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Serological comparison of three strains of Aedes aegypti
Comp. Biochem. PhysioL, Vol. 60B, pp. 257 to 260 © Pergamon Press Ltd 1978. Printed in Great Britain
0305-0491/78/0615-0257502.00/0
SEROLOGICAL COMPARISON OF THREE STRAINS OF AEDES A E G Y P T I EMMANUEL C. IGBOKWE* and AYLWARD E. R. DOWNE Department of Biology, Queen's University, Kingston, Ontario, Canada (Received 25 July 1977)
Abstract--Young adults of three strains of the .mosquito, Aedes aegypti, were compared serotogically by means of the double-immunodiffusion technique. 1. Strains and sexes were serologically distinguishable. 2. Differences in antigenic composition were evident among the strains and sexes. 3. Degree of intraspecific serological relationship varied with sex.
Electrophoresis The method of polyacrylamide gel electrophoresis described by Clark (1964) was employed in the fractionation of the mosquito extracts. For each extract, 0.2 ml was fractionated. Interpretation of the precipitin reactions were based on the principles established by Korngold (1956), Wilson & Pringle (1956), and Ouchterlony 0958).
INTRODUCTION In research on mosquitoes, the precipitin serological technique has been used to differentiate the sexes (Chao, 1969), to assess molecular and systematic relationship (Downe, 1963), and to compare metamorphic antigens (Smith & Silverman, 1966). Also, strains of other insects have been differentiated successfully by means of this technique (Fox, 1949; Nigam & Musgrave, 1964). The studies reported here compared three strains of Aedes aeoypti with reference to the serological characteristics of saline extracts obtained separately from adult male and female mosquitoes It was hoped that the comparison would provide a parameter for gauging the biochemical divergence in this mosquito.
Disc-immunodiffusion test Disc-immunodiffusion test was carried out as described by Seto & Hokama (1964). Following the fractionation of each mosquito extract by disc electrophoresis, the cylindrical polyacrylamide gel was placed directly in a matching agar trough. The challenge antiserum was placed in another trough, constructed parelled to that holding the polyacrylamide gel. Agar plates were prepared from 1%
MATERIALS AND METHODS Mosquito strains Three Ae. aegypti strains, designated WT, MYS and AO respectively, were investigated. The adults were easily distinguished by their morphological characteristics. WT originated from the National Institute of Health (U.S.A.) stock, and has been maintained in our laboratory for several years. MYS was the hybrid of Ae. aegypti and the closely related Ae. mascarensis. AO originated from an Algerian population of Aedes aegypti. Both the MYS and AO strains were supplied to us by Dr A. W. A. Brown (then at the University of Western Ontario, Canada) who obtained these from Dr G. B. Craig, Jr, of the University of Notre Dame (U.S.A.). All three strains were maintained in our laboratory by mass culture in population cages. Three-day old adults fed only 10% aqueous sucrose solution were collected for use in these studies. Mosquito extraction At least 500 individuals of each sex were pooled and weighed. The mosquitoes were homogenized in a predetermined volume of buffered physiological saline (0.13M NaC1 in 0.01 M phosphate buffer, pH 7), and extracted for 24 hr at 4°C. For extraction, 3 ml of saline were used per 0.4 g mosquito weight. The homogenate was centrifuged at 3600 rev/min for 30 rain. Each extract was sterilized by seitz filtration, bottled and frozen until required for use.
*Present address: Department of Biology, Rust College, Holly Springs, MS 38635, U.S.A. 257
G
d
b
C
i f
g
Fig. 1. Precipitin patterns obtained for three strains of Aedes aegypti with (A) anti-~3'WT-serum, and (B) anti~WT-serum, in Ouchterlony test.
258
EMMANUEL C. |GBOKWE and AYLWARD E. R. DOWNE
Bacto-Agar (Difco Co., Detroit, Michigan) in phosphatebuffered, physiological saline (0.26 M NaCI in 0.01 M phosphate buffer, pH 7.0). Agar gel plates were stored at 4°C for at least 48 hr before use. The tests were incubated for 19 days in a chamber maintained at 12°C and 80% relative humidity. The resultant precipitin patterns were observed and recorded as described for the Ouchterlony test, and interpreted in accordance with the immunoelectrophoretic principles described by Hirschfeld (1960).
