8/10/2019 Rearing Mosquitoes
1/124
Pubiished III.
,UlERICAS MXQU I-0 COY I-ROL ,ISSOC I_ITIO~, 15-C.
8/10/2019 Rearing Mosquitoes
2/124
MaLlLLal
for
MOSQUITO REAKIKG AND EXPERIMENTAL TECHNIQUES
E~GESE J. GERBERC
Insect Control and Research. Inc.
IIII Sorth Roiling
Road, Baltimore, Jlcl. zrzz8
Bulletin 30. 3
Bulletin SO. 5
is a\.ailable from:
T. G.
R.\LLY,
Executive Secretnry, ;IMCX
P.O.
Box 278, S&m, California 93662
Price 53. j0
8/10/2019 Rearing Mosquitoes
3/124
CONTENTS
FOREWORD-KESUETH i_. KTISHT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , . . , .
v
_1CKSOTYLEDGAIESTS
. . . . . . . . . . . . . . . . . . . . . . . . . . .
Ti
ITTRODL7CTIOT . . . . . . . . . . . . . .
xll
I. THE ITSECT_ \R1-
...........................................................
I
Construction of room>.
.....................................................
1
Temperature and humldlt equipment. . . . . . .
2
Lighting ,,............................................................... 3
SeiuritJ- and safet>- medrure>.
. .
-1. Faslllt> requirement< .
I. Building . . . . . . . .
2. Room layout . . .
. .
.
B. Labor&tory
and rearing prcxedures.
.....
I. Eggs ...........................
2.
Larl-at and
pupae.
................
3. _klults .........................
5
. .
5
C. SIiscellaneow requireme nts an d recom menda tion\
5
II. SI.U?XET_1SCE OF MOSQ L-IT0 COLOTIES. . .
;
Equipm ent for handling adult>. . . . . . . . . . . .
I.
;Ispxators or wition deliies.
. . .
2. cage> . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.Adult food . . . . . . . . . , . I. .
.......................
_irtificial feeding techniques. . .
.......................
Induced rndtmp . . . . . . .
....................... . .
Colleiting and handling eggs .. . . . . . . . . . . . . . . .
1;
Larl al rearing ttihnia~ueh dncl tquipnmt
...............
Counting larvae
...............................
Rearing tra -s ..................................
Handling larl ae ...............................
Equipm ent for feecling 1arTde.
Lxval toocf ancl ieedm:
....... ............
_ .
c ..............................
Handling.
counting. separating and sexing pupa e.
........
.
. . .
. .
Sterile
culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 j
I II . PROC EDURES FOR L_~BOR_~TO RYL- RE-ARISG OF SPECIFIC MOSQ UTOkS. .
. .
. . . .
. . . .
. . . .
. .
. . .
. . . .
. . . .
.
.
. .
.......
-
....... _ij
....... ;_j
....... qj
.......
46
.......
ir
.......
45
.......
50
. .
. . *
. . .
. .
. . . . . . . .
.
.
. . .
. . .
. . .
. .
. . . . .
.
.
. .
P~rJi.Op/lOi.,7
. . . . . . . . . . . . . . . . -. . . +. e .. . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50
i
8/10/2019 Rearing Mosquitoes
4/124
Pnge
Et.etnupodites
. . . . . . . . . . . . . . . . . . . . . .
. , .
. * . ,
j2
dedes
. . . . . .
. .
. . .
a....,.....
. . . . . . . . . . . . . . . .
*... >3
Aiwigere~
. . . . . . . . 8 . . . . , . . . . .
.,...*.....
. .
. . . .
64
Hcienzngogm . ...,,..*.. . *. . . . . . .a
. . . .
65
Opifes . . . . .
. . . . . *
. ..I.......
. ..a .a a....
. . . .
66
Clllijetn . . . .
. . . . . .
a.. . . . . . . . .
,....*.....
* . . . . * .
68
Crrles
. . . . . . . .
. . . . . . . . . . .
. . .
.a. . . . .
. . . . I . .
69
Deirtocerite_i
. . . . . . . . . . . . . , . . . , . . . . . . . .
* . , .
78
APP END IX A. List of mosq uitoes and mosquito colonies maintained b>- laboratories, . . . . . . . . ig
.IPPEN DIX B. Tables of measu res and equivalents.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . , , . 85
APP END IX C. Relative humidity tables.
. . . . . . . . . . . . . . . . . . . . . , . . . . . . . , . . . . . . . . . . . . , .
89
REFE RENC ES . . . . . . . . . . . . . . . . ..~...~............................................ gI
ii
8/10/2019 Rearing Mosquitoes
5/124
LIST OF ILLUSTRATIONS
FIG. I.-Interior of insectary at V-alter R eed _k-m - Institute of Resea rch--US. _km y.
FIG. Z.-Interior or insectary at Insect Control k Rese arch . Inc.-R. Stacks. Baltimore
FIG. 3.--Mo rlan s
colony cage..-CDC.
.....................................
FIG. J.-Gerbergs collapsible cage-R. C. Ruehl. Jr.: Cornell C s E Co.
...........
FIG. 5.-Barraud cage-P. S. Barraud.
.......................................
FIG. 6.-Gillett cag e-J . D. Gillett
...........................................
FIG. 7.-Plastic cage-L7 .S. *Arm .............................................
FIG. 8.--hicCra -s escape-proof colon , cage -CDC .
..........................
FIG. 9.-Pollard cage -D. G. Pollard.
........................................
FIG. I o.-_knimal restrainers in
more Sun . . . . . . . . .
mass feeding of
. . . . . . . . . .
mosquitoes on
. . .
Sun
.
. . .
.
.
. .
chicks-R. Stacks.
. . . . . . . . . . . . .
Pnge
.Co\-er I
. . .
I
. .
8
. . .
9
. . .
IO
Balti-
. . . . . 12
FIG. II.--Apparatus for mem bran e feeding of mosqu itoes--U.S. ;irmy.
. . . . . . . . . . . . .
14
FIG. I z.--_\ppara tus for mem bran e feeding. detail--US. -Arm >-.
. . . . . . . . . . . . . . .
I 5
FIG. r3.-*kliquot container for mosquito larvae-R. Stacks. Baltimore Sun.
........
FIG. I _I.--\Vhite enam el tra s for rearing larvae-L.S. _Lrmy .
...................
FIG. IT.----SYhite en amel dishes for rearm g larv ae-K.S.D._L.
.................... .
. .
I9
. . 20
. . .
?I
FIG. 16.-Construc tion of gall-anized metal rearing tra --Morlan. . . . . . . . . . . . . . . . . . .
21
FIG. r;.-Mass rearing of msoqu itoes in
galvanized tray s-R. Stacks. Baltimore Sun. . . . . . 22
8/10/2019 Rearing Mosquitoes
6/124
All Metal Collapsible Cage
(See Fig. 4, Page 9, This Bulletin)
The Standard Cage in Major Laboratories
All aluminum construction with plastic ham mock
Metal construction eliminates mold and contamination
Hinged for easy collapsing and storage
Cages easily disassem bled for cleaning
Escap e-proof feeding ham moc k enables e xposu re of animal for blood feeding with-
out mosquito escapage
Sucrose solutions on cotton balls can be fed mosquitos through hammock ; sugar
does not com e in contact with metal parts.
Surgical stockinet sleeve provides easy access inside cage
Econom ical in cost - efficient in operation
Available in three
convenient sizes
Gl 12 x 12 x 12
G2 24 x 24 x 24
G3 177/8x 18~ 22-1116
For further information and prices write-
LOW-COST AUTOMATIC HUMlDlFlCATION
Whe ther for mosqu ito-rearing or experimen tal conditions, there
is a Standard Humidifier available to provide accura te, auto-
matic humidification for your needs . Famo us Standard units
are engineere d for long, trouble-free service an d easy installa-
tion, and are priced surprisingly low. All units are fully
guaranteed.
Wfite for 4-page
free brochure
a
Model 42L
Humidifier
State de tails of your
project for promp t
guaranteed
recom-
mendation
8/10/2019 Rearing Mosquitoes
7/124
8/10/2019 Rearing Mosquitoes
8/124
On behalf of the members of the American Mosquito Control Association and
of mosquito workers everywhere, I first thankfully add this fine publication to my
library of mosquito literature and secondly extend sincerest thanks to its autho r
for his devotion to its preparation.
KENNETH
L.
KNIGHT,
Head
Department of Entomology
N. C. State University
Raleigh, N. C. 27607;
Chairman, Editorial Board of
Mosquito News, Journal of the
American Mosquito Control Association
vi
8/10/2019 Rearing Mosquitoes
9/124
DEDICATION
This manu al is dedicated to Mrs. Helen Louise Trembley Durkee,
who devoted a large part of her time and effort to producing the
original B ulletin 3, entitled Mosquito Cu lture Techn iques and Experi-
mental Procedures. Her work has served for many years as a guide
to entom ologists and other w orkers in the field of culicidology.
ACKNOWLEDGMENTS
The preparation of the present manual was originally intended to be the work of a
committee. However, committees perform best when they can meet, discussand work
together. Because distance and individual research problems made effective commit-
tee work very difficult and subject to long delays, I decided to tackle the problem
alone, with whatever local assistancecould be obtained. I was fortunate in having
Capt. James W. Gentry, U.S.A. (Ret.), join our staff in 1967. He spent many eve-
nings gathering data. Dr. Ronald A. Ward, at the Walter Reed Army Institute of
Research, was extremely helpful in securing literature and locating mosquito rearing
laboratories. This, then is the working Committee to whom I offer my sincere thanks.
Dr. Ross H. Arnett, Jr. kindly reviewed this manuscript and used his grammati-
cally minded red pencil with justifiable abandon. My sincere thanks and appreciation
are offered-any errors remaining are mine, not his.
To the many other culicidologists that assisted n one way or another, I give my
grateful acknowledgment for help and advice.
