
nuously.
Results and Discussion
Patch culture
As shown in Figures 1 and 2, the kinetics of the carbohydrates
consume and tt}c pll of the fcrmentated medium are important par ame
ters to stablish growth and sporulation phases of B. thuringiensis
in batch culture. These results were utilized as operational con
.250,
ditions for controlling the continuous fermentation.
Continuous Fermentation
The results obtained in this step are summarized in Tables 1,
2, 3 and 4.
Variation of pH
The pH value tends to be stable on 6,0 at the end of the fer
mentation step, independently of the initial pH of the conti
nuous fermentation .
Carbohydrates utilization
At higher dilution rates (0,15-0,30 h"1) it is observed that
there is a great percentage of glucose remaining in the medium.
It indicates incomplete fermentation and that these conditionsare
not satisfactory for the development of the Bacillus thuringiensis
For lower dilution rates the microrganism has a good growing
rate, as can be observed on Table 3.
Growth and sporulation
The results of analysis of optical density (DO) and -dry cel
lular mass (MS) indicate good production of biomass corresponding
to lower dilution rates (0,10-0,05 h ). These are reinforced by
The data of Table 4, for microscopic examination of the final me
dium fermentated.
The system with a descontinuous fermentor at the end showed
good results for all the parameters' analysed.
.251.
glucose (UA)
I
200
ISO
100
50
O 5 10 20 25 30
Fig. 1- Kinetics of glucose utilization in batch fermentation of
Bacillus thuringiensis
time (hr)
log phase sporulation
pH
6,0
5,0: i
time (hr)
10 20 25 30
Fig. 2- Variation of pH in batch fermentation of Bacillus thuringiensis
.252.
Table 1. Continuous ferscntation of Buciilu» thuringicinis
D • 0.1S-0.30 h'1 (dilution rate)
Tia»
(fcr)
0
2
S.S
21.S
30.2
pH
«.0
6.0
S.6
S.6
S.6
S.4S.S
s.s
S.7
S.S
s.s
flucose
(U.AM
21
2t
31
43
JS
52SO39
62
62
62
D.O.
<U.A»)
S.2
3.2
4.6
4.2
4.2
S.O4.1S.l
S.O
S.O
7.5
M.ST"
(t/1)
3.S
3.6
1.2
;.i
1.4
1.3
1.0
1.0
3.9
2.S
2.9
Minifer»
Ist
2«d
Ist
2nd
l»t
1"2»d
U»t
i "
2»d
l.St
Table 2. Continuous fermentation of Bacillus thuringicmis
D . 0,10- 0 « n"1 dilution rate)
TiM
(hr)
0
2
6
23.S
pH
6.S
6.4
S.7
6.0
6.0
6.0
flucote
CU.A»)
20
25
SO
70
4»
3S
D.O.
(U.A")
4.6
7.0
7.0
4.4
4.1
S.2
M.S!"
((/I)
4.2
...
...
...
...
6.1
Minifer».
1lt
l»«
1st
1ft
2nd
3rd
{*) 1 g jHcosc/1 . I.2S UA
(•') 1 f bio«ass/l . 0.(6 UA
(•••) MS • dry cel l , bioMfS
. 2 5 3 .
Table 3. Continuous fiTBcnt.ition of Bacillus t n u n m i c m n
D • COS h*1 (dilution rate)
T i n
(hr)
0
2.5
29
S3
57
7.0
6.0
6.0
6.0
6.S
S.S
6.0
6.0
S.S
6.0
6.5
ll lKOM
(U.A.#)
23
22
S2
23
34
20
19
27
t l
19
IS
0.0.(U.A**)
10.4
10.0
6.6
10.1
7.S
7.0
10.0
7.S
7.S
I.S
1.0
M.St"
(1/1)
. . .
. . .
. . .
—
. . .
. . .
—
6.3
S.O
6.1
7.0
Miaifera.
l "
l "
i "2»d
l M
2»d
3 r d
i "
2»d
:»»t
(*) 1 f (lucose/1. »,2S UA
f ) 1 g bioMSi/1 . 0,8» UA
(•••) MS . dry c e l l , biouss
Tablt 4. Microscopic examination of the fermented nediua of
Bacillus thurintiensi» obtained on a continuous
fermentation
Tible Hinifermentor Presence of spores
l "
2nd
list
i"
,rd
i "
2nd
3 r d
last
. no spores
. some »porcs
. 2 5 4 ,
Conclusions
Although the alimentation of fresh medium, at continuous fer
mentation of B. thuringiensis ,could be initiated at any pü, the
value 6,0 to 6,5 is more convenient for minimization of op-ratio
nal time.
Also the kinetics of the carbohydrates utilization is an im
portant parameter on caracterization of fermentation of Bacillus
thuringiensis.
The continuous fermentation under the studied conditions runs
well for multiple stages system, where the last fermentor is a
descontinuous one, for spore maturation.