Preparation of antisera Antisera to females and males of the WT strains were prepared separately in New Zealand white rabbits. A series of three consecutive 1 ml doses of extract was injected subcutaneously into a rabbit at 24-hr intervals. The rabbit was bled from the marginal vein of the ear 7 days following the final injection, and again on each of the following 2 days. Each sample of blood was refrigerated at 4°C for 30 min, and centrifuged at 3600 rev/min for 30 min at room temperature. The antibody titre of the antiserum was determined by the interfacial precipitin-ring test (Downe, 1962). Where necessary, antiserum was concentrated by dialysis against aqueous 30% solution of polyvinyl pyrrolidone. Only antisera with antibody titre of 1:640 were used in these studies. The antisera were bottled and frozen until required for use.
T
Double-immunodiffusion tests Ouchterlony test. The mosquito extracts were directly compared in agar immunodiffusion plates purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio) and Pentex Incorporated (Kankakee, Illinois). The plates were supplied with prepunched reagent wells. Each plate (50 mm dia) contained a central circular well (5 mm dia) and 6 peripheral circular wells (4 mm dia) located regularly and concentrically at a radius of 7 mm from the edge of the central well. These plates were stored at 4°C until required for use. Immunodiffusion tests were incubated for 11 consecutive days in an illuminated chamber maintained at 12°C and 80% relative humidity. Line drawings of the precipitin bands were made from visual inspection of the plates against an indirectly illuminated background. RESULTS A N D DISCUSSION
Ouchterlon y test
The precipitin patterns obtained from the Ouchterlony test are shown in Fig. 1. No changes in these patterns were observed following longer periods of incubation. Furthermore, identical precipitin patterns
DISTAL ~PROXIMAL L.. W T
g~
I.
I
I
1 wT
I
I MY S $~-
I
l
I
J
I
L AO ~-
I [
ANTI- ~ WT- SER.
ANTI- @@
WT- SER.
-I
MYS g g
W T - SER.
~--AO g@
Fig. 2. Precipitin patterns obtained for three strains of Aedes aegypti with anti-~3WT-serum in Discimmunodiffusion test.
Serological comparison of three strains of Aedes aegypti
T
259
DISTAL ~ PROXIMAL
!
.]
WT ~-£
,]
ANTI- 2@
WT-SER.
I
I
] MYS ?~£
I
j ANTI- -~@ WT-SER.
I
I MYS
I
.].. Ao ~-~
I__
]
I
WT-SER.
.,J
Fig. 3. Precipitin patterns obtained for three strains of Aedes aegypti with anti-~WT-serum in Discimmunodiffusion test. were obtained with lots of antisera obtained from different rabbits. Although indicating strain and sex differences, the patterns also suggest varying degrees of serological relationship among the strains. Intraspecific differences in the number of precipitin bands were observed. All sexes and strains shared identical antigens represented by fractions a (Fig. 1A) and f (Fig. 1B)~ Band a developed more slowly and less intensely against both sexes of AO, less so against AO males. The corresponding thickness and gel location of bands a and f suggest that they probably represent an antigen or a composite of similar antigens present in the highest concentration and characterized by the smallest molecular size in the mosquito extracts. A similar relationship among antigens was described by Korngold & van Leeuwen (1957). In reference to anti-g~WT-serum (Fig. IA), fraction b was common to females of MYS and AO, c to males of MYS and AO, and d to both sexes of WT. All three fractions, b, c and d, correspond partially to one another. Against antigens of females of MYS and AO, only fractions a and b were obtained for each strain. Fraction e was observed only in MYS male. Three fractions were obtained for each sex of
WT. In reference to anti-~WT-serum (Fig. 1B), fractions f and g were common to females of WT, MYS and AO. However, females of WT and AO shared the additional fraction h. Among the males, WT and MYS shared fraction i which corresponds partially to fraction # in the females of all three strains. Fraction l in AO male corresponds partially to fractions g and i in both sexes of other strains Fractions j and k were observed only in MYS male. On the basis of these results, varying degrees of qualitative serological relationship were deduced among the strains. The precipitin reaction between WT strains and the corresponding antiserum was used as reference in assessing serological relationship among the strains. In the test with anti-c~c~WT-serum, MYS and AO in both sexes shared a closer relationship. With anti-9~WT-serum, AO and WT shared a closer relationship among females, whereas WT and MYS were closer among males. Disc-immunodiffusion test
The precipitin patterns obtained in the discimmunodiffusion test are shown in Figs 2 and 3.