EUGENE J. GERBERG
vii
8/10/2019 Rearing Mosquitoes
10/124
INTRODUCTION
The need for information on mosquito rearing has been intensified by the increased
interest in mo squito biology, control, chemoltherapy of mosquito borne diseases, and
in other investigations in which mosquitoes are used as experimental animals. Since
the publication of Helen Louise Trembleys Mosquito Culture Techniques and
Experimental Procedures
in 1955 , published literature in this field has increased like
salt marsh mosquitoes after a high tide.
The acquisition elf knowledge concerning proper con trol of mosquitoes and mos-
quito borne diseasesrequires that studies be made of the biology, p hysiology, anatomy,
genetics, taxonomy and ecology of the insect. Insecticide resistance, biological, chemi-
cal, and integrated control must be investigated.
Research on mosquito genetics
may have far-reaching consequences in the control of m osquitoes and m osquito-borne
diseases. Th e use of m osquitoes as screening agents for pesticides or chemothera-
peutic compounds requires large numbers of mosquitoes. All phases of mosquito
research usually use individual or quantity rearings of mosquitoes.
Mass rearing of
mosquitoes for control purposes may become, in the near future, as common as mass
production of pesticides.
The purpose of this man ual is to provide information on the rearing of mosquitoes.
A thorough search of the literature p rovided many useful references. The great
amou nt of literature made this no easy task. Undoubted ly, articles were overlooked,
but it is hoped that no serious omissions have occurred. An apology is offered to
those authors so neglected.
Author credit-lines omitted in the text are included in
the list of references at the end of the rearing description for the species.
For some
species, particularly those reared in the authors laboratory, the technique is described
in more detail. Often this sam e technique will apply for related species as well.
No method for rearing mo squitoes is guaranteed. Perhaps the most important
requirem ent for successful rearing o f mosquitoes is attention to detail. Mosquito
rearing to be successful requires attention 24 h ours a day, 7 days a week.
The basic
rules are: avoid over-crowding mosquitoes, and overfeeding larvae; a void pesticide
contamination, observe temperature and humidity requirements, standardize rearing
methods and avoid nonstandardized food.
Observance of these principles should
produce uniform animals.
The following guide to mosquito insectary construction and rearing practices is
presented step by step with no elaboration of theory. Much of this theory m ay be
yearned by reference to the literature cited.
.
I.111
8/10/2019 Rearing Mosquitoes
11/124
8/10/2019 Rearing Mosquitoes
12/124
the room.
Whenever possible use coved wall floor junctures and caulk and seal the
cracks and crevices n the walls.
The best insectariesare windowless with low ceilings
(not more than 8 also painted white or light colored. If possible, use recessed
lighting fixtures that are flush with the ceiling.
Smooth floors without a floor covering are recommended. A light colored epoxy
floor paint can be used to improve the appearance. The doors of the rearing area are
best constructed of metal and painted white or light colored. Weatherstripped door
frames and jambs are necessary for a tight fit. Overlapping marquisette curtains
hung on the inside of the door frame provide additional mosquito barriers (see sec-
tion on mosquito security).
Hot and cold running water and a sink with ample draining surfaces are required
in the rearing room. A water blending tank r installed above the sink facilitates
the filling of trays, separation of pupae and other tasks that require water to be a
certain temperature. Several water outlets around the room will avoid the clutter of
hoseson the floor.
Movable tables, benches, chairs, storage cabinets, etc., are preferable, since, in
general, permanently installed furniture prevents new arrangements to meet changing
conditions. Some laboratories have all furniture mounted on casters or wheels so
that it can be readily moved.
TEMPERATURE AND HUMIDITY EQUIPMENT
Temperature and humidity controls are probably the most important factors in the
successful earing of mosquitoes. The simplest method of providing regulated heat
and humidity is the use of a small electric light bulb and a wet towel draped over
the cage. Th
e most elaborate is a complex environmental chamber with programmed
electronic controls of temperature, humidity, and photoperiod.
The size of the
insectary will regulate the type of temperature and humidity control system required.
The proper design of equipment for the production, control, and recording of tem-
perature and humidity may require the services of an engineer.
If a large installation is planned, it is better to have
2
smaller heating and cooling
units than one large one.
For example if it is determined that 6 tons of air condition-
ing is
required, use two S-ton units rather than one 6-ton unit.
Install the heating
and air conditioning system of the peripheral area so that if all the inner area systems
fail, the peripheral system can be shunted to the inner area. When there is a possi-
bility of contamination from pesticides from adjoining laboratories, install air condi-
tioning equipment to insure that the outside air intake does not pick up contaminated
exhaust air.
Automatic thermostat controls for the heating and air conditioning
system are best.
Humidity regulation is usually provided by comparatively small commercially avail-
able units.2
If a source of clean compressed air is available, the air supply and water
supply can be connected and regulated by solenoids and humidistats. The water is
forced out through fine nozzles installed in a hose and produces a fine mist. Steam
units are available which will provide warm moisture. Various improvised humidi-
1 Sarco Co., Inc., Allentown, Pa. Blender Type DB.
2 Walton Laboratories, Inc., Union, N.J., Standard Engineering Works, Pawtucket, R.I., Bete Fog
Nozzle, Inc., Greenfield, Massachusetts.
2
8/10/2019 Rearing Mosquitoes
13/124
fying methods have been described, including such methods as using wet towels,
blowing a fan through layers of wet excelsior or gauze; steam from boiling water or
from radiators; wet sand; chemicals in water.
In determining the type of humidifier unit that should be used in a rearing room,
certain factors must be taken into consideration:
(I) The relative humidity desired and what variation would be permissible; (2)
Room size-length, width and height; (3) A
verage temperature; (4) Heat factors,
i.e., number of people or animals in the room; number of lights and wattage, other
sources of heat, and heat loss; (5) Construction of room-walls, floors, ceiling, parti-
tions, windows; (6) Openings to other rooms; (7) Exhaust fans-capacity. As in
temperature control, it is better to have two smaller units than one large one.
Use a recording hygrothermograph
for a permanent, continuous record of tem-
perature and humidity conditions.
Humidity can be checked with a sling psychrom-
eter, a bulb psychrometer, or a Honeywell hygrometer with electronic sensors which
will show the relative humidity at the exact location desired without disturbing air
movements. Although it is recognized that the critical factor is the rate of evapora-
tion, usually an indication of the relative humidity is sufficient for successful earings.
A maximum-minimum thermometer will indicate the total range of temperature
change for any desired period.
References-
Barlow (1961); Bertram and Gordon (1939); Buxton (1931); Flitters (1964); Haufe (1964);
MacPhee and Patterson (1958)
;
P
rovost et al. (1965); Wharton and Kniille (1966); Winston
and Bates (1960).
LIGHTING
The photoperiod and light intensity affect the development of the various stages
in the life cycle of the mosquito and are the subject of a great many papers (see
references). In the insectary, a cycle of 14 hours of light and IO hours of darkness
appears to allow the best and most uniform development, without interfering with
normal working hours of the staff.
Some speciesmay require a crepuscular period to swarm and mate. The automa-
tion system described by Levin, Kugler and Barnett (1958) coordinates the numerous
pieces of electrical equipment that regulate temperature, humidity, and light. This
type of equipment can be readily modified to dim lights gradually for dusk effect,
turn off all lights for darkness, then turn up lights gradually for dawn and turn on
full lighting for daylight.
The standard fluorescent lights usually supply adequate light for daylight.
Some
especially made fluorescent lamps generate light at wavelengths approaching day-
light, when required by exacting experimental work.
Incandescent lights connected
to a variable transformer can be used for gradually diminishing or increasing light
intensity. A bl
ue or blue-green light stimulates swarming of some species (Bates
949; TheOdOr and ParSOnS 1945).
Commercial light meters are used to measure light intensity. Evans (1961) de-
scribes a portable visual photometer for measuring very low light intensities. The
1 Bendix Corp., Environmental Science Division,
Baltimore,Maryland,21204-Hygrotllermograph
Model NO. 594,
Belfort Instrument Company, Baltimore, Maryland 21202.
3
8/10/2019 Rearing Mosquitoes
14/124
scale of a regular Weston light meter used for photograph y can be converted e asily
to foot-candles by multiplying the reading by
2.
References- I
Bates (1941)
;
Belton et al. (1967) ; Brennan and Harwood (1935)
;
Callot (1965)
;
Eldridge
(1963); Evans (1961); Flitters (1964); Fowler et ctl. (1958); Haufe (1962); Hubert et al.
(1954); Jobling (rg35), (1937); Killough and Weidhaas (1963); Levin et al. (1958); Nielsen
and Haeger (1955); Omardeen (1958); Parker and Rozeboom (1960); Seaton and Lumsden
(1941) ; Tate and Vincent (1932) ; Theodor and Parsons (1945).
SECURITY AND SAFETY MEASURES
The escape of possible insect vectors of disease, like the release of pathogenic
organisms cannot be tolerated.
The responsibility of the entomologist rearing mos-
quitoes is similar to the responsibility of the bacteriologist cultivating pathogenic
bacteria. Certain routine sec urity measu res are practiced by laboratories involved in
the rearing of medically important arthropods.
The Comm ittee l on Experimental Use of A&s aegypti of The En tomolog ical So-
ciety of America has developed a set of recomm endations on mosquito security and
surveillance that serves as a guide for mosq uito rearing laboratories. The follolwing
recomm endations closely follow, but are not an exact copy of the ESA report.
A.
FACILITY REQUIREMENTS
I.
Building
Window less construction, if possible, is preferred. If window s exist, keep locked.
A sign perman ently fastened near the lock indicating that the windows always should
be locked, will help assu re this protection.
Glass brick can som etimes be used to
replace windows.
Screened windows norm ally are not to be used, but if for some
specific reason they are required, double screens with
22
mesh screening and an addi-
tional outside protective screen of heavy hardw are cloth will provide the required
security.
2. Room layout
a. When the rearing room and laboratory are not one and the same, then the
rearing room should be an annex of the laboratory room and open into it,
If another
door exists or is required for safety reasons it shou ld be mark ed for emergency use
only .