Bibliography
CAPALBO, D.M.F. (1982) "Contribuição ao estudo da fermentação con
tínua com Bacillus thuringiensis ", MSc Thesis, Campinas
State University, Brasil
FREIMAN, V.B. & CHUPIN, A.A. (1973) "Aspects of the continuous
cultivation of spore-forming microbes from the group
Bacillus thuringiensis ", Advances in Microbial Engineering
Part I, Biotechnology and Bioengineering Symposium,number 4,
pages 259-265
MORAES,I.0. (1973) "Obtenção de inseticidas bacterianos por feí
mentação submersa", MSc Thesis, Campinas State University ,
Brasil
.255.
EFFECT OF AVERMECTIN IN THE COHTROL OF PhyllocoptAuta oteÁvotã (ASHHEAD.1879) (ACARI-ERIOPHYIDAE) AND BUtvifXllpuA photnicii (CE1JSKES. 1939) (ACARl-TENUIPALPIDAE) ON CITRUS
0 . NAKANO ; C. OMOTO; M.J.FORNAZIER
Department of Entomology
Universidade de São Paulo - ESALQ
INTRODUCTION
Within the many pes t s of c i t r u s , two of them can be h ighl ighted
because of the frequency and damage that they cause , i . e . c i t r u s rust mite
- PhyttocopViuta oleÃvoia. and leprosis mite - &ie.vipalpui phoinicii. Even
though both mite species cause russeting or irreversible lesions on leaves
and frui ts , the leprosis mite damage is wors.e because of the premature
drops of leaves and fruits and kill ing of branches; when attacked fruits
are harvested on time, they have an unpleasant taste being rejected by the
consumer.
With the discovery of a new acaricide called Avermectin, a
natural lactone obtained from the fungus, kcAinomyczò aveAiwcXÍJLLb, new
perspectives are available for the control of both mites. This i s due to
the prpven efficacy of Avermectin in the laboratory and ifs low toxicity
considering the quantity utilized.
With the aim to investigate the effect of this product on both
mite species, under field conditions, two trials were carried out at the
locality of Piracicaba (SP), util izing the Avermection;in the formulations
0.36? S.L. and 1.87. E.C. at different rates and with/without mineral spray
o i l , in comparison to the officially recommended standard products.
MATERIALS AND METHODS
The f i r s t t r i a l cons i s ted of 6 treatments with four r e p l i c a t e s .
The experimental plot had 5 p l a n t s , with a f o l i a r area of 35 m' each. The
treatments and rates per 10,000 m1 of f o l i a r area are described below:
. 2 5 6 .
1. Avermectin 0.36Z S.L. • Triona B at 1,400 a l • 1,000 ml
2. Avermecrin 1.80Z E.C. + Triona B at 280 ml + 1,000 ml
3. Avermectin 1.80Z E.C. at 280 ml
4.'Averaectin 1.80Z E.C. at 560 ml
5. Acardifon 22Z E.C., Dicofol 16Z + Tetradifon 6Z at 1,000 ml
6. Untreated control
The treatments were sprayed on 1/14/83 with a motorized knapsack
sprayer "HATSUTA". The volume of spray was 1.4 1/plant. Because of low
mite infestations, only two evaluations were carried out. The first
evaluation occurred after 60 days and consisted of taking 40 leaves per
experimental plot,'located between the third and fourth nodes considering
the tip of the branch. The leaves were cleaned in the laboratory by a
mite brushing machine. The mites were collected on appropriate dishes and
the countings were done under a microscope of 15 x magnification. The
second evaluation occurred after 123 days by sampling of fruits. The fruit
analysis was done using a ring of 1 m in diameter and placing it over the
4 sides of a tree. The total number of fruits wirh or without mite damage
fallen within the area of the plastic ring were counted. The porcentagens
of efficacies were calculated by the Abbott's formula and the results aro
found in Tables I and II.
The second trial consisted of 7 ticotmentj with 4 replicates.
The experimental plot had 2 plants, with an approximate foliar area of IS
m2 each. The treatments and rates per 10,000 m2 of foliar area were as
follows:
1. Avermectin 0.36Z S.L. 4 Triona B at 1,400 ml + 3,300 ml
2. Avermectin 1.80Z E.C. + Triona B at 280 ml • 3,300 ml
3. Avermectin 1.80Z E.C. at 280 ml
4. Avermectin 1,80% E.C. at 560 ml
5. Dicofol 18.5Z E.C. at 2,660 ml
6. Bromopropylate 50Z E.C. at 1,000 ml
7. Untreated control
The treatments were sprayed on 3/2/83 with a manual knapsack
sprayer "JAC" V . The volume of spray was 2 I/tree. Two evaluations for
each site species were carried out by counting the mite poprlations
present. In the casa cf citrus rust mites, 20 leaves were collected after
.257.