260
EMMANUEL C. IGBOKWE and AYLWARD E. R. DOWNF
These indicate a greater antigenic complexity among the strains than was obtained in the Ouchterlony test. In the test with anti-3~WT-serum (Fig. 2), MYS and AO shared a closer resemblance to their patterns. This was found to be in accord with the order of relationship established through the Ouchterlony test. The strongest resemblance in all three strains was found among the distal, elongate precipitin bands, collectively labelled 1 in tests with both reference antisera. Although differing in shape between the strains and sexes, these bands appeared to represent a common antigen. These bands were convex toward the antigen trough in A O and WT, but concave in MYS. According to Korngold & van Leeuwen (1957), the convexity of a precipitin band toward the antigen well in a double-diffusion test suggests a higher diffusion rate and a lower molecular weight for a given antigen as compared to its corresponding antibody, and vice versa for bands concave toward the antigen well. Differences in number and length of bands were observed between the sexes. The bands labelled 2 and 3, although differing in length in both sexes of MYS, probably represent corresponding antigens. The differences in band-length was taken to suggest a higher concentration of this antigen in MYS male as compared to the other strains. In both sexes of AO, the bands numbered 4 probably represent immunologically similar but electrophoretically dissimilar antigens, hence their location at slightly different positions. Among the three strains, more bands were obtained in W T in the distal half of the precipitin patterns, that is, among the faster-moving electrophoretic fractions. With anti-'~WT-serum, but not a n t i - ~ W T - s e r u m , resemblances within strains were stronger than resemblances between strains (Figs 2 and 3). However, among all three strains, W T and A O shared closer resemblance in both sexes, as also indicated by the results of the Ouchterlony test. It is not known how the genetic variability and other known sex differences in this mosquito bear upon the serological variability observed in these studies. Inherited protein polymorphism and other genetic differences have been described in Aedes aegypti (Craig & Hickey, 1967; Trebatowski & Craig, 1969; Trebatowski & Haynes, 1969). Similarly, sexspecific proteins (Freyvogel et al., 1968; Fuchs et al., 1969; Townson, 1969) and antigens (Allen & West, 1966) have been reported in this mosquito. In any event, the reproducibility of the serological precipitin pattern obtained in these studies is considered as adequate justification for the use of pooled homogenates in the serological comparison of the mosquito strains described here. Acknowledgements--We wish to thank Dr A. S. West for several useful suggestions in the course of this study.
We gratefully acknowledge the National Research Council of Canada for providing funds (Grant No. A 2368 to Dr A. E. R. Downe and Grant No. 97 to Dr A. S. West) in support of this investigation.