Plan exits from the laboratory roo m o r rearing room (if not an annex to the
laboratory room ) through an ante-room , i.e. a small vestibule with entry or exit doors
or through another small room.
A com pletely empty ante-room is best, with white
or light colored walls and ceiling.
Post signs to be sure one door is closed before the
other is opened.
Protect the entrance to the ante-room from the laboratory with
some protective barrier such as close weave nylon netting or an air curtain.a
b. Paint the walls and ceiling of the laboratory, rearing room, ante-room and other
work areas with a light shade of paint (gloss ename l, epoxy, etc.), preferably white.
Light colored ceramic tile may be substituted for paint.
1
Committee members: G. Craig, Jr., E. J. Gerberg, C. Judson, D. W. Micks, M. W. Provost, I-I.
F. &hoof, C. N. Smith.
2 Ecco Aire, Inc., New Castle, Pa.; Dynaforce Corp., Queens Village, N.Y. 11428; Penn Ventilator
Co., Inc., Philadelphia, Pa., 19140; Neico Products, Inc., San Francisco, Calif. 94124.
4
8/10/2019 Rearing Mosquitoes
15/124
c. Screen all floor drains even though they enter a closed system. It is best to
drain into a tank or dry well in which insecticide is regularly introduced.
d. Screen or filter all air vents to the outside.
B. LABORATORY ND REARING PROCEDURES
I. Eggs
(a) Store eggs in a container in the rearing room.
(b) Treat surplus unhatched eggs with hot water or chemicals to kill them. If
possibleburn the old egg papers.
2. L.uruae and Pupae
a. Larvae
( I) In laboratories that do not maintain
containers with tight fitting lids to
adults. In mass-rearing laboratories,
several larval rearing racks are housed
b. Pupae
( I) Collect pupae routinely, so that no
containers.
T-day-week operations, rear larvae in
prevent the escape of any emerging
normally on a T-day-week operation,
in enclosures nside the rearing rooms.
adults emerge in the larval rearing
(2) Place pupae in special containers inside cages. Provide each container with
a lid so that the container can be closed after the pupae have emerged. It
should be removed from the cage through a sleeve.
(3) Treat unneeded larvae, pupae, and discarded rearing water by heating or
with chemicals so that only dead waste materials enter the sewage system.
3. Adults
a. Use sturdy cages, preferably of metal, fitted with a surgical stockinette sleeve.
When used properly the tight-fitting sleeve will allow manipulation of objects within
the cage. Th
e mosquitoes obtain their blood meals by feeding on the host through
the screening of a nylon mesh hammock.
This is preferable to introducing the verte-
brate host animal into the cage.
b. Keep the rearing room neat and clean.
Remove excess sugar pads, fruits, etc.
c. Hosts for blood feeding.
(I) Provide animal rooms separate from the rearing room. Check host animals
to be sure they are free of mosquitoes.
d. Transfer of adults.
All insects ransported from one secure laboratory to another must be housed within
a screened, securely closed container.
C. MISCELLANEOUS EQUIREMENTSAND RECOMMENDATIONS
I. Train employees to handle mosquitoes and make them aware of the potential
hazard of escapees.
Loose mosquitoes endanger the entire project. Even one
loose mosquito must be killed or caught immediately.
A sign in the rearing room
indicating the name of the individual responsible for the mosquito security helps
to maintain vigilance.
Replace careless workers or technicians with competent,
conscientious personnel.
2. Do not remove any living stage of the insect from security conditions without
taking proper precautions.
5
8/10/2019 Rearing Mosquitoes
16/124
3. Place surveillance oviposition-traps (Fay and Perry, 1965 ; Fay and Eliason, 1 966)
in the laboratory, rearing rooms and adjoining rooms. Numb er each trap and record
on a map. Keep a record of the weekly check.
Place additional surveillance oviposi-
tion-traps outside within 25 feet of the laboratory bu ilding, at approx imately r5o-foot
intervals. Rem ove all potential breeding containers from the surveillance area. (For
detailed information on preparation of surveillance oviposition-traps, and selection of
sites, see Ovipos ition trap reference handbook , CD C, Aedes aegyptt Handboo k series
No. 6, 1967.)
4. Personnel
Limit access o rearing and laboratory facilities to authorized personnel who under-
stand the need to retain all live s tages under security conditions.
.References-
Bertram
(1958) ; Fay
and Eliason (1966);
Fay
and Perry (1965); Hocking,
B. (1960)
;
Pratt
and Jakob (1967).
8/10/2019 Rearing Mosquitoes
17/124
8/10/2019 Rearing Mosquitoes
18/124
this is done the significance of the DR S can be ascertained for any species of m os-
quitoes. Preliminary research indicates that a DR S of 1.8 gives good results.
Many cages for holding and/or mating mosquitoes have been described. McKiel
(1957 ) describes a cage 30 wide x
20
long x
20
high (RS= 12,90 0 sq. cm.) for
4000 Aedes aegypti adults.
This would provide a DR S of 3.2. Weathersby (1962 )
used a cage 18x18~18 for
Aedes togoi
and
Armigeres
sp. Blakeslee et
al., (1962)
used a cage 24x24~24 for Culex erythrothorQx. Kitzmiller and Micks (1954) cage
is 12X12x12
and 24X24X24
cages for Culex
pipiens.
Hayes and Morlan ( 1957)
used a similar size cage for Aedes
triseriatus.
Williams ( 196 2) employed a 12x9s x
14 cage for the same species.
None of these authorrs mention the density per cage.
McLintocks (1952 ) cage is 12x12~2 2 (RS= 6812 )
f or 1500-2000 Culiseta inornata.
This gives a DR S of approximately 3.4-4.5. Casanges et al. (1949 ) describe a
22x.36x24 cage (RS-17 957) f
or
3
000-5000 Anopheles quadrimaculatus, or a DRS
of 3.6-6. Aedes aegypti have been kept in many types of cages.
Christophers (1960)
descriibes a numb er of cages for this species including one type 8 2x8 2x8 1/z and
another 14x8~/~~12.
Liles et aZ. (1960) employed a cage 12x8~9 for IOO A. aegypti.
Wall is (1954) cage is 36x36x30
for 10-350 female A. aegypti and concludes that
the density of ovipositing females did not increase the percentage of eggs produced.
Roth (1948 ) used an 11x11~15 cage for his work on aegypti.
Morlan et al. (1963)
indicate that their colony cages (Fig. 3)
consist of approximately 10,00 0 adults in
MOSQUITO COLONY CAGE
ASSEMBLY
FIG. 3.-Mm-hs colony cage.
8
8/10/2019 Rearing Mosquitoes
19/124
FIG. q.-C &be rgs collapsible cag e.
a
22x22~22
cage, (RS-12,487 ) or a DRS of 1.2. Thus, there is no standardized
size, type or construction material or population density.
A standard size cage may be of value for rearings for research purposes.
To this
end a light weight aluminum cage has been developed 1 that is now widely used
(Fig. 4). This cage is constructed of pressed-form .032 inch alum inum frame 7s
thick by 3/4
wide and is screened with 18 x 22 mesh aluminum screening. The
screening is held in place by a thin rubbe r sp line, for easy replacem ent of screening
when necessary.
The floor of the cage is constructed of either .090 or .03 2 inch
aluminum sheeting depending upon the model of the cage. A hamm ock constructed
of a fine mesh nylon is an integral part of the top of the cag e.
Anesthetized animals
such as guinea pigs with shaved backs can be used for blood feeding.
Sucrose-soaked
cotton balls, fruit, and su gar, can be placed in the hamm ock for o ther types of feed-
ings. The
cage is fitted w ith a 24-inch long su rgical stockinette sleeve which fits
a 9l/2x97/s opening.
The size of the cages may be
12~12x12
(3ox3ox3ocm) or
8/10/2019 Rearing Mosquitoes
20/124
; 1
I 1
I
I
:
I
;, I
,I
_--
----___
,._. _----
_ -q
BARRAUD
CAG E
Barraud,F?J.(1929)
FIG. 5.-Barraud cage.
GILLET CAGE
Gillet, J. G.(1962)
PIG. 6.-Gillett cage
:
-LJ
L
POL LARD
CAGE
,-
Pollard, D.G,(1960)
FIG, 8,-&kCrays escape-proof colony cage,
FIG. g.-Pollarcl cage.
IO
8/10/2019 Rearing Mosquitoes
21/124
24x24~24~
(6ox6ox6ocm ) ( h
own in Fig. 4 as G-I, G-2, and G-3, respectively), or
other combinations.
All cages are collapsible for ease of storage and can readily be
cleaned by steam or hot water.
These cages have been successfully used with many
species of mosquitoes in most of the major mosquito laboratories.
Many kinds of cages for special purposes have been described. Perhaps one of
the most useful cages for field work is the Barraud Cage, Fig. 5, (Barraud, Igag),
a cage with a wire frame over which is hung netting. Lantern globe cages, made
from the glass globes of kerosene lanterns have been widely used (Fig. 6) (Trembley
195 5; Gillett 1962 ). Others have modified glass cylinders (Burgess and Young
rg46), glass jars (Eldridge and Gould 196 0) and plastic containers (Fig. 7) (Yolles
FIG.
7.-Plastic cage.
and Knigin 1943; Young and Burgess 1946; Barnett 1955; Gerberg et al.
1967;
Alger
1968 ). Escape proof cages have been described by Bar-Zeev and Ga lun (1960 )
and McC ray (Fig. 8) (1963 ), b u are not practical for mass rearings. Pollard (1960 )
describes a cage (Fig. 9) that minimizes the escape of mosquitoes and is still easy to
References-
Alger (1968); Barnett ( 1955) ; Barraud ( Igag) ; Bar-Zeev and Galun ( 1960) ; Blakeslee et al.