26 days per experimental plot, following the same procedure described for
Trial N9 1. The other evaluation carried out after 87 days of the
application allowed the percentages of fruit damage to be determined by
collecting 20 fruits per experimental plot. The data concerning these two
evaluations are found in Tables III and IV. For the leprosis mite, the
number of lesions found on 20 fruits per experimental plot were counted.
These fruits were randomly picked after 26 days after rhe application. In
another evaluation the number of leprosis mites present on fruits were -
counted 48 days after the application. The data related to these two
evaluations are summarized in Tables V and VI.
RESULTS AND DISCUSSIONS
Table I. Number of citrus rust mites found on leaves per experimental plot,
total number of mites, and Z of efficacy (Z E), 60 days after the
application. Piracicaba, 3/S/83..
Treat
1
2
3
4
5
6
A
24
0
24
0
36
312
Replicates
B
72
48
12
0
60
540
C
0
48
0
0
24
216
D
36
12
48
12
96
888
Total
132
108
84
12
216
1956
Z E
93,25
94,47
95,70
99,38
88,95
.258.
Table II. Percentages of fruit russeting by citrus rust mites per ex-
perimental plot, total damage, mean damage, and Z of efficacy
(X E), 123 days after the application. Piracicaba, 3/17/3.
Treat
1
2
3
4
5
6
Table
Treat
1
2
3
4
5
6
7
III.
A
12
36
0
0
Q
0
12
A
0
0
0
0
25
50
Replicates
B
10
0
0
0
10
75
C
0
0
0
0
0
25
D
10
10
0
0
10
50
Total
20
10
0
0
45
200
Mean
5.
2.
0.
0.
11.
50,
0
.5
.0
.0
.25
.0
Z E
90.0
95.0
100.0
100.0
77.5
Number of citrus rust mites on leaves per experimental plot,
per treatment, and % of
application.
1
B
60
0
12
12
0
12
192
. Piracicaba
Replicates
C
12
12
24
0
0
12
0
eff icacy
, 3/28/83
D
0
0
36
12
12
24
156
« E),
•
26 days after
Total
84
48
72
24
12
48
360
the
Z E
76.66
86.67
80,0
93.34
96.67
86.67
—_..
.259.
Table IV. Number of fruits attacked by citrus rust mites per experimental
plot, total per treatment, and % of efficacy (% E), 87 days
after the application. Piracicaba, 5/21/83
Treat.
1
2
3
O
O
O
O
1
O
O
Replicates
C
0
0
0
0
0
0
2
D
0
0
3
0
0
0
0
Total
0
0
3
0
1
0
2
% E
Table V. Number of lesions produced by leprosis mites on fruits per ex-
perimental plot, totla per treatment, and 7, of efficacy % E) ,
26 days after the application. Piracicaba, 3/28/83.
Treat
1
2
3
4
5
6
7
105
204
151
33
99
49
85
15
27
13
8
84
31
29
Replicates
C
81
86
12
19
6
183
29
D
1
24
25
136
19
4
18
Total
202
341
201
196
208
267
161
% E
.260.
Table VI. Number of leprosis mites found on fruits per experimental plot,
total per treatment, and % of efficacy (% E), 48 days after the
application. Piracicaba, 4/22/83.
Treat
1
2
3
4
5
6
7
A
0
0
0
0
0
0
0
B
0
0
0
0
0
1
0
Replicates
C
0
0
0
0
0
0
5
D
0
0
0
0
0
0
2
Total
0
0
0
0
0
1
7
% E
100.0
100.0
100.0
100.0
100.0
85.71
Table VII. Number of predatory mites found on fruits per experimental plot,
total per treatment, 48 days after the application. Piracicaba,
4/22/83.
Treat.
1
2
3
4
5
6
7
A
2
3
0
2
1
0
8
Replicates
B
10
0
7
5
2
31
3
C
2
7
3
5
6
12
4
D
2
5
40
1
0
0
3
Total
16
15
50
13
9
43
18
In trie first trial, Table I clearly shows the efficacy of
Avermectin by protecting the leaves against citrus rust mites for 60 days
after the application. Both formulations of Avermectin at the minimum
.261.
rate of 5 g active ingredient/10.000 m2 of foliar area, with and without
mineral spray oil were efficaceous. The standard product used, Acardifon,
was also efficaceous, but with a greater population of mites. After 123
days from the application, the evidence of efficacy was still present
because Avermectin offered an optimum fruit production (Table II). More-
over, the standard product was considerably affected by mites, including
the untreated control with near 50Z of fruits with russeting damage.
In the second trial, Table III also proves the results obtained
in the first trial related to citrus rust mites, because good control was
obtained after 26 days. By comparison of Tables I and III, the effect of
Avermectin seems to be slow but steadily increasing and reaching the best
performance at 60 days. On the other hand. Table IV did not allow to
conclude anything related to the efficacy of the products because the
mites disappeared including the untreated control plots, with the end
results of little fruit damage.
