Plant Research Institute and Entomology Research Institute, Canada Department of Agriculture,Ottawa, Canada
Accepted November 14, 1964
Tsodiametric particles 28 my in size were observed in thin sections of honey beelarvae showing sacbrood disease symptoms . Particles were not occluded in polyhedraor granules . They were either randomly distributed or arranged in crystalline arrays .Some particle aggregates occurred in vesicles, while others were free within the cyto-plasm. Such particles were not observed in sections from healthy bee larvae .
INTRODUCTION
election microscope . The observatiorns re-corded are presented in this paper .Saebrood, a disease of honey bee larvae
(Apis mettifera Linnaeus) is caused by avirus. White (1917) was the first to suggestthe viral nature of sacbrood. Steinhaus(1949) and Break et at . (1963) observedspherical particles from water extracts ofinfected larvae with particle sizes of 60 muand 30 Into, respectively . However, it wasBailey et at . (1964) who correlated an iso-diarnetric particle measuring 28 mg to theinfectivity of preparations from sacbroodlarvae, thus presenting unequivocal evidencefor the establishment of sacbrood as a virusdisease .
Larvae infected with sacbrood succumbin a few days, and the drastic changes whichoccur both in external and internal morphol-ogy of diseased larvae are suggestive ofrapid pathologic breakdown in the host cellsof diseased larvae. Nothing is known aboutthe course of infection and of the method ofviral replication . Moreover, there is no evi-dence that sacbrood virus particles are inpolyhedra or granules, as are many of theinsect viruses . Some information on thesequestions was obtained by studying the vi-rus in situ in infected larval tissue with the
MATERIALS AND METHODSNaturally infected larvae showing ad-
vanced symptoms of sacbrood were removedfrom brood cells and injected with a chrome-osmic acid solution (Galton 1955) . Theywere held overnight at 5 ° and minced intosmall pieces the following day . It was notpossible to identify organs from infectedlarvae due to the advanced disintegration ofinfected insects . Following fixation, the tis-sue was washed in distilled Hz0 and dehy-drated in a graded series of ethyl alcoholwith two changes in absolute alcohol . Aftertwo rinses in propylene oxide, they wereembedded in Epon resin according to Luft(1961) . Embedded material was polymerizedat 45 ° overnight, followed by 1 day at 63 °° .Sections were cut with a diamond knife us-ing a Porter-Blum microtome . Sections weremounted on Formvar-carbon grids, stainedfor 15-30 minutes in an aqueous solution ofuranyl acetate to enhance contrast, andrinsed in distilled H,0. They were examinedin a Philips model 100 electron microscopeor a Siemens Elutiskop I . Photographs weretaken on the latter instrument at 20,000 X
I Joint contribution from the Plant, Research or 40,000 X magnifications .Institute (Contribution no . 403) and the Entomol-
Healthy larvae of a similar size to dis-ogy Research Institute, Research Branch, Canada eased ones were prepared for sectioning con-Department of Agriculture, Ottawa .
currently for comparison,387
388
Fic. 1 . Crystalline inclusion of saebrood virus which appears to he enclosed by a membrane (M) .
OBSERVATIONS
Isodiametric particles 28 my in size werefound in infected tissue from diseased honeybee larvae, but not in tissue from healthylarvae. The particles were not found inpolyhedra or granules, although in somesections they were found in large vesicles
LEE AND FUILCALA
(Fig . 1) . In other sections, the particles werefound in a cytoplasmic matrix without asurrounding membrane or vesicle (Figs . 2and 3) .The arrangement of particles was either
random or packed in crystalline arrays, andin some inclusions both random packing and
SACBROOD VIRUS IN SITU 383
FIG . 2 . Large inclusion lying free in the cytoplasmic matrix . Arrow points to particles showing iso ,diametric, shape . Particles in highly compacted arrays form a crystal lattice with linear pattern andperiodic spacing of 30 mg (eirclc) .
390 LEE AND FURGALA
Fiu. 3 . Particles distributed randomly in cytoplasm .Frn. 4 . Inclusion showing particles in crystalline array with cross-hatched pattern, as well as parti-
cles randomly oriented .