(1962)
;
Burgess and Young (1946); Casanges et al. (1949); Christophers (1960); Eastwood
and Jamieson (1965); Eldridge and Gould (1960) ; Gerberg et al. (1967) ; Gillett (1962) ;
Hayes and Morlan (1957); Kitzmiller and Micks (1954); Lennox (1959); Liles et al. (1960);
McCray (1963); McKiel (1957); McLintock (1952); Morlan et al. (1963); Pollard (1960);
Reynolds (1960); Roth (1948); Trembley (1955); Wallis (1954); Weathersby (1962);
Williams (1962); Yolles and Knigin (1943);
Youngand Burgess1946).
8/10/2019 Rearing Mosquitoes
22/124
ADULT FOOD
Although the female mosquito usually must ingest a blood meal for ovarian devel-
opment, adu lts of both sexes require carbohydrate foods in addition. Carbohy drates
are generally supplied as a sugar solution.
Although sucrose and g lucose in concen-
trations of from 37/o to 20% have been used,
10%
sucrose, made by dissolving
IOO
gms of ordinary household white sugar in one liter of water appears to provide
the best results.
Other forms of suga r, such as corn syru p, honey, various fruit
juices, raisins, apple slices, and bananas , have also been used.
FIG. Io.--Animal restrainers in use; mass feeding of mosquitoes on chicks.
Soake d cotton balls are the easiest method of providing the suga r solution to ad ult
mosquitoes. Th
e cotton balls are soaked in the
IO~~
sugar solution, the moisture is
squeezed out and the balls are then placed on the top of the cage. Usually 4 cotton
balls, changed daily, are sufficient for a 30x30~30 cm
(1x1~1
cage. The cotton balls
must be changed daily. Coluzzi (1964) used a tank and a side cup trough. The
liquid in the cup maintains its level and keeps a piece of filter paper wet continuously.
Porter et al. ( 196 1) describe a small solution feeder for adult mosquitoes m ade from
a ro-dram plastic vial in which is inserted a wick made of a small roll of blotting
paper.
The vial is filled with the feeding solution and the mosquitoes feed through
12
8/10/2019 Rearing Mosquitoes
23/124
small holes punched through the wall of a plastic vial. Eliason (1963) used a tech-
nique of feeding mosquitoes on solid sugar.
Blood meals are provided by supplying animals that have been anesthetized or
restrained, and placed on the adult cage, rather than in the cage.
This prevents loss
of mosquitoes, hazards to the technician, and destruction of mosquitoes by the blood
donor.
Animals may be anesthetized by the use of I grain nembutal (see appendix)
administered intraperitoneally at the rate of
0.2
ml for each I lb (0.5 kg) of body
weight. Various types of restrainers are available commercially and are described
by Porter et al. (1961); Gerberg et al. (1967) (Fig. IO); Morlan et al. (1963);
Trembley (1955); Dunn (1932); Jones and Scheltema (1952).
The more commonly used sources of blood meals, other than a technicians bared
arm, are guinea pigs,
rabbits, mice, rats, hamsters, monkeys, and chicks.
Blood
meals may also be supplied by artificial means (see artificial feeding techniques).
Shave the animal prior to presentation to the mosquitoes.
Some prefer shaving or
clipping the hair from the back of animals, and others the underside. Animal clip-
pers have proved very useful for this purpose.
References-
Dimondet al. (1955); Dunn (1932); Eliason 1963); Gerberg t
al. (1967);
Greenberg
1951);
Knierim et al. (1955); Lang and Wallis
(1956);
L 1es
et
al. (1960); Morlan et
al.
(1963);
Porter et al. (1961); Roy (1936);
T rembley
1955); Woke (1937);
Woke et al. (1956).
ARTIFICIAL FEEDING TECHNIQUES
Xmembrane feeding technique is one approach for artificially providing a blood
meal to mosquitoes.
This technique, used with other haematophagous arthropods
prior to the successfulfeeding of mosquitoes, consists of a membrane-covered con-
tainer, such as a glass tube or test tube, containing the liquid mosquito food. The
container is placed so the membrane side is accessible o the mosquitoes for feeding.
The liquid should b
e several degrees warmer than the ambient temperature to induce
certain species to feed. Tarshis (1958)
review of artificial feeding techniques
includes the various types of membranes used, and describes various techniques and
materials.
Knowlton and Rowe (1935)
used animal mesentery bags to feed mos-
quitoes on
suspensionsof diseased (equine encephalitis) brains. Woke (1937) fed
A&es
aegypt;
on whole blood through rat skin membranes. Yoeli (1938) gave
Anop&des elutus blood through rabbit skin membranes. Bishopp and Gilchrist
(1944, 1946) induced feeding by Aedes aegypti on blood through chicken skins.
Greenberg (1949,
1951)
reports the feeding of Aedes aegypti on a variety of artificial
fluids through Baudruche (a bovine
intestinal preparation) capping membranes.
Eyles ( 1952)
used hog-gut sausage casings, and Trembley (1952) used Baudruche
capping membranes
and chicken-skin membranes.
Kartman (1953) fed Aedes
aegypti
blood through hog-gut sausage casings.
Bar-Zeev and Smith (1959) in
studying the action of repellents, gave A.
aegypti titrated blood through a membrane
made from the outermost layer of ox caecum.
Tarshis (1959) discusses ive different
1 Oster Mfg. Co., Milwaukee, Wise. Model A2 Clipper, No. 40 blade.
8/10/2019 Rearing Mosquitoes
24/124
memb ranes and an apparatus for feeding Culex
tarsalis.
Collins et al. (1965 ) used
the Baud ruche (untreated) memb rane to feed Anopheles mosquitoes Plajmodium
falciparum
infected blood.
FIG. I I.-Apparatus for me mb rane feeding of mosq uitoes.
Rutledge et
al. (
1964)
d
escribe in detail their techniques for mem brane feeding
of mosquitoes, Figs. I I, 12.
The feeder is mad e of heat res istant glass or stainless
I4
8/10/2019 Rearing Mosquitoes
25/124
Mosquito Membrane Feeder
Inlet
FIG. I x--Apparatus for
detail.
membrane feeding,
steel. Circulating warm water controls
the temperature of the blood. Accord-
ing to them, chick skin is superior to
the Baudruche membrane, and chick
blood is more acceptable than erythro-
cyte extract or serum.
Blood meals can be supplied by means
of preserved blood absorbed on a cotton
pad.
Russell (
193 I )
reports feeding
mosquitoes blood distributed over net-
ting.
Others (MacGregor and Lee
1929; Knowlton and Rowe 1935; Shiavi
and Franc0 1949; McL,intock 1952;
Dimond et al. 1955; Trembley 1952;
Liles et al. 1960) mix blood with honey,
sucrose or glycerine.
Knierim et al.
(1955) used frozen titrated beef blood plus 10% honey (or
without honey) heated
to 35-38 c.
References-
Bar-Zeev
and Smith (1959)
;
Behin (1967)
;
Bishop and Gilchrist (1944, 1946)
;
Collins (1963)
;
Collins et al. (1965, 1966); Co11
ns et al. (1964)
;
Collins et a/. (1964)
;
Dimond et al. (1955) ;
Eyles (1952) ; Galun (1965) ; Greenberg (1949) ; Grifliths and Gordon (1952) ; Jones (1956) ;
Kartman (1953) ;
Lang
and Wallis (1956) ;
Liles et al. (I
960) ; MacGregor (I
930) ; MacGregor
and Lee (1929); Mattingly (1964); McConnell (1956); Msangi (1956); Ogden (1961);
Ogunba (1967); R
ass (1956) ; Russell (1931) ; Rutledge et al. (1964) ; Schiavi and Franc0
(1949) ; Shambaugh (1954); St. J h n et d. (1930); Tarshis (1957, 1958, 1959); Trembley
(1952); Willis (1958); Woke (1937); Yoeli (1938).
INDUCED MATING
Aedine mosquitoes are normally difficult to maintain in the laboratory but Mc-
Daniel and Horsfall (1947)
used efficient procedures for stimulating copulation and
obtaining Aedes stimulans and Aedes vexans eggs. Horsfall and Taylor (1967)
modified the technique and used it successfully to mate over 40 species of mosquitoes.
Copulation is induced by manipulating an immobilized female into contact with a
decapitated male.
This technique consists of using at least 3 day old mosquitoes.
The female is anesthetized lightly with chloroform, and is picked up with a suction
pipette and manipulated to make contact with the male. The dorsum of the thorax
of living decapitated males is cemented with a non-toxic glue to a small piece of
glass slide. Contact between the two immobilized mosquitoes is accomplished
manually under a
20x
stereomicroscope.
The female is placed in the mating position
with the ventral side up and the head directed away from the male.
Apexes of the
two abdomens are then brought together, venters uppermost. The male is stimu-
lated by touching the claspers several times with the female. Insemination is usually
completed within a 5-50 second period.
Usually 3-4 males are thus prepared so
that if one male is unresponsive,
others are immediately available. Frizzi (1958)
used the induced copulation technique on Anoptieles macdipennis with variable
8/10/2019 Rearing Mosquitoes
26/124
results, and Carvaglios (1961 ) using the technique without decapitation of males
maintained a colony of Anopheles Zabranchiae.
After mating, the females are transferred to a feeding cage for recovery from
anesthesia.
A blood meal is provided after 12 hours. Bake r et L$ (1962 ) obtained
IOO O/~ fficiency in single pair matings of A.
maculipennis
by induced copulation.
Anopheles punctipennis, A. quadrimaculatus, A. freeborni, and A. albimanus were
maintained in the laboratory by the above authors, using the induced copulation
technique.
Wheeler (1962 ) modified the technique by gluing cold-anesthetized mosquitoes to
the heads of insect pins.
The female-bearing pin is inserted into a cork glued to a
microscope slide so that the mosqu itos body axis is in a horizontal plane. The
male-bea ring pin is inserted into an adjustable pinned insect holder an d then
oriented until the genitalia meet. Ow Yang et al.
(1963)
simplified the technique
by using 3-6 day old males caught w ith a fine pipette attached to a suction device, and
pinned latera lly through the thorax using a minuten pin fixed into the end of a 6-
long soft wooden stick. The females are blood-fed 2 days after emergence, and are
collected in individu al tubes the next m orning.