With respect to the leprosis mites, Table V cannot be used as
an indicator of control because the existent lesions could have been
produced before the sprays. However, this table shows the evidence of
mite presences, an important factor in the obtention of the results. The
lesion» found in the treatments show a certain uniformity of damage.
The efficacies obtained in the different treatments, Table VI,
where most of the leprosis mites were found almost exclusively in the
untreated control plots, show the good performance of Avermectin at
different rates and formulations, as well as the standard products Dicofol
and Bromorropylate, 48 days after the application.
With respect to predatory mites, Phytoitíu&UÂ spp. and other
species, Table VII Shows some negative effect produced by Dicofol 18.5Z
at 2.66 1/ha and Avermectin 0.56 1/10.000 m* of foliar area. The addition
of mineral oil also could have affected somehow the predatory mites.
CONCLUSIONS
Based on the results obtained, it can be concluded that:
1. Avermectin in both formulations, 0.36% S.L. and •1.8/S.E.C. is highly
efficaceous in the control of mites, citrus rust mites and leprosis
.262.
mites at the rate of 5 crams of active ingredient per 10.000 m2 of
foliar area.
2. The rate here defined protects the developing fruits for near 120 days
in the case of citrus rust mites and 48 days in the case of leprosis
mites as a preventive treatment.
3. There was no elimination of predatory mites where Avermectin was
applied at the rate of 5 grams of active ingredient per 10.000 m2 of
foliar area.
.263.
REFERENCES
ALMEIDA, S.L.; CR.CORTE; A.A.MORAIS; L.CS.GALHARDO; T.J.FEKETE & F.A.M.
MARICONI, 1981 - Defensivos químicos e o fungo H-íjL&utitZa thomp&onü.
(Fisher, 1950) pulverizados contra PhylZocoptAuta oleÁvofia (Ashm.,
1879) (Acaros da fa lsa ferrugem dos c i t r o s ) . 0 Solo , Piracicaba, 7 j
(2):11-17.
BETOLOTTI, S.B.; S.DODO; CM.OLIVETTI; O.NAKANO, 1976 - Ensaio visando o
controle do ácaro da leprose - S*eiM.palpu& photnicii (Geiyskes, 1939)
(Acari-Tenuipalpidae). 0 Solo 68(1) :47-51 .
BLEICHER, E . ; F.S.PÜLS; N.L. DOMIC IANO; J.F.FRANCO; CR.KIRYU; J.B.MIRANDA
F9 & F.A.M.MARICONI, 1975 - Combate ao ácaro PhyttocopViuta olzivoia
(Ashm., 1879) causador da "mulata" das laranjas . An. Soe. Ent. Bras i l ,
Jaboticabal, 4O):98-103 .
FEKETE, T.J . ; A.A.MORAIS; R.L.SOARES; S.L.ALMEIDA; CR.CORTE; L.CS.GA-
LHARDO; H.B.ARRUDA & F.A.M.MARICONI, 1983 - Ensaio de combate ao ácaro
PhyltocopÜuUa oteÀVOta. (Aíhm., 1879) em laranje iras , em pulverização.
0 Solo , Piracicaba, 25(2):51-55.
IDACAW\, T . ; N.T.MURAI: W.T.SANADA & F.A.M.MARICONI, 1974 - Ensaio de com-
bate ao "ácaro da falsa ferrugem" PhyCLocopÜuUa olelvona (Ashm., 1879)
em c i t r o s . 0 Solo, Piracicaba, 66(1):14-17.
MARCONATO, J.R.; S.TAVARES; H.C.BRUNELLI JR.; R.FAGAN; J.C.OLIVEIRA F9; J.
C.CARVALHO & F.A.M.MARICONI, 1980 - Cotbate químico ao ácaro da falsa
ferrugem - VhytlocopVluta oltivona (Ashm., 1879). O Solo, Piracicaba,
7 2 ( 0 : 5 3 - 5 6 .
MARICONI, F.A.M.; H.C.BRUNELLI JR.; R.FAGAN; J.R.MARCONATO; S.TAVARES; J.
C.CARVALHO; J.C.OLIVEIRA F9 & C.L.SOUZA JR., 1979 - Inibidores de for-
mação de quit ina, inset ic idas e acaríerdas pulverizados contra o ácaro
Vhyttocopttoita. olÚVOla (Ashm., 1879). 0 Solo, Piracicaba, 7_K2):23-
28.
MURAI, N.T.; T.IDAGAWA & F.A.M.MARICONI, 1973 - Pulverização e a l t o volu-
me contra o ãcaro da falsa ferrugem dos c i t r o s - PhyfjtocoptAuta oleÁvo^
ia (Ashm., 1879). 0 Solo, Piracicaba, 6 5 ( 0 : 2 7 - 2 9 .
NAKANO, 0 . ; L.A.SANTOS; H.SUGUINO & J.M.M.ARR DA, 1977 - Controle químico
experimental visando o ácara da fa lsa ferrugem - PhyllccoptAiLta ottÁvo_
na (Ashm., 1879) em c i t r o s . Livulg. Agronom., S .P . , 42.: 16-20.