REFERENCES ALLEN J. R. & WEST A. S. (1966) Some properties of oral secretion from Aedes aegypti. Expl. Parasit. 19, 124-131. CHAO J. (1969) Agar gel immunodiffusion and immunoelectrophoresis studies with the extracts of male and female adult Culex tarsalis. Mosquito News 29. 4 6. CLARK J. Z. (1964) Simplified "disc" (polyacrylamide gel) electrophoresis. Ann. N . Y Acad. Sci. 121,428-436. CRAIG G. B. t~ HICKEY W. A. (1967) Genetics of Aedes aegypti. In Genetics ~f Insect Vectors of Disease, 67 131. Elsevier, Amsterdam. DOWNE A. E. R. (1962) Serology of insect proteins. Preliminary studies on the proteins of insect cuticle. Can. J. Zool. 40, 957 967. DOWNE A. E. R. (1963) Comparative serology of four species of Aedes [Ochlerotatus). Science 139, 1286-1287. Fox A. S. (1949) Immunogenetic studies of Drosophila melanooaster. The development of techniques and the detection of strain differences. J. Immun. 62, 13-27. FREYVOGEL T. A., HUNTER R. L. & SMITH E. M. (1968) Nonspecific esterases in mosquitoes. J. Histochem. Cytochem. 16, 765-790. FUCHS M. S., CRAIG G. I. t~¢ DESPOMMIERD. D. (1969) The protein nature of the substance inducing female monogamy in Aedes aegypti. J. Insect Physiol. 15, 701-709. HIRSCHEELD J. (1960) Immunoelectrophoresis--procedure and application to the study of group-specific variations in sera. Sci. Tools 7, 18 25. KORNGOLD L. (1956) Immunological cross-reactions studied by the Ouchterlony gel diffusion technique. J. Immun. 77, 119-122. KORNGOLD L. t~ VAN LEEWENG. (1957) The effect of the antigens molecular weight on the curvature of the precipitin lines in the Ouchterlony technique. J. lmmun. 78. 172-177. NIGAM P. C. & MUSGRAVEA. J. (1964) Effect of DDT and starvation on the antigenic composition of Sitophilus granarius (L.) (Coleoptera), GG strain, and a comparison with other strains and species, using the Ouchterlony technique. Can. J. Zool. 42, 1041-1048. SETOJ. T. & HOKAMAY. (1964) Disc electrophoresis analysis of Sialidae from influence virus. Ann. N.Y. Acad. Sci. 121, 640-650. SMrm S. & SILVERMANP. H. (1966) Metamorphic antigens of mosquitoes. Mosquito News 26, 544-551. TOWNSONH. (1969) Esterase isozymes of individual Aedes aegypti. Ann. trop. Med. Parasit. 63, 413-418. TREBATOWSKI A. M. & CaAIG G. B. (1969) Genetics of an esterase in Aedes aeoypti. Biochem. Genet. 3, 383-392. TREBATOWSKIA. M. & HAYNES J. F. (1969) Comparison of enzymes of twelve species of mosquitoes. Ann. ent. Soc. Amer. 62, 327 335. WILSON M. W. & PRINGLE g. H. (1956) Cross reactions in the Ouchterlony plate; analysis of natural and halogenated bovine serum albumins. J. Immun. 77, 324-331.
0305-0491/78/0615-0257502.00/0
SEROLOGICAL COMPARISON OF THREE STRAINS OF AEDES A E G Y P T I EMMANUEL C. IGBOKWE* and AYLWARD E. R. DOWNE Department of Biology, Queen's University, Kingston, Ontario, Canada (Received 25 July 1977)
Abstract--Young adults of three strains of the .mosquito, Aedes aegypti, were compared serotogically by means of the double-immunodiffusion technique. 1. Strains and sexes were serologically distinguishable. 2. Differences in antigenic composition were evident among the strains and sexes. 3. Degree of intraspecific serological relationship varied with sex.
Electrophoresis The method of polyacrylamide gel electrophoresis described by Clark (1964) was employed in the fractionation of the mosquito extracts. For each extract, 0.2 ml was fractionated. Interpretation of the precipitin reactions were based on the principles established by Korngold (1956), Wilson & Pringle (1956), and Ouchterlony 0958).
INTRODUCTION In research on mosquitoes, the precipitin serological technique has been used to differentiate the sexes (Chao, 1969), to assess molecular and systematic relationship (Downe, 1963), and to compare metamorphic antigens (Smith & Silverman, 1966). Also, strains of other insects have been differentiated successfully by means of this technique (Fox, 1949; Nigam & Musgrave, 1964). The studies reported here compared three strains of Aedes aeoypti with reference to the serological characteristics of saline extracts obtained separately from adult male and female mosquitoes It was hoped that the comparison would provide a parameter for gauging the biochemical divergence in this mosquito.