The female is anesthetized with
ether and tipped out on her back onto a clean white table. Copu lation is achieved by
bringing the pinned male down at about a 45 angle to the female. When copula-
tion is affected the pair can be lifted together, with the fema le firmly clasped by the
inale, and gently dropped into a container, by removing the male from the pin.
The
mating procedure is carried ou t under a microscope at a room temperature of 26-28
C. and RH of about 80%. The authors were successful in maintaining a laboratory
colony of
Anopheles maculatus
by using this technique.
Coluzzi (1962) maintained Culex
impudicus
and C.
territans
by induced mating.
Hors fall (1964 ) states that species of Aedes, Anopheles, Culex, Culiseta and Psoro-
phora have been effectively insemina ted in his laboratory. He recomm ends that
adults of the same age be held for a 60-72 h ours premating interval at 80 + 7; RH .
They are fed on a carbohydrate diet (honey diluted I :I w ith water). Mating is
carried out in a room with a temperature of 26-28 C. and about 85% RH. One
technician prepares the males, the second prepares the females and joins the two in
copula, at the rate of 40 matings per hour.
The males are collected into individual
tubes and the open vial is inverted over a Buchn er funnel through which flows a
gentle stream of CQ2.
Immediately upon knockdow n the male is plucked from
the funne l with forceps and is glued ventral side upw ard to a glass slide (casein
glue is used).
Four or five males are placed in a row. Decapitation may precede
gluing, or delayed for longer surviva l until just prior to the act of copulation, w hich
will not take place unless the male is decapitated. The male survives longer if
decapitation is delayed.
Initiate the mating procedure only after full recovery of the
males (2-5 minutes).
The females are anesthetized with chloroform only to the
point of relaxation and are then picked up with a vacuu m p ipette made from a 5-mm
glass tube, bent at right angles and d rawn to an open tip small enough to apply to
the anterior slope of the mesonotum.
A vacuu m pum p with a bleeder tube or valve
is used. Th
f
emale is held ventral side uppermost and broug ht into contact with
the claspettes.
Manipulate the female so that her genital opening is brought proximal
to the phallosome of the male.
Proper positioning is prerequisite to bringing abo ut
complete union.
For
Aedes
an angle of about 120 between the .?bdomens will
16
8/10/2019 Rearing Mosquitoes
27/124
permit proper union.
For Culex the angle must be nearer to 180 Horsfail blood-
feeds on the day following mating
(6-12
hours between time of anesthesia and
feeding).
Baker (1964) feeds th
e males, prior to copulation, with honey on a cotton ball,
and the females with water on a saturated sponge. Generally he provides a blood
meal for the female shortly before mating. Baker does not anesthetize the males
but collects them with an aspirator and softly blows them between layers of cotton
wool. This holds them until pinned laterally through the thorax using a dissecting
needle fixed into the end of wooden stick
7.5-15
cm (3-6) long. If it is advan-
tageous to keep the male alive, he can be held with a vacuum pipette.
Baker reports
success with Culex t~salis using this technique.
Esah and Scanlon ( 1966) estab-
lished a colony of Anopheles b. balabacensis by induced mating.
References-
Baker (1964)
;
Baker and Kitzmiller (I 961)
;
Baker et al. (1962)
;
Burcham (1957)
;
Caravaglios
(1961); Coluzzi (1962); Esah and Scanlon (1966); Frizzi (1958, 1959); Gopal and Wattal
(1962) ; Hayes, D. E. (1968) J Hayes, R. 0. (1953); Horsfall (1964); Horsfall and Taylor
(I 967) ; McDaniel and Horsfall ( 1957) ; Ow Yang et al. ( 1963) ; Wheeler (I 962) ; Wheeler and
Jones (1963).
COLLECTING AND HANDLING EGGS
The eggs of mosquitoes are usually deposited on the surface of the water, or on
surfaces that are subsequently inundated. To obtain mosquito eggs, a reasonable
substitute must be provided for the oviposition site.
A plastic container approximately rgxrox5cm (6~4x2) has proved quite successful
for anophelines.
A layer of cotton balls is placed in the bottom, covered with a
sheet of filter paper or paper toweling and then flooded with water so that the water
surface barely covers the filter paper (Gerberg et al. 1968). Some investigators use
bowls or pans of open water as oviposition sites. Giglioli (1947) used a white glazed
china finger bowl (II cm in diam. 7 cm in depth) half filled with water. Others
have lined the oviposition container with paper to prevent the anopheline eggs from
sticking to the sides of the container. Kepler et al. (1964) employed a water-filled
petri dish, the bottom lined with a strip of filter paper. Yoeli and Bone
(1967)
provided small cork rafts and protruding stones on which the females alight prior
to laying eggs.
Trembley (1955) reports that some Anopt2eZes preferred dark colored
containers or containers lined with darker paper toweling.
Species of the genus CuZex usually lay their rafts of eggs on open water, in pans,
bowls, petri dishes or other type containers. Krishnan (1964) claims that more egg
rafts were obtained in trays containing hay infusion than in those containing tap
water.
Jupp and Brown (1967)
confined individual gravid females in laying tubes
r in. in diameter containing a little water and a strip of filter paper down the side
which provided a suitable surface for the mosquito to rest on. The tubes are closed
with nylon gauze on which is placed a cotton pledget soaked in sugar solution.
Takahashi (1968) bt
ained eggs of C. portesi by placing an inverted clay flower
Fat, with an additional I
side entrance hole in a white enamel dish containing
water.
Inside the flower pot and standing in the water is a black-painted g ass
oviposition jar.
7
8/10/2019 Rearing Mosquitoes
28/124
Laurence (1960) collects Mansonia eggs from floating discs of plastic-impregn ated
&aft paper in an oviposition chamber.
Usually aedine eggs can withstand desiccation and are collected on a moist sub,
strate.
The most comm on method for the collection of A. mgypti eggs is to use a
3
wide paper toweling, lining the interior of a pint ice cream carton o lr a glass jar.
Fill the carton with enough water to cover approximately I in. of the paper toweling.
The eggs are
conditioned by leaving the egg paper in the container for an addi-
tional 24 hours, after removing the co,ntainer from the cage.
The egg paper is then
air dried (at 80 F. and 80% RH) for
4
d
a
y
s and then stored for future use. Other
A&es species may be induced to lay eggs on various types of m oist surfaces.
Cotton
wrapped in cheesecloth, porous sponges, sphagnum moss wrapped in cheesecloth,
large-pored plastic materials, and other types of rough or porous materials have
been used as oviposition sites.
Reynolds (1960 ) describes an escape proof m osquito
egg harvester, for use under maximum mosquito security.
Anoplieles, Culex and
Culiseta eggs
are not readily stored and are usually hatched
in the water where laid. Cond itioned aedine eggs can be stored. To hatch these
eggs, place in water with a reduced oxygen content.
Many methods for deoxygena-
tion have been described, since the most uniform method of hatching is by imm ersing
the egg papers in deoxygenated water.
To make deoxygenated water, submerge
quart Mason jars in boiling water and cap under water.
The jars can then be stored
a t 27 C . ( 80 OF.).
When an egg paper is to be used, the jar is uncapped and the
egg paper is immediately placed in the w ater and the cap replaced.
Eclosion usually
begins in minutes.
Some egg papers can be used over again as all the eggs do not
always hatch after the first immersion.
Tho irgh it is possible to count aedine eggs on the egg paper, for mass rearing
purposes this is not feasible.
Instead an aliquot sam pling m ethod is used on the
newly hatched larvae.
References-
Burgess (1959); Elzinga (1961); Fay and Perry (1965); Giglioli (1947); Gjullin et
al. (1941);
Horsfall (1958); J d
son (I 960) ; Jupp and Brown (1967) ; Keppler et al. (1964) ; Krishnan
(I 964) ; Laurence (I 960) ; Lavoipierre (1953) ; Reynolds (I 960) ; Shute (1933) ; Takahashi
(1968); Trembley (1955); Williams (1962); Yoeli and BonC (1967).
LARVAL REARING TECHNIQUES AND EQUIPMENT
Counting Lmuae
1
Newly hatched larvae may be counted individually using a hand lens and a hand
counter.
For large rearing trays or mass rearing purposes, the aliquot sample method,
Fig. 13, (Gerberg et OZ. 196 8) is useful. Pour the first instar larvae into a gallon
battery jar and fill to 3500 ml of 27O C. (80 F.) water. The battery jar has 5
random ly placed outlets (at 5 levels).
A variable speed stirrer, mo unted on a ring
stand, is immersed in the water.
The stirrer is operated at approximately 250 rpm,
to suspend the larvae
uniformly in the water.
Twenty-five samples of I ml each are
withdrawn through the random outlets and the mean number of larvae per sample
(ml) is determined.
It is then possible to withdraw a given qu antity of water
containing a known number of larvae.
Morlan et al. ( 1963)
d
escribe and figure an autom atic dispenser for obtaining
18
8/10/2019 Rearing Mosquitoes
29/124
Fig. r3.-Aliquot container for mosquito larvae.
large volumes of dispersed mosquito larvae. This equipment is made from a modi-
fied agitator type washing machine and a rain gaug e type of tripping bucket.
Bar-Zeev (19 62) describes a simple technique for obtaining standard numbers of
newly hatched mosquito larvae.
Rearing Trays
A wide variety of containers capable of holding water may be used as rearing trays.
Trembley (1955) suggests white enam el photographic developing trays 16xgf/2x2,
8/10/2019 Rearing Mosquitoes
30/124
or containers of 3 quart capacity, 2-3 inches deep, preferably, but not necessarily,
opaque . Containers of these types are shown in Figs.
14
and 15. For mass rearing
of A&es aegypti, Morlan et al. (1963)
used a galvanized metal tra y 2x10~ 72.