ROSSETTI, V. & A.A.SALIBE, 1959 - Pxperíêncía sobre o controle da leprose
. 2 6 4 .
IArq. do Inst . Bio lógico , 26:119-130
SUPLICY FILHO, N. ; A.F.CINTRA; I.MYAZAKI; D.A.OLIVEIRA & J.TEOFILO SOBR.,
1977 - Experimentos sobre o uso de praguicidas contra o âcaro da ferru
gem dos c i t ros - Phy&Locopttiuta oltÂVona (Ashm., 1879). O Biológico ,
São Paulo, «3(7-8):151-156.
, 2 6 5 .
BIOENGINEERING
Coordinator
Dr. KENJI
Heart Institute, University of S.Paulo
Clinical Hospital
São Paulo
DEVELOPMENT OF EMISSION COMPUTED TOMOGPV *. IN JAPAN
E. TANAKA
National Institute of Radiolr . >i Sciences
ABSTRACT
Single photon emission computed tomography(SPECT) using
gamma-camera rotating systems or ring-deteotor type SPECT
devices is gradually spreading in Japan as an important tool
in diagnostic nuclear medicine. The quality of the SPECT
images has been improved, but the quantitativeness is still
unsatisfactory mainly due to inadequate compensation for
photon attenuation in patients. We have developed new
image reconstruction algorithms based on filtered back-
projection, which provide fairly quantitative images with
slight modifications of the existing algorithms.
Positron emission computed tomography(PCT) has advan-
tages of the better quantitativeness and wider availability
of radio-pharmaceuticals. At present, eight institutions
are working on PCT in Japan. We have developed two PCT
devices, one is for head and the other for wholebody. A
high resolution PCT device for animal study is now under
construction. The spatial resolution is expected to be 3-4
mm in full-width at haIf-maximum.
.266.
INTRODUCTION
Nuclear medicine has become a successful routine method for
the diagnosis of disease in important organs such as the thyroid,
skelton, lung, heart, liver, spleen and kidney. The Anger
scintillation camera (gamma-camera) is most widely used for two-
dimensional imaging of radionuclide in patients. At present,
about 1100 gamma-cameras are working in Japan.
Great efforts have been paid for realizing three-dimensional
imaging since Kuhl and Edwards first described the transaxial
tomography in 1963[1], in which a computer was not used yet to
reconstruct images. Arrival of X-ray computed tomography
brought about a great advance in the image reconstruction
algorithms, and the fundamental concept of the recent single
photon emission computed tomography (SPECT) has been established.
In addition, SPECT systems using rotating gamma-camera have been
commercialized, and this technique is becoming popular as an
important tool in the diagnostic nuclear medicine. At the end
of 1963, about 145 rotating camera systems are installed and
being used clinically in Japan.
Special purpose SPECT devices with multi-detectors arranged
on a circular ring have also been developed, which provide higher
sensitivity and resolution 'Shimadzu Co. Headtome II)[2], The
device has a special rotating collimator inside the detector ring.
The collimator consists of hundreds of tungsten strips which are
circularly arranged in equal spaces but the angle of each strip
to the radial direction gradually varies in such a way that a
detector sweeps the entire field of view by means of the colli-
mator rotation.
.267.
However, the quanta, tati veness of the SPECT images is still
unsatisfactory. The main reason for this is the difficulty of
adequate compensation for the attenuation of gamma-rays in the
object Although various correction methods have been reported
including iterative correction procedures[3], convolution back-
projection methods with simple attenuation correction are widely
used because of their simplicity assuming that tissue attenuation
is uniform within a known body contour. The one is the "pre-
correction method" in which the correction is applied to pro-
jection data before backprojection[4], and the other is the
"post-correction method" in which the correction is applied to
reconstructed images[5]. However, these simple methods are not
accurate enough for relatively large objects.
Tretiak reported an analytical method "inversion of attenu-
ated Radon transform"[6], but the method is not practical because
it yields large statistical noise in the images. To overcome
this difficulty, we have developed two new methods of attenuation
correction. The outline of these methods will be described in
the next section.
RECONSTRUCTION ALGORITHMS WITH ATTENUATION CORRECTION FOR SPECT
The methods are basically a filtered backprojection with
some modifications. The first method, "weighted backprojection
(WBP) method", consists of three steps: normalization of observed
projections, modified convolution, and weighted backprojection[7,
8]. In the first step, a set of normalized projections, p (x),
is generated from observed projections, p(x) by the equation:
Pn (x) = p(x)exp(wyb) (1)
where (x,y) is a rotating coordinate system taken at each view
angle around a fixed coordinate origin, yb the y-coordinate of
the object boundary and y the attenuation coefficient of gamma-
rays. The modified convolution is expressed by
Pf(x) = [pn(x)F(x) * g(x)]/F(x) (2)
where g(x) is a convolution filter, F(x) is a correction function
defined later and the asterisk denotes convolution. Finally,
the filtered projections, p.(x), are backprojected on to an image
plane with a weighting function, W(y):
I = [ Pf(x)W(y) (3)e
W(y) = exp(kyy)/cosh[(l+k)yy] (4)
where I is the image density at a point (x,y) and k (0^1) is an
arbitrary constant named "reconstruction index".