Disc-immunodiffusion test Disc-immunodiffusion test was carried out as described by Seto & Hokama (1964). Following the fractionation of each mosquito extract by disc electrophoresis, the cylindrical polyacrylamide gel was placed directly in a matching agar trough. The challenge antiserum was placed in another trough, constructed parelled to that holding the polyacrylamide gel. Agar plates were prepared from 1%
MATERIALS AND METHODS Mosquito strains Three Ae. aegypti strains, designated WT, MYS and AO respectively, were investigated. The adults were easily distinguished by their morphological characteristics. WT originated from the National Institute of Health (U.S.A.) stock, and has been maintained in our laboratory for several years. MYS was the hybrid of Ae. aegypti and the closely related Ae. mascarensis. AO originated from an Algerian population of Aedes aegypti. Both the MYS and AO strains were supplied to us by Dr A. W. A. Brown (then at the University of Western Ontario, Canada) who obtained these from Dr G. B. Craig, Jr, of the University of Notre Dame (U.S.A.). All three strains were maintained in our laboratory by mass culture in population cages. Three-day old adults fed only 10% aqueous sucrose solution were collected for use in these studies. Mosquito extraction At least 500 individuals of each sex were pooled and weighed. The mosquitoes were homogenized in a predetermined volume of buffered physiological saline (0.13M NaC1 in 0.01 M phosphate buffer, pH 7), and extracted for 24 hr at 4°C. For extraction, 3 ml of saline were used per 0.4 g mosquito weight. The homogenate was centrifuged at 3600 rev/min for 30 rain. Each extract was sterilized by seitz filtration, bottled and frozen until required for use.
*Present address: Department of Biology, Rust College, Holly Springs, MS 38635, U.S.A. 257
G
d
b
C
i f
g
Fig. 1. Precipitin patterns obtained for three strains of Aedes aegypti with (A) anti-~3'WT-serum, and (B) anti~WT-serum, in Ouchterlony test.
258
EMMANUEL C. |GBOKWE and AYLWARD E. R. DOWNE
Bacto-Agar (Difco Co., Detroit, Michigan) in phosphatebuffered, physiological saline (0.26 M NaCI in 0.01 M phosphate buffer, pH 7.0). Agar gel plates were stored at 4°C for at least 48 hr before use. The tests were incubated for 19 days in a chamber maintained at 12°C and 80% relative humidity. The resultant precipitin patterns were observed and recorded as described for the Ouchterlony test, and interpreted in accordance with the immunoelectrophoretic principles described by Hirschfeld (1960).
Preparation of antisera Antisera to females and males of the WT strains were prepared separately in New Zealand white rabbits. A series of three consecutive 1 ml doses of extract was injected subcutaneously into a rabbit at 24-hr intervals. The rabbit was bled from the marginal vein of the ear 7 days following the final injection, and again on each of the following 2 days. Each sample of blood was refrigerated at 4°C for 30 min, and centrifuged at 3600 rev/min for 30 min at room temperature. The antibody titre of the antiserum was determined by the interfacial precipitin-ring test (Downe, 1962). Where necessary, antiserum was concentrated by dialysis against aqueous 30% solution of polyvinyl pyrrolidone. Only antisera with antibody titre of 1:640 were used in these studies. The antisera were bottled and frozen until required for use.
T
Double-immunodiffusion tests Ouchterlony test. The mosquito extracts were directly compared in agar immunodiffusion plates purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio) and Pentex Incorporated (Kankakee, Illinois). The plates were supplied with prepunched reagent wells. Each plate (50 mm dia) contained a central circular well (5 mm dia) and 6 peripheral circular wells (4 mm dia) located regularly and concentrically at a radius of 7 mm from the edge of the central well. These plates were stored at 4°C until required for use. Immunodiffusion tests were incubated for 11 consecutive days in an illuminated chamber maintained at 12°C and 80% relative humidity. Line drawings of the precipitin bands were made from visual inspection of the plates against an indirectly illuminated background. RESULTS A N D DISCUSSION
Ouchterlon y test
The precipitin patterns obtained from the Ouchterlony test are shown in Fig. 1. No changes in these patterns were observed following longer periods of incubation. Furthermore, identical precipitin patterns
DISTAL ~PROXIMAL L.. W T
g~
I.
I
I
1 wT
I
I MY S $~-
I
l
I
J
I
L AO ~-
I [
ANTI- ~ WT- SER.
ANTI- @@
WT- SER.
-I
MYS g g
W T - SER.
~--AO g@
Fig. 2. Precipitin patterns obtained for three strains of Aedes aegypti with anti-~3WT-serum in Discimmunodiffusion test.