SACBROOD VIRUS IN SITU
Fic. 5 . A few particles in crystalline formation with others randomly distributed .Fw . 6 . Inclusion with particles densely compacted but in random distribution .
391
392
LET AND FFItUALA
crystalline lattices were observed (Figs . 1-4) .The formation of a crystalline array doesnot appear to be dependent on the abun-dance of particles. In Fig. 5, only a fewparticles appear in an orderly fashion; whilein Fig. 6, the particles, although numerous,are packed in random fashion .
The external morphology of the individualparticles is evident in Fig . 2 . The hexagonaloutline is clearly shown in particles locatedin Itie central area of the micrograph . How-ever, fine structure of the particles is notdiscernible in any of the micrographs .
in Figs . 2 and 4, some particles are tightlycompacted in crystal lattices to give a linearpattern with a periodic spacing of 30 orµ(Fig. 2), or a cross-hatched pattern (Fig . 4) .The differences in pattern arc most likelythe result of different cutting planes in thetwo inclusions .
DISCUSSIONThe size and shape of the particles ob-
served in infected tissue font bee larvaeshowing sacbrood symptoms is in agreementwith the size and shape of the particles ofsacbrood virus isolated by Bailey et ad .(1964) .
The appearance of random packing andcrystalline lattices of virus particles withina single inclusion may he significant . Thephenomenon suggests that a random distri-bution of particles not only precedes crys-talline formation, but may also be a featureduring the breakup of a crystalline aggro-gale .
The absence of typical polyhedra or gran-ule inclusions in the tissue examined setssacbrood virus apart front the majority ofinsect viruses which have been studied . Al-though it was not possible to determine theorgans sectioned in this study, the finestructure of the infected tissue resembledthat of insect fat body, and the randomdispersal of groups of virus particles through-out the tissues examined was at least inpart located in the ground cytoplasm of thecells .
Tipula iridescent virus (TIV), a largeicosahedral particle (Williams and Smith,in Smith, 190:3) and sacbrood virus aresimilar in some respects : (1) Both virusescause rapid pathologic breakdown of theirhosts resulting in the release of fluid frominfected larvae which is a rich source ofvirus inoculum . (2) Neither virus is occludedin polyhedra or granules . (3) Sacbrood viruslike TIN' can be found in abundance wheninfected larvae are thin-sectioned and ex-amined in the electron microscope . (4) Bothviruses may be found in aggregates with orwithout a surrounding membrane or vesicle,and the particles of either virus are eitherarranged randomly or in crystalline arrays .These similarities are of interest, althoughfurther investigations have to be undertakenbefore the nature of sacbrood virus can beascertained .
ACKNOWLRUUttIENTS
One of us (P. E . L .) wishes to thank Dr . EricSmith, Mines Branch, Mines and Technical Sur-veys, Ottawa, for use of the 9iemens 1 .1minkop felectron microscope, and also Miss J . Ng Yelimfor assistance in the preparation of Forntvar-carbon substrates .
REFERENCES
BAILEY, L ., (:BAS, A. J . . and Woons, 12 . 1) .(1964) . Saehromcl virus of the larval honey bee(ipis mc1(ifera l .innaeus) . Virology 23, 42 -
Ba56K, J ., SYOBOUA, J., and KnA, .trc, O. (1903) .Electron microscopy investigation of sacbroodof the honey bee . .1 . Insect Pathul- . S, 385399 .
GALrIN, A . .! . (1955) . A chrome-osrniurn fixativefur electron inieroseopy . Ann t . keeor/ 121, 281 .
Lrx-r, J . Ii . (1961) . Improvements in epoxy resinembedding materials . J. Biophgs. Biochem .('21eal . 9, 409-414 .
Sem'ru, K. M . (1963) . The cytoplasmie virus dis-eases. In "Insect Pathology" (E . A . Sleinhans,ed .), Vol . 1, pp . 457-497 . Academic Press, NewYork .
SrE,NHAi's, E. A . (19x9) . Nomenclature and clas-sification of insectt viruses . Boctea-iod . Her . 13,20:3-223 .
Ww're, ( : . F. (1917) . Sachruod . 1' . S. belt,Igr .1301 .-431, 1-55 .