The relative contributions of two opposing projections can
be controlled by the reconstruction index, k, in such a way that
the statistical noise in the image is low. The method enables
to form an image with larger weights on the front views than on
the rear views when k > 0. This results in the improvement in
signal-to-noise ratio in off-center area compaired with the
conventional averaging method of two opposing projections.
The second method is a simplified version of the WBP method
with k»0. In this algorithm, the image density, I, is expressed
by
I - H-MwR) I [pB(x)F(x) • g(x)] (5)
wheree n
H(pR) • (1/w) F(i)Rcos8)cosh(pRsin8) de (6)Jo
H(pR) i s a function of radial distance, R, from the coordinate
.209.
I origin. In this method, an image is formed by a constant-weight
backprojection followed by a multiplicative correction with an
< object-independent function, H(uR), which can be pre-stored in a
: computer memory as a correction matrix. This method is named
"radial post-correction (RPC) method". The image forming per-
formance is similar to that of the WBP method with k=0.
In both the methods, WBP and RPC, the correction function,
P(x), and the filter function, g(x), play an important role in
reducing image density distortion for extended sources. We found
empirically that the following fuctions provide satisfactory
results for objects having a diameter up to 35 cm for Tc-99m (u=
0.15 cm" 1).
! F(x) « texpfCi (nx)2} + C 2]/U + C2) (7)
Í g(x) - g o(x)U + C 3 ( P X )2 ] / [ 1 + Cf(wx)M (8)
where go (x) is the Shepp-Logan filter and Ci^Ci, are constants.
I The constants were optimized by a computer iteration program
: (see Table 1).
Statistical noise of a reconstructed image depends on the
position of the coordinate origin and the value of k. The best
signal-to-noise ratio is obtained when the origin is at the
noisiest point in the image, and the peripheral noise is reduced
I by using positive k-value(0.25^1). Since the RPC method yields
. an image similar to that of WBP method with k=0 in a shorter com-
l putation time, the RPC method may be preferable when the signa1-
I to-noise ratio in the peripheral area is not very important.
Some results of simulation studies are shown in Figs. 1*3.
These two methods improve the quantitativeness of SPECT with only
slight modifications of the existing methods.
.270.
POSITRON EMISSION COMPUTED TOMOGRAPHY
Another field of three-dimensional imaging is positron
computed tomography (PCT). This method is based on coincidence
detection of pair photons produced by annihilation of positrons,
and it has inherent advantages over the SPECT in the good
quantitativeness and the wide availability of useful radio-,
pharmaceuticals. The radionuclides used are positron emitters
such as C-ll, N-13, 0-15, and F-18 which are produced by a small
cyclotron, or Ga-68 and Rb-82 which are obtained by "milking"
from their parent nuclides, Ge-68 and Sr-82, respectively.
With increasing recognition of medical usefulness of the PCT
technique, the instrumentation has progressed very rapidly in
this s A yal years. In Japan, the PCT devices and the baby
cycleii %i are commercially available, and seven institutions are
work i i ith the PCT and one has a plan to install a PCT system
in tftr rear future.
•*jor requirements to the PCT systems are high detection
sen* tivity, high spatial resolution, good image quality and
qua,)' itativeness and high speed data acquisition capability.
Variias detector configurations have been investigated, but the
circular ring system using bismuth germanate (BGO) scintillation
detetVors is the best choice for high resolution applications.
Me have developed two PCT devices along this line. The first
device, POSITOLOGICA I, was completed in 1980(9,10]. The device
is a single slice machine for brain strdy. It has 64 BGO detec-
tors on a circular ring having a diameter of 44 cm. To obtain an
adequate linear sampling, th.e detectors are arranged on a circle
at non-uniform spacings and the whole detector ring is rotated
.271.
continuously at a speed of one rotation per second. The detector
position on the circle was determined by a computer so as to
yield a uniform sampling density(ll). The spatial resolution is
about 7-9 mm in full-width at half-maximum (FWHM).
The second device, POSITOLOGICA II, is a multi-slice whole-
body machine, which was completed in 1983 under the sponsorship
of the Ministry of International Trade and Industry, Japan[12,13].
The device has three detector rings (85 cm in diameter), and it
c*n provides 5 slice images (3 in-layer images and 2 cross-layer
images) simultaneously. The slice thickness is 2.4 cm in full
width.