Serological comparison of three strains of Aedes aegypti
T
259
DISTAL ~ PROXIMAL
!
.]
WT ~-£
,]
ANTI- 2@
WT-SER.
I
I
] MYS ?~£
I
j ANTI- -~@ WT-SER.
I
I MYS
I
.].. Ao ~-~
I__
]
I
WT-SER.
.,J
Fig. 3. Precipitin patterns obtained for three strains of Aedes aegypti with anti-~WT-serum in Discimmunodiffusion test. were obtained with lots of antisera obtained from different rabbits. Although indicating strain and sex differences, the patterns also suggest varying degrees of serological relationship among the strains. Intraspecific differences in the number of precipitin bands were observed. All sexes and strains shared identical antigens represented by fractions a (Fig. 1A) and f (Fig. 1B)~ Band a developed more slowly and less intensely against both sexes of AO, less so against AO males. The corresponding thickness and gel location of bands a and f suggest that they probably represent an antigen or a composite of similar antigens present in the highest concentration and characterized by the smallest molecular size in the mosquito extracts. A similar relationship among antigens was described by Korngold & van Leeuwen (1957). In reference to anti-g~WT-serum (Fig. IA), fraction b was common to females of MYS and AO, c to males of MYS and AO, and d to both sexes of WT. All three fractions, b, c and d, correspond partially to one another. Against antigens of females of MYS and AO, only fractions a and b were obtained for each strain. Fraction e was observed only in MYS male. Three fractions were obtained for each sex of
WT. In reference to anti-~WT-serum (Fig. 1B), fractions f and g were common to females of WT, MYS and AO. However, females of WT and AO shared the additional fraction h. Among the males, WT and MYS shared fraction i which corresponds partially to fraction # in the females of all three strains. Fraction l in AO male corresponds partially to fractions g and i in both sexes of other strains Fractions j and k were observed only in MYS male. On the basis of these results, varying degrees of qualitative serological relationship were deduced among the strains. The precipitin reaction between WT strains and the corresponding antiserum was used as reference in assessing serological relationship among the strains. In the test with anti-c~c~WT-serum, MYS and AO in both sexes shared a closer relationship. With anti-9~WT-serum, AO and WT shared a closer relationship among females, whereas WT and MYS were closer among males. Disc-immunodiffusion test
The precipitin patterns obtained in the discimmunodiffusion test are shown in Figs 2 and 3.
260
EMMANUEL C. IGBOKWE and AYLWARD E. R. DOWNF
These indicate a greater antigenic complexity among the strains than was obtained in the Ouchterlony test. In the test with anti-3~WT-serum (Fig. 2), MYS and AO shared a closer resemblance to their patterns. This was found to be in accord with the order of relationship established through the Ouchterlony test. The strongest resemblance in all three strains was found among the distal, elongate precipitin bands, collectively labelled 1 in tests with both reference antisera. Although differing in shape between the strains and sexes, these bands appeared to represent a common antigen. These bands were convex toward the antigen trough in A O and WT, but concave in MYS. According to Korngold & van Leeuwen (1957), the convexity of a precipitin band toward the antigen well in a double-diffusion test suggests a higher diffusion rate and a lower molecular weight for a given antigen as compared to its corresponding antibody, and vice versa for bands concave toward the antigen well. Differences in number and length of bands were observed between the sexes. The bands labelled 2 and 3, although differing in length in both sexes of MYS, probably represent corresponding antigens. The differences in band-length was taken to suggest a higher concentration of this antigen in MYS male as compared to the other strains. In both sexes of AO, the bands numbered 4 probably represent immunologically similar but electrophoretically dissimilar antigens, hence their location at slightly different positions. Among the three strains, more bands were obtained in W T in the distal half of the precipitin patterns, that is, among the faster-moving electrophoretic fractions. With anti-'~WT-serum, but not a n t i - ~ W T - s e r u m , resemblances within strains were stronger than resemblances between strains (Figs 2 and 3). However, among all three strains, W T and A O shared closer resemblance in both sexes, as also indicated by the results of the Ouchterlony test. It is not known how the genetic variability and other known sex differences in this mosquito bear upon the serological variability observed in these studies. Inherited protein polymorphism and other genetic differences have been described in Aedes aegypti (Craig & Hickey, 1967; Trebatowski & Craig, 1969; Trebatowski & Haynes, 1969). Similarly, sexspecific proteins (Freyvogel et al., 1968; Fuchs et al., 1969; Townson, 1969) and antigens (Allen & West, 1966) have been reported in this mosquito. In any event, the reproducibility of the serological precipitin pattern obtained in these studies is considered as adequate justification for the use of pooled homogenates in the serological comparison of the mosquito strains described here. Acknowledgements--We wish to thank Dr A. S. West for several useful suggestions in the course of this study.
