Edman & Haeger:
1977
\Ve thank
J. Med. Entomol.
Schmid, Sherwood Evans, Charles Hansen and Hilda Lynn for technical assistance. LITERATURE CITED
Bidlingumyer, W. L. 1967. A comparison of trapping methods for adult mosquitoes: speciesresponse and environmental influence. J. Med. Entornol. 4: 200-20. Edman, J. D. 1971. Host-feeding patterns of Florida mosquitoes. I. Aedes, Arzopheles, Coquillettidia, Mansonia and PsoroPhora. J. Med. Entornol. 8: 687-95. 1974. Host-feeding patterns of Florida mosquitoes. IV. Deinocerites. J. Med. Entomot. 11: 105-07. Edman, J. D. & W. L. Bidlingmayer. 1969. Flight capacity of blood engorged mosqutoes. Mosq. News 29: 386-92. Lumsden, W. H. R. 1958. A trap for insects biting small vertebrates. Nature, London 181: 819-20. O'Meara, G. F. & J. D. Edman. 1975. Autogenous egg production in the salt-march mosquito, Aedes taeniorhynchus. Bioi. Bull. 149: 384-96. Seabrook, E. L. & T. E. Duffey. 1946. The occurrence of Megarhinus rutelus Coq. in S.E. Florida. Mosq. News 6: 193-94. Srihongse, S. & C. M. Johnson. 1965. Wyeomyia subgroup of arbovirus: isolation from man. SciCllce 149: 863-64. 1967. The isolation of Ilheus virus from man in Panama. Am. J. Trop. Med. Hyg. 16: 516-18.
Lawrence \Vebber, Amelia
24 December
Vol. 14, no. 4: 479-483
1977
ISOZYMES OF CULEX
II.
PIPIENS QUINQUEFASCIATUS (DIPTERA: CULICIDAE) Genetics of an alcohol dehydrogenase I
By Sudhir Narang2,
Satish C. Bhalla3, Neelam. Narang2 and Miriam. Tavares-Souza4
Abstract: Three zones of ADH activity were observed in the mosquito Culex PiPims quinquefasciatus. Variants were found only in zone I. Genetic studies on these variants suggest that the codominant alleles Adh-l S and Adh-l F are responsible for the 3 phenotypes observed (slow band, fast band and the 3banded pattern). The Adh-Ilocus is located in linkage group II. The gene order is Adh-I-y-ru. The average recombination in the Adh-I-y segment is 9.4 ± 1.01. Gene frequency studies showed that Adh-l S was the more abundant allele in all natural populations tested. Most of the laboratory populations reared for over 3 years showed fixation of this allele. ADH isozymes
'This work was supported by grants from the National Council of Scientific and Technological Development-CNPQ, Brazil and the National Institutes of Health, U.S.A. (Grant No. AI-I 1725). "Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil. 'University of Maryland School of Medicine, International Health Program, 660 West Redwood Street, Baltimore, Maryland 21201, U.S.A. 'University of Bahia, Salvador, Brazil.
show relatively low substrate specificityto various alcohols tested.
The mosquito Culex pipiens quinquefasciatus Say (=Cx. p.fatigans Wiedemann) is an important vector of filariasis in the tropics and the subtropics. In recent years a fair amount of genetic information has become available for this species. However, genetics of the polymorphic enzyme systems in this mosquito have been neglected. Outside of a few general studies on enzyme polymorphism (Miles 1974) and specific studies on esterases, malate dehydrogenases and phosphoglucomutases (Bullini & Colluzi 1974, Cheng & Hacker 1976, Garnett & French 1971, Narang & Narang 1975, Narang et al. 1977, Pasteur & Sinegre 1975, Simon 1969, Stordeur 1976), genetic studies of isozymes are practically nonexistant. In view of the lack of
Downloaded from http://jme.oxfordjournals.org/ by guest on June 7, 2016
species had consistently low engorgement rates, even lower than those observed among other autogenous forms, i.e., Aedes taeniorhynchus and Deinocerites cancer, in the same forest (Edman 1974). However, it should be noted that Wyeomyia were captured in suction traps while actively flying, rather than with aspirators while at rest, as in the case of Ae. taeniorhynchus and De. cancer. The engorgement rate of Wy. vanduzeei was similar to that for Wy. mitchellii. Thus, these field data are not indicative of the different blood requirements observed in the laboratory. Since Wy. mitchellii appears to be more abundant in inland rather than coastal forests, thc question arises as to whether its poorer success in coastal areas might be related to lower host populations (O'Meara & Edman 1975), coupled with its apparent inability to develop eggs without a blood meal. Extensive investigations on the bionomics of Wyeonryia in Florida, which should answer these and other questions, are currently in progress at the FMEL. Ackllowledgmm/s:
479
Florida Wyeornyia host-feeding patterns
J. Moo.
480
+
Adh-I F Hybrid
ADHI [
Adh-IS
-----
ADHm
----
FIG. I. Schematic drawing showing isozyme pattern in Culex pipims quinquifasciatus larvae. The 3 zones of ADH activity are shown on the left and the 3 bands (fast, hybrid and slow) of Adh-l locus are shown on the right. Solid lines=intensely staining, dashed lines = faintly staining.
sufficient genetic markers with high penetrance and uniform expressivity in this species, a need for more biochemical genetic markers can hardly be overemphasized. The zymogram techniques, in addition to providing new genetic markers, provide a vary handy tool to uncover the hidden (cryptic) variability in natural populations. This, in turn, can provide important baseline data of great potential value in any future genetic control strategy of the vector species (Narang et al. 1977, Sakai et al. 1973). Efficient use of the zymogram technique involves the scoring of alleles at loci encoding particular enzymes, rather than mere comparison of numbers and positions of bands of enzyme activity on gels. This approach requires formal progeny studies to establish the number of loci and to support genetic interpretation of zymogram patterns (Wagner & Selander 1974). These studies were undertaken to investigate the following: (1) various isozyme patterns of alcohol
Vol. 14, no. 4
dehydrogenase, (2) the genetic basis of various electrophoretic forms, and (3) the isozyme variability in natural populations of Cx. p. quillqueJasciatus. MATERIALS
AND METHODS
The following strains of Culex p. quinqueJasciatus were used for genetic studies. (I) URUGUAI: This strain was established in 1972 from a locality of the same name in Salvador, Brazil. A subs train homozygous for Adh-l F (fast) allele was isolated from this strain; (2) Y TU: This strain was isolated by inbreeding mosquitoes collected in Salvador, Brazil (Bhalla & Tavares-Souza 1975). A substrain homozygous for Adh-l S (slow) allele was isolated from this strain. Single pair matings were set up and both parents of each cross were subjected to electrophoresis after the female had oviposited