Each detector ring is composed of 40 units of "quad-BGO
detectors", each of which consists of four BGO crystals (15x24x24
mm3) coupled to two photomultiplier tubes. The inner two crystal
are optically coupled each other while the outer crystals are
isolated from the inners, in such a way that the crystal detect-
ing a photon is easily identified from relative proportion of
pulse amplitudes of the two photomultiplier tubes. The continu-
ous rotation scan with non-uniform detector arrangement is also
used. The spatial resolution is 9-11 mm FWHM and the detection
sensitivity is about 28 Tccps/pCi/ml for in-layer images and 38
Jccps/yCi/ml for cross-layer images for a uniform cylinder water
phantom of 20 cm in diameter.
At present, a high resolution PCT device is under develop-
ment for animal study. The target resolution is 2-3 mm FWHM.
A major obstacle to attain such a high resolution lies in the
fact that a small size photomultiplier tube with good timing
characteristics is not commercially available. To overcome
.272.
this difficulty, we have developed new detector units under the
cooperation of Hamamatsu Photonics Corp. This unit con sits of
two BGO crystals coupled to a snail rectangular photomultiplier
tube[14,15]. The crystal size ii 4 an wide, 10 mm high and 20 mm
long. The photomultiplier tube has a 13 mm square photocathode
and has a small grid electrode inside the tube near the photo-
cathode (see Pig. 4). The grid controls collection of photoelec-
trons released from a half area of the photocathode. This allows
a photomultiplier tube to couple two crystals and to identify the
fired one of them.
The animal PCT device under construction has a single
detector ring composed of 64 detector units (128 BGO crystals)
as shown in Fig. 5. The crystals are arranged on a circular
ring of 265 mm in diameter at unequal spacings and the ring
is rotated 360 degrees repeatedly clockwise and counterclockwise
at an angular velocity of 360 degrees per minute, resulting in
uniform sampling and high detector redundancy. A wedge-shaped
slice collimator is adopted to increase sensitivity lowered
by offering an effective slice thickness as thin as 3 mm. The
collimetor has a gap 5 mm at entrance of 135 mm in diameter and
12 mm at exit. The system sensitivity is estimated to be about
1.2 kcps/itCi/ml for a uniform source in a 10 cm diameter cylinder
water phantom, and the 'system intrinsic resolution is less than
3 mm in n m except the blurring effect due to the finite range
of positrons in objects. The device is expected to be useful
in developing and testing new radio-pharmaceuticals labelled with
positron emitters.
.273.
REFERENCES
1. Kuhl DE, Edwards RQ: Image separation radiolsotope scanning. Radiology80:653-61, 1963
2. Hlrose Y, Ikeda Y, et al: A hybrid emission CT - Beadtoae II: IEEE TrasHud Sei NS-29: 520-3, 1982
3. Budinger TP, Gullberg GT: Transverse section reconstruction of gamma-ray emitting radionuclides in patients. In: Reconstruction Tomographyin Diagnostic Radiology and Nuclear Medicine. Ed. by MM Ter-Pogossian,et al., Baltimore, university Park Press, 1977, pp 315-42
4. Sorenson JA: Quantitative measurement of radioactivity in vivo bywhole-body counting. In:Instrumentation in Nuclear Medicine, Vol.2. Ed.by GJ Bine and JA Sorenson, New York, Academic Press, 1974, pp 311-48
5. Chang LT: A method for attenuation correction in radlonudlde computedtomography. IEEE Trans Nucl Sci NS-25:638-43, 1978
6. Tretiak 0J, Delaney P: The exponential convolution algorithm foremission computed axial tomography. In: Review of Information Process-ing in Medical Imaging. Ed. by AB Brill and RR Price, Oak RidgeNational Laboratory Report ORNL/BCTIC-2, 1978, pp 266-78
7. Tanaka E: Quantitative image reconstruction with weighted backproject-ion for single photon emission computed tomography. J Comput AssistTomogr 7:692-700, 1983
8. Tanaka E and Toyama H: A generalized weighted backprojection algorithmfor single photon emission computed tomography. Proc. VIII-th Intern.Conf. on Inform. Processing in Med. Imaging, Brussels, 1983
9. Nohara N, Tanaka E, et al: Posltologica: A positron ECT device with acontinuously rotating detector ring. IEEE Trans Nucl Sci NS-27:1128-36,1980
10. Tanaka E, Nohara N, et al: Rotational positron computed tomographs.Medical Radionucllde Imaging 1980, Vol 1, IAEA: pp 165-72, 1981
11. Tanaka E, Nohara N, et al: "Positology"-The search for suitabledetector arrangements for a positron ECT with continuous rotation.IEEE Trans Nucl Sci NS-26:2728-31, 1979
12. Tanaka E, Nohara N, et al: Analytical study of the performance of amultilayer positron computed tomography scanner. J Comput Assist Tomogr6:350-64, 1982
13. Takiai K, Tanaka E, et al: Performance study of whole-body, multi-slice positron computed tomograph. IEEE Trans Nucl Sci NS-30:734-8,1983
14. Murayama H, Tanaka E, et al: Twln-BGO detectors for high resolutionpositron emission tomography. Nucl Instr Meth 221:633-40, 1984
15. Yamashita Y, Uchida H, et al: Recent developments in detectors forhigh spatial resolution positron CT. IEEE Trans Nucl Sci NS-31:424-8,1984
.274.