We gratefully acknowledge the National Research Council of Canada for providing funds (Grant No. A 2368 to Dr A. E. R. Downe and Grant No. 97 to Dr A. S. West) in support of this investigation.
REFERENCES ALLEN J. R. & WEST A. S. (1966) Some properties of oral secretion from Aedes aegypti. Expl. Parasit. 19, 124-131. CHAO J. (1969) Agar gel immunodiffusion and immunoelectrophoresis studies with the extracts of male and female adult Culex tarsalis. Mosquito News 29. 4 6. CLARK J. Z. (1964) Simplified "disc" (polyacrylamide gel) electrophoresis. Ann. N . Y Acad. Sci. 121,428-436. CRAIG G. B. t~ HICKEY W. A. (1967) Genetics of Aedes aegypti. In Genetics ~f Insect Vectors of Disease, 67 131. Elsevier, Amsterdam. DOWNE A. E. R. (1962) Serology of insect proteins. Preliminary studies on the proteins of insect cuticle. Can. J. Zool. 40, 957 967. DOWNE A. E. R. (1963) Comparative serology of four species of Aedes [Ochlerotatus). Science 139, 1286-1287. Fox A. S. (1949) Immunogenetic studies of Drosophila melanooaster. The development of techniques and the detection of strain differences. J. Immun. 62, 13-27. FREYVOGEL T. A., HUNTER R. L. & SMITH E. M. (1968) Nonspecific esterases in mosquitoes. J. Histochem. Cytochem. 16, 765-790. FUCHS M. S., CRAIG G. I. t~¢ DESPOMMIERD. D. (1969) The protein nature of the substance inducing female monogamy in Aedes aegypti. J. Insect Physiol. 15, 701-709. HIRSCHEELD J. (1960) Immunoelectrophoresis--procedure and application to the study of group-specific variations in sera. Sci. Tools 7, 18 25. KORNGOLD L. (1956) Immunological cross-reactions studied by the Ouchterlony gel diffusion technique. J. Immun. 77, 119-122. KORNGOLD L. t~ VAN LEEWENG. (1957) The effect of the antigens molecular weight on the curvature of the precipitin lines in the Ouchterlony technique. J. lmmun. 78. 172-177. NIGAM P. C. & MUSGRAVEA. J. (1964) Effect of DDT and starvation on the antigenic composition of Sitophilus granarius (L.) (Coleoptera), GG strain, and a comparison with other strains and species, using the Ouchterlony technique. Can. J. Zool. 42, 1041-1048. SETOJ. T. & HOKAMAY. (1964) Disc electrophoresis analysis of Sialidae from influence virus. Ann. N.Y. Acad. Sci. 121, 640-650. SMrm S. & SILVERMANP. H. (1966) Metamorphic antigens of mosquitoes. Mosquito News 26, 544-551. TOWNSONH. (1969) Esterase isozymes of individual Aedes aegypti. Ann. trop. Med. Parasit. 63, 413-418. TREBATOWSKI A. M. & CaAIG G. B. (1969) Genetics of an esterase in Aedes aeoypti. Biochem. Genet. 3, 383-392. TREBATOWSKIA. M. & HAYNES J. F. (1969) Comparison of enzymes of twelve species of mosquitoes. Ann. ent. Soc. Amer. 62, 327 335. WILSON M. W. & PRINGLE g. H. (1956) Cross reactions in the Ouchterlony plate; analysis of natural and halogenated bovine serum albumins. J. Immun. 77, 324-331.