(Fig. 16). Th
e ra was coated with a thin layer of paraffin. Each tray had a j/4
y
copper-tube outlet to facilitate draining and was stacked on a metal rack mad e of
i/sx 3/4
3/4 I1
angle iron. Each
rack held 24 rearing trays. They used 7 liters of
water in each tray to rear 7000 larvae. This system provides for 1.4 larvae/sq. cm.
of surface area and 1.4 larvae/ml of water. Fay et al. (1963) reared
8000-14,000
larvae per tray (2~10x 72) with no significant difference of percentage of yield or
adult vitality.
In the IC R laboratories, mosquitoes are mass reared in paraffin-coated, galvanized
trays r38x7 6x5cm (54x20x2), (Fig. 17). Each tray is filled to a depth of 2cm
( g) with water and approximately 15,000 larvae are introduced. Gahan (1967)
suggests 100 -250 Amp/&es larvae for a polyethylene pan 12 in diameter.
For small scale rearings, white polyethylene pan s 13I/4x1ox2 have been used very
successfully.
Handling Larvae
If larvae have to be moved or transferred, Trem bley (195 5) suggests a 50 ml.
volumetric transfer pipette shortened to 8-10 and fitted with a rubber bulb. We
have found very useful a 12-18
length of rigid clear plastic tubing covered with a
FIG. Iq.-White enamel trays for rearing larvae.
20
8/10/2019 Rearing Mosquitoes
31/124
FIG.
IS.-White enamel dishes for rearing larvae.
LARVAL REARING TRAY
28 GA. GALVANIZED
SHEET METAL
314. LCl 1.062 WALL)
COPPER TUBING -SILVER
SOLDER TUBING FLUsn
RIVET AT EACH CORNER
ROTE: MAKE ALL SEANS WATER TIGHT.
FIG. 16.-Construction of galvanized metal rearing tray.
21
8/10/2019 Rearing Mosquitoes
32/124
FIG. IT.-Mass rearing of mosqu itoes in galvanized trays.
fine mesh cloth at one end and fitted into a rubber bulb. Also useful for picking
up larvae or pupae is a piece of 20-40 mesh stainless steel screening 27x75m m
(1x3)
and bent so that approximately
I
inch is at a 45 angle to the remaining 2
Equipment for Feeding Larvae
Food
can be premeasured into small containers and then dispensed over the water
surface.
Some technicians prefer a salt shaker and can judge the amount of food
22
8/10/2019 Rearing Mosquitoes
33/124
by the number of shakes. Heal and Peregrin (1945) recommend sheet metal spoons.
Hunt and DaveY (1947) used reversed pen nibs. Morlan et al. (1963) used a
plastic spoon of appropriate capacity. Regardless of the method of measurement, it
is imperative that a rigid feeding schedule be followed and the type and amount of
food be standardized if uniform and consistent results are desired.
Refevences-
Bar-Zeev (1957 , 196 2); Fay et al. (1963); Galun (1967); Gerberg et al. (1968); Heal and
Peregrin (1945); Long and Breland (1956); Morlan et al. (1963); Soderstrom and Levitt
(1967); Trembley (1955).
LARVAL FOOD AND FEEDING
Mosquito larvae are fed everything from a highly refined chemical diet to a witchs
brew made from guinea pig droppings, mud, and the ground-up skins of water
boatmen. Perhaps one of the greatest reasons for variability in the results of rearing
mosquitoes is the lack of uniformity or standardization of larval food. An abundance
of literature refers to larval foods, both natural and chemically defined. Asahina
(1964)
provides an excellent review of food material and feeding procedures for
mosquito larvae.
Some of the early investigators (Bacot 1916) report that bacteria and yeasts are
essential items of diet.
MacGregor
(1915)
increased the bacterial and protozoan
population by the addition of guinea pig feces. Boyd et al. (1932) used hay infusion
to provide protozoan food for the larvae. Weyer (1934) added dried blood and
powdered calfs liver.
Crowell (1940) was probably the first to use commercial dog
biscuits as a complete and somewhat standardized larval food. Perhaps the most
widely used food and feeding schedule is the one developed at the Communicable
Disease Center (Morlan et al. 1963). The larvae are fed dog chow (5% crude fat)
ground to pass through a 40 mesh screen.
This food is primarily for A&es aegyptz
but will work for other speciesas well, with the following schedule:
Day o (day of hatching) o.zmg/larva
I
0.3mgJarva
2
o.qmg/larva
3-7
o.6mg/larva
Note that the addition of a live yeast mixture (0.7 gm/I liter of water/Io,ooo
larvae) on day 3 improves adult production.
Trembley (1955)
used the following procedure for Anopheles quadCmacuZatus,
using a 16x9 l/*x2
tray for 300 larvae.
12 hours before day o
25 mg powered dried brewers yeast
and 25 mg Bacto-brain heart infusion.
Day o
IOO
mg of
I :I
powdered yeast and
powdered dog food
Day
I
50 mg
Day 2-3
100 mg
Day 4-14
150 mg
Gerberg et al. (1968)
use the following schedule for Anopheles stephensi.
23
8/10/2019 Rearing Mosquitoes
34/124
The larval food consists of a 50:5 0 mixture of dog chow a nd pig liver pow der
ground to 40 mesh.
Day o-Day 2 .I 3 mg/larva
Day 3 .20 mg + .045 mg Fleischman ns
dried brewers yeast/larva
Day 4 .26 mg/larva
Day 5 .40 mg/larva
Day 6-9 .53 mg/larva
The nutritional requiremen ts of mos quito larvae were investigated by Trag er
(1935, 1937, 1953), de M 11
l on et al. ( 1945), Goldberg ( 1947), Goldberg and de
Meillon ( 1948), Lea et al. ( 1956), Lea and DeLong ( 1956), Singh and Brown
(1957) and Akov (1962).
References-
Akov (1962);
Asahina
(1964); Racot (1917);
Boyd et al. (1932): Buddington (1941);
Crowell (1940) ; de Meillon et al. (1945) ; Frost et al. (1936) ; Gerberg ez ai. (1968) : Golberg
(1947); Goldberg and de Meillon (1948); Hinman (1930, 1932, 1933); Lea et al. (1956);
Lea and DeLong (1956); MacGregor (1915); Morlan et al. (1963); Singh and Brown (1957);
Trager (1935, 1937, 1942, 1953); TrembleY (1955); WeYer (1934).
HANDLING , COUN TING, SEPARATING AND SEXING PUPAE
Pupae may be picked up by means of pipettes or by small lifters made of screening.
The comm ercial m edicine dropp er with the open end filed off to ma ke a wide m outh,
or Kom ps modification of a syringe (Trem bley 195 5) may be used. Plastic pipettes
have become popular.
The rubber bulb normally supplies adequate suction but
others have relied on the use of vacuum pumps or water-operated suction pumps
(Johnson 194 7) to increase the efficiency of operation.
Lifters can be mad e from small pieces of metal screening, 20-4 0 mesh, 25x75c m
(1x3). Individual pupae can be lifted by means of small wire loops.
Pupae may be counted by manually picking them out with a pipette or lifter.
They can also be counted vo lumetrically.
A volumetric counter can be made from
a piece of plastic tubing with an ID of 3/s-2, approxim ately 6 long with one
end covered with fine wire screening. A know n num ber of one size pupae are added
to the tube and the level of pupae is marked on the tube. From then on pupae
can be added to the m ark, the tube inverted and the pupae washed out by flushing.
Pup ae can be separated rapidly from larvae by the ice water techniques of Ram a-
krishnan et QZ. 1963), Weathersby ( 1963 ) and Hazard ( 1967). Larvae and pupae
are concentrated in a sieve and im mersed in ice water.
The larvae sink immediately
and the pupae float and are poured off.
Bar-Zeev an d Ga lun ( 1961 ) used a rather
unique method of separating larvae from pupae by magnetic means.
Iron dust is
added to the rearing trays and the culture exposed to a magnetic field. Only the
larvae, which have ingested the iron p articles, are held b ack by the mag netic field.
Fay and Morlan (1959) describe a mechanical device not only for separating larvae
from pupa e, but ca pable of sexing the pupae of some species.
The principle of sepa-
ration and sexing is based on size differences.
The culture of mixed larvae and
pupae is
polured through a funnel into a wedge-shaped space between two slightly
separated and adjustable glass plates.
The female pupae, being largest are retained
24
8/10/2019 Rearing Mosquitoes
35/124
in a line at the upper levels of the plates, the smaller male pupae are held below
the females and the even smaller larvae are retained towards the bottom. The
smallest larvae drain out. The authors claim the separation of IOOO pupae in 20
minutes. Grose et al. ( 1966) made improvements on Fay and Morlans design.
McCray (1961) d escribes a simple separating device made from an aluminum
sheet with stamped-out 0.039 slits. The larvae and male pupae wash through
and the female are retained. The author claims that 30,000 individuals can be
processed n 5 minutes. Gentry and Gerberg are developing a small separating and
sexing device that uses removable comb-like plates in a trough arrangement. The
plates are interchangeable and the proper size plate is inserted depending upon the
species to be separated and the size of the pupae and larvae.
The sorted pupae are placed in containers of convenient sizes and the containers
placed inside cages to await adult emergence. Various devices have been used to
aid the emergence of adults. Some workers use small floating objects, such as small
pieces of cork, (Trembley 1955, Christophers 1960) or twigs to assist the adults in
leaving the water.
Smith and Whitlaw (1963) d escribe an emergence disc of wide
mesh dacron cloth that is claimed to reduce the mortality of emerging adults.
References-
Bar-Zeev and Galun (1961); Christophers (1960); Fay and Morlan (1959); Gillett (1955) ;
Grose et al. (I 966) ; Jones (1957) ; Lowrie and Gubler (I 968) ;Hazard (I 967) ; Johnson (I 947)
McCray (1961); Ramakrishnan et
al.
(1964): Smith and Whitlaw (1963); Trem bley (1955);
Weathersby (1963).
STERILE CULTURE
Rearing of mosquitoes under aseptic conditions may be required for
mosquito
nutrition investigations for in vitro studies, and other types of research.