- 1
Table 1. Optimized constants in Equations (7) and (8)
IA
#B
k
0.000.250.501.00
0.000.250.501.00
•Ci C2 C3 Ci,
0.34 0.16 0.21 0.190.38 -0.05 0.11 0.210.40 -0.21 0.01 0.200.43 -0.34 -0.12 0.16
0.28 0.13 0.27 0.200.30 -0.11 0.19 0.220.33 -0.23 0.09 0.210.38 -0.39 -0.09 0.16
#A is for wRm<2.5 and IB ft for uR11<3.0, where R,is the largest distance of source distribution fromthe coordinate origin.
Dimmttir(cm) 20x1S
WBPlk<0)
25 «20 30* 24 35x28
RPC
FIGURE 1. Images of mathematical phantoms reconstructed bythe WBP method and RPC method. Attenuation coefficientis 0.15 cm"1 (Tc-99m).
.275.
Oiamtltr (cm) 20 «1»
RPC
25x20 30x24 35x28
Pf t -Cerrtctien
Post-Corrtctwn
FIGURE 2. Images of mathematical phantoms reconstructed by
the RPC method, the pre-corection method with arithmetic
mean[4], and post-correction method[5]. Attenuation
coefficient is 0.15 cm'1.
Thmatom
FIGURE 3. Comparison ot images reconstructed from projections
having statistical noise. The phantom diameter is 30 cm.
Attenuation coefficiet is 0.15 cm*1. Total number of
counts is 10 6.
(A) RPC method (The coordinate origin is 4 cm off-centered)
(B) Pre-correction method with arithmetic mean[4]
(C) Post-correction method[5]
(D) Inversion of attenuated Radon transform[6]
.276.
r
B60 SaturnGrid Photomuttiptiw
tube
U
FIGURE 4. A twin-BGO detector unit for high resolutionpositron computed tomography[14,15]
12b bCO 4 i r y . H l »(4x111x21) mm)
FIGURE 5. Detector configuration of the high resolutionpositron computed tomograph for animal study.
.277.
ELECTRICAL STIMULATION IN EXPERIMENTAL SPEUDARTHROSIS* **
A.B. Rodriguez Fuentes and S. Mascarenhas
The use of electrical stimulation to activate bone foraatlonin fractures and pseudarthrosis healing is part of a recent, lineof research. In the present study we investigated the effect ofelectric stimulation with direct constant current on inducedpseudarthrosis in the radius of adult dogs. Two types of- surgicaltechniques for inducing pseuâarthrosis in the lower third of theradius of adult dogs are described. Out of a total of 57 animals,41 were electrically stimulated and 16 were used as controls.Statistically significant data showed endochondral ossificationand bone union by electric stimulation. On the basis of ourresults, we conclude that » simple linear osteotomy in thedistal third off the adult canine radius induced pseudarthrosisin 71% of the animals investigated; 2) resection osteotomy(cylindric section 3 mm wide) caused pseudarthrosis in 95% ofthe animals; 3) osteogenesis can be stimulated by electriccurrents (continuous 4uA current) and by placing the negativeelectrode on the lesion site.
* -' Prof.Assist.Doutor da FMRP-USP.** - Prof.Titular do instituto de Física e Química de São
Carlos - USP.
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SPACESCIENCE AND TECNOLOGY
Coordinator
Dr. NELSON DE JESUS PARADA
National Institute for Space
Research, São José dos Campos, SP
A C K N O W L E D G E M E N T
The Organizing Ccmnittee of the Fourth Japan-Brazil Symposiumon Science and Technology expresses its grateful recognitionto:
- The COMISSÃO NACIONAL DE ENERGIA NUCLEAR for its veryvaluable collaboration
- Academia Brasileira de Ciências, Escola Superior deAgricultura "Luiz de Queiroz", Fundação Oswaldo Cruz,Faculdade de Medicina de Botucatu, Instituto de Físi-ca da USP. Instituto de Macromoléculas da UFRJ, Instituto Nacional de Pesquisas Espaciais, Instituto dePesquisas Energéticas e Nucleares, and Instituto dePesquisas Tecnológicas for permitting the use of theirAuditorium
- CESP-CPFL-ELETOOPAULO, Maquinas Agrícolas JACTO, Ban-co America do Sul, Câmara de Comércio e Industria Ja-ponesa do Brasil, Sociedade Brasileira de Cultura Japonesa, Aliança Cultural Brasil-Japão, BeneficiênciaNipo Brasileira de São Paulo Nikkey Palace Hotel. andFINEP for financial aids.
A special thanks are due to the office staff of the Academia deCiências do Estado de São Paulo for its extraordinary dedica -tion that enabled smooth running of the Symposium.