Eggs may be surface sterilized.
Techniques described by Atkin and Bacot (1917)
use 0.5% lysol; Barber (1927)
dripped 80%
alcohol for 2-3 minutes over A.
mgypti
eggsand Roubaud and Colas-Belcour (1929) surface sterilized eggs using
hydrogen
peroxide
and also mercuric chloride. Whites (193 I) solution, consisting of mercuric
chloride 0.25 gm, sodium chloride 6.5 gm, hydrochloric acid, 125 ml, ethyl alcohol
.
250 ml and distilled water 750 ml, was more effective against bacterial contamination
than against other types of contaminants.
Hinman (1932) disinfected eggs in hexyl-
resorcinol.
Trager (1935, 1937)
used 5% castile soap and then Whites solution.
Lea et ul. ( 1956)
and Jones and DeLong (1961) surface sterilized eggs by
washing
the eggs in
70% alcohol for 5 minutes, transferring eggs by means of flamed forceps
to a 0.87% sodium hypochlorite solution for 2 minutes, then to a second 70% alcohol
rinse for 4 minutes,
and then to a final wash in autoclaved distilled water. Mar-
tignoni and
Milstead (1960) sterilized A. aegypti eggs on paper by placing one-
month-old eggs in a petri dish containing an autoclaved IO:/, solution of benzalko-
nium chloride for 30-60 minutes,
then washing in sterile water, followed with
immersion for
15-30
minutes in 80/~ alcohol and then two more washings in sterile
water. Akov (1962) describes a method of obtaining sterile larvae of uniform age
by placing
the sterile eggs in a desiccator and reducing the pressure for 10-20 minutes.
Trager (1935, 1937); de Meillon et al. (I945), Lea et al. (I956), Lea and DeLong
(1956), Nayar (1966)~ Singh
and Brown (I957), Akov (1962) reared larvae on a
sterile chemical media.
8/10/2019 Rearing Mosquitoes
36/124
Pupae can be washed to surface sterilize them, and adults may be fed on sterile
diets.
Thou gh it may be superfluous to mention, it is important to see that all wo rk in
rearing aseptic anim als is condu cted under com pletely aseptic cond itions.
References-
Akov (1962, 1964) ; Atkin and Bacot (1917) ; Barber (1927) ; Boorman (1967) ; Butt and Keller
(1961); Hinman (1930, 1932); Jonesand DeLong (1961); Lea et al. (1956); Lea and DeLong
(1956); de M ei on1 et al. (1945)
;
Lichtenstein (1948)
;
Martignoni and Milstead (1960);
Nayar (1966)
;
Roubaud and Colas-Belcour (1929)
;
Singh, K. R. P. and Brown (1957) ;
Trager (1935,
1937);
White (1931).
26
8/10/2019 Rearing Mosquitoes
37/124
III. PROCEDURES FOR LABORATORY REARING OF
SPECIFIC MOSQUITOES
Information gleaned from literature has been compiled to present a rearing tech-
nique for individual species,or in some casesa genus.
Usually, for each genus there
is a detailed description for at least one species hat could serve as a guide for rearing
other members of the genus.
The arrangement of genera and nomenclature is based on Stone, Knight and Starke
(1959).
SYSTBMATIC
ARRANGEMENT OF THOSE GENERA IN
ARE AVAILABLE
Subfamily Anophelinae
Genus
Anopheles
Subfamily Toxorhynchitinae
Genus Toxorhynchites
Subfamily Culicinae
Tribe Sabethini
Genus
Thchoprosopon
Genus
Wyeomyia
Genus
Sabethes
Tribe Culicini
Genus
Coquiltettidia
Genus
Mansonia
Genus
Uranotaenia
Genus
Ohopodomyia
Genus
Psorophora
Genus
Eretmapodites
Genus
Aedes
Genus
Armigeres
Genus
Haemagogus
Genus Opifex
Genus
Culiseta
Genus Culex
Genus
Deinocerites
ANOPHELES
WHICH REARING TECHNIQUES
Eggs may be collected in a small container, usually r5cm (6) in diameter or
15xrox5cm
(6x4~2).
Cotton balls, or an absorbent cotton pad, are laid inside the
container and then barely covered with water.
Filter paper or paper toweling is
then laid over the cotton pads so that the water surface is even with the surface of
8/10/2019 Rearing Mosquitoes
38/124
8/10/2019 Rearing Mosquitoes
39/124
coarse-ground mixture of equal parts of Kelloggs concentrate, wheat germ and live
yeast. At the 2nd instar they are transferred to clean pans with fresh water and food
(Keppler et al. 1964). Gilotra ( 1966)
reared 200-250 larvae in enamel pans
I I .5x7.5x2
containing I l/2 liters dechlorinated tap water, and fed on powdered
Purina laboratory chow supplemented with Fleischmanns yeast. The larval period
usually lasts
7-14
days when reared at 27 C. (80 F.).
Pupae
The
1936).
Ad&S
pupae are collected once a day and placed
The pupal stage lasts 30-33 hours.
in cups of fresh water (Rozeboom
The adults are maintained in cages goxgoxgocm (2.5x2.5x2.5. They are fed on a
5% honey solution or sugar solution. The females are provided a blood meal at
dusk. A rabbit is placed in the cage for 2 hours. The best temperature appears to
be 28'
C.
(SIF.) (D
owns and Arizmendi 1951). Adults are also maintained in
cages 6ox6ox6ocm
(2x2x2'),
and are fed on sugar water, and various fruits. The
females are provided with a blood meal (human arm) once a day. Cages r2xgxg
maintained at 65% RH are also used for adults. A guinea pig provides a blood meal
and adults are maintained on honey-saturated balls of cotton wool (Keppler et al.
1964). Adult longevity is approximately 30 days.
References-
Coluzzi (1964) ; Dow ns and Arlzmendi (1951); Gilotra (1966) ; Ikpplcr rt 4. (1964) ; Roze-
boom (1936).
Anopheles albitarsis
Lynch Arribalzaga
Referrnces-
Barrett0 and Coutinho (1943); Galvao and Grieco (~9~3) JGalvao et nl. (1944).
Anopheles at-gy&arsis
Robineau-Desvoidy
Refeyences-
Galvao et al.
(1944).
Anopheles axtecus Hoff mann
See methods used for A. quadrimaculatus and A. freeborni.
References-
Downs et
al. (1948);
Dow ns and Arizmendi ( 1 g5 I) ;
Wallis (I 955 ).
Anopheles ba.Zabacensi.s
Baisas
Eggs are collected on filter paper discs kept in contact with moist cotton pads.
The mean number of eggs per female is 163.
Eggs are removed from the filter
paper discs to enamel trays containing 2,500 ml of seasoned tap water over a layer
of sterilized stream sand.
The eggs hatch in 48-72 hours.
8/10/2019 Rearing Mosquitoes
40/124
8/10/2019 Rearing Mosquitoes
41/124
Anopheles crucians Wiedemann
See A. quadrimacuZatus techniques.
References-
Boy1 (I
926) ; Boyd et al. (1935).
Anopheles culicifacies
Giles
Reference-
Jayewickreme ( 1952)
;
Pal ( 1945).
Anopheles darlingi Root
Eggs are collected on moistened filter paper (Freire and Faria 1947) or in white
glazed fingerbowls (diam.
I I
cm, depth 7 cm) half filled with water. The eggs
are allowed to hatch in these bowls. Eggs hatch in 24-48 hours at 27 C. (80 F.).
Larvae
The larvae are reared in containers 49x7oxIocm or in white enamel basins (28 cm
in diam, and 8 cm deep). App
roximately
IOO
larvae are placed in the basin.
The
larvae are fed on an infusion made by mixing toasted fish and toasted bread in pro-
portions of I :2 plus a trace of brewers yeast. This mixture is suspended in water
4-5 days at 27O C. (80 F.), and should have a pH of 7.o-7.8. Giglioli (1947)
feeds larvae on dried brewers yeast or poultry laying mash or combination of both.
No special attempt is made to regulate light, temperature (70-M F.) or relative
humidity (66-95% RH). Th
e water used is clean, fresh, with a pH of
larval stage ranges from 6-12 days.
6-7. The
Pupae
The pupal period is approximately 2 days. The pupae are collected
placed in containers in the adult cage.
daily and
Ad&s
The adults are kept in cages 4ox4ox5ocm in an insectary maintained at
and 80-90~/~ RH. The time required from egg to imago is IO days. The
__ _
26-2a" c.
adults are
fed hony on cotton pads.
Adult longevity is 35-45 days. A cage 3 m high with
a 1x1-m base is required for mating (Freire and Faria 1947).
Giglioli ( 1947) main-
tains adults in a cylindrical cage 60 cm high and 35 cm in diameter, consisting of F
cotton mosquito gauze sleeve stretched over a IO gauge wire frame. The adults
are supplied with cotton wads soaked in cane sugar solution and changed daily.
Blood meals (human) are provided daily for l/2 hour in the morning. A tempera-
ture of 80 F. and a relative humidity of 85% seems optimal.
The first oviposition
usually occurs 6-8 days after emergence.
References-
Bates (I
947) ;
Freire and Faria (I
947) ;
Giglioli (I 947).
31
8/10/2019 Rearing Mosquitoes
42/124
8/10/2019 Rearing Mosquitoes
43/124
Larvae
The larval stage lasts from g-10 days.
Pupae
The pupal stage lasts for
I-I%
days.
Adults
Mating will occur in an insectary.
A minimum of
2
days after emergence is
required by adults before mating.
Eggs are laid
2
days after a blood meal. Females
will feed on man, and animals. Adults feed on fruit juices. Females will live for
51 days at 85% RH when provided with blood meals.
References-
J&m (1945); Perry (1946).
Anopheles fluuiatilis James
-
Eggs are collected in earthen pots, lined inside with mud and containing water.
The eggs are transferred to large enamel basins and dishes containing water. The
eggs are floated inside paraffined cork rings.
Larvae
The larvae are fed on hay infusion, plus a small quantity of a mixture of 2 parts
litmus milk and
I
part dehydrated blood serum.
Dried brewers yeast may be sub-
stituted for the above.
The water in the basin is aerated vigorously once each day.
Relatively larger amounts of yeast are added to the rearing basins when the larvae
reach the fourth instar.
Pupae
The pupae are removed from the larval rearing basins, placed in a bowl of clean
water and placed inside the colony cage.
A few blades of grass are floated in the
bowl to provide a foothold for the emerging adults.
Adults
The colony cage measures
2x2~2
and is placed inside a larger cage with solid
wooden sides and glass top.
Humidity is maintained by suspending pieces of cloth
in a saturated solution of common salt. Adults are fed on
IO~
glucose on cotton
wool. A rabbit provides nightly blood meals. Mating takes place only in the
presence of a blue
light.
Egg laying occurs 48-72 hours after a blood meal. Len-
gevity is
17-18 days at 27 C. and 60-80*/~ RH.
References-
Mohan (1945); Pal (1943); Sin& and M&an (1951); Viswanathan et al. (1944).
Anopkeles ft-eeborni Aitken
Qxs
Eggs are collected in a glass crystallizing dish containing distilled water.
Approxi-
mately 100-200 eggs are laid per female. The eggs hatch in 3 days at 220 C.
8/10/2019 Rearing Mosquitoes
44/124
8/10/2019 Rearing Mosquitoes
45/124
The larvae are reared in enameled iron pans 25cm (IO) in diameter in water
about r2.5-17mm (s-3/4) deep,
(A
rmstrong and Bransby-Williams
1961).
The
water is boiled, cooled stream water.
Approximately 150 larvae are added to each
pan. Powdered meat meal is provided as food. Powdered blood and yeast suspen-
sion used by Shute (1956)
seemed to produce heavy scum.
Wegesa ( 1964) com-
pares meat meal and yeast and finds adult emergence and survival rates significantly
increased by using meat meal.
A fine mesh tea strainer is used for applying the
food.
Larvae are reared in an enamel tray 35x3ox5cm at 26.5 C. and require
7-13
days to pupate.
Two hundred newly hatched larvae are placed in 2 liters of distilled
water. They are fed
0.2
g meat meal on alternate days.
Pupae
Pupae are collected daily and 300 placed in a 7.5cm (3) diameter aluminum pot
with a minimum of larval water.
Distilled water is added to bring the water level
within
r2.5-r7mm (l/2-3/4) of the top. Pupae are maintained at 26.5 C. & 0.5 OC.
Adult emergence is complete in about
2
days.
Eight hundred pupae are placed
in a cage.
Adults
Adults are kept for the first 7 days in cages 3ox3ox3ocm
(1x1~1') and then
transferred to larger stock cages 75x38x4ocm (30x15~16). The adults are main-
tained in an insulated, temperature controlled (26.5 C.) (70-90% RH) darkened
room.
The adults are maintained on sugar solution or corn syrup on cotton wool and
are offered a blood meal on the day after emergence, and on alternate days after
that. Rabbits are used as a source of blood meals. The back of the rat&it
is shaved
and the unanesthetized rabbit is placed in a box in the cage. Mating occurs at dusk
or in a darkened room or cage.
Maximum mating occurs 3-5 days after emergence
(Shute
1956).
Fertilized females take a blood meal more readily
than unfertilized
females.
Approximately 15.6/, survive 20 days (Wegesa 1964).
References-
Armstrong and Bransby-Williams (1961); Causey et al. (1943); Coluzzi (1964); Gillies
(1961) ; Goma (1959)
;
Haddow and Ssenkubuge (1962)
;
Jones,M. D. R. et al. (I
967)
; Mathis,
C. (1936); Mathis, M. (1935); Moores (1953); Muirhead-Thompson (1948); Philip (1930);
Shute, G.
T.
(1956); Wall (1953); Wegesa (1964).
Anopheles kymanus
Pallas
Anopheles jamesii Theobald
-
Eggs
are
collected in petri dishes 1% in diameter.
Larvae
Larvae are reared on a medium (pH 7.1)
consistingof mud water and tap \lrater.
Larvae are fed on brown bread powder.
Pupae
Pupation occurs IO days after oviposition.
8/10/2019 Rearing Mosquitoes
46/124
Adz&s
The adults are maintained in Barraud cages 6x6~6. They are fed water-soaked,
split raisins, placed on top of the cage and covered with a pad of dam p cotton w ool.
The adults emerge in 24 hours.
Reference-
Jayewickreme1952).
Anopheles labranchiae Falleroni
Anopheles labranchiae subsp.
atroparvus
Van Thiel
Eggs are collected on damp filter paper. The filter pape r is placed on wet cotton
in small plastic cups or in petri dishes. Eggs are left on damp filter p aper for at least
24 hours but not more than 72 before hatching. The eggs are removed with the
filter paper for hatching purposes.
Larvae
Approximately 300-40 0 larvae are placed in a white enamel or polyethylene bowl
34cm (14) in diameter, containing water to a depth of approximately 3-4 cm (I s").
The water temperature should be about 27 C. (80 F.). Mu d or a piece of grass
turf is used in add ition to artificial food. The use of these variables leads to non-
standardized mosquitoes.
Ground dog biscuits, grain foods and liver powder are
used successfully.
Pupae
Pupae are usually hand picked though mechanical means of separation may be
used.
Adults
One thousand or more adults are maintained in a 2x2~2' cage. The pup ae are
placed in the cage in sm all containers and the containers covered or rem oved after
adult emergence.
The adults are maintained on 10% sucrose soaked cotton wool or
cotton balls. Restrained guinea pigs are laid on top of a cage, or a human arm is
inserted into the cage to provide a blood m eal.
References-
DpAlessandrot al. (1961); Bertram and Gordon (1939); Coluzzi (1964); Meller (1962);
Shute,P. G. (19 36).
Anopheles maculatus Theobald
Each female lays about 80-100 eggs on moist filter paper, in individual tubes or
in paper cups,
(Ow
Yang
et al. 1963).
At the ICR laboratories the fertilized females
are placed in 1~1x1
alum inum screened cages and lay eggs in plastic containers.
Larvae
Jayewickreme (1952) reared larvae in a medium consisting of 20 parts hay infu-
36
8/10/2019 Rearing Mosquitoes
47/124
sion to 80 parts tap water.
75 cc of hay infusion was added daily. Larvae were
reared in an earthenware dish with a diameter of 20 cm and a surface of 314.2 sq cm
containing 500 cc of media.
Larvae were fed on the brown bread powder after the
4th day.
Pupae
Maximum pupation occurred on the 13th day after oviposition.
Adults
/
Maximum adult emergence occurs on the 15th day after oviposition. The adults
are maintained in Barraud cages 6x6~6 and are fed water-soaked raisins placed on
top of the cage and covered with a pad of damp cotton wool.
Jayewickreme (1952)
does not mention mating or egg production. Ow Yang et
al.
(1963) were able to
maintain a colony by artificial mating.
Sexed pupae (by size) emerged in separate
cages,
1~1x1
They were maintained at 27 C. and 70-90% RH and fed on 5%
glucose solution. Two days after emergence the glucose was removed and a guinea
pig was placed in the cage overnight. The following morning engorged females were
collected in individual tubes.
Males 3-6 days old were used for the artificial mating
(see techniques section).
References-
Jayewickreme
(1952); Ow
Yang etd. (1963).
Anopheles pharoensis Theobald
Eggs are collected in petri dishes containing water. The embryonic period is 2-4
days, depending upon temperature. The percentage of egg fertility varies from
35-95% with an emergence of 70% (Theodor and Parsons 1945).
Larvae
The optimum density of larvae is roe-150 first stage larvae and 50 third or fourth
stage larvae in a bowl of 15 cm diameter.
The rearing technique of Theodor and
ParSOnS 1945)
consistsof placing IOO first stage larvae into the white enamel bowl
containing a layer of mud I cm in depth and about 4 cm of water. The larvae are
daily fed a few drops of bakers yeast in water.
At the second stage, they are fed
powdered biscuits,
sprinkled on the surface of the water-sometimes 3 or 4 times
a day. The larval period is I 1-12 days at 28-29 C. At 23 C. the larval period
is 23 days and at 27O C. about 18 days.
The pupae are removed every morning and placed in a bowl inside the cage.
The
pupal period lasts 3-4 days at
20~
C.
The adults are maintained in a large cage 6ox6oxIoocm.
Humidity is maintained
by covering the sides and front of the cage with a moist blanket. The adults are
fed a sugar solution on a soaked cotton pad or sponge. A rabbit was first used for
blood meals, but a marked reluctance to feed was observed. Human blood is
readily
37
8/10/2019 Rearing Mosquitoes
48/124
acceptable. Swarming and
white light is turned on.
mating takes
place in blue light and
ceases
when a
References-
Abdel-Malek et al. (1966)
;
de Meillon et al. (1963); Theodor and Parsons (1945).
Anopheles pseudopunctipennis Theobald
References-
Downs and Arizmendi
(1951);
Hardman
1947).
Anopheles punctipennis (Say)
Reference-
Hardm an (I 947).
Anopheles punctulatus Donitz
References-
Backhouse
(1937);
MacKerras and Lemerle
(1949).
Anopheles quadrimaculatus Say
-
Eggs are collected in a pan or bow l of water, or on damp filter paper or toweling.
Avoid desiccation of the eggs. Gah an (1967 ) states that whenever a surplus of eggs
is obtained, the excess supply may be wrapped in damp filter paper o r toweling and
may be stored in a refrigerator for IO days to 2 weeks. Eggs are usually deposited
at night. Rep lace the egg collecting container daily. Eggs are concentrated and
introduced to larval rearing trays or the eggs are left to hatch and then co unted by
the aliquot method and placed in the rearing trays. The eggs hatch in 30-40 hours.
Larvae
Trays of various sizes
and composition are used.
White enamel
(14x10~4")
or white
plastic trays are used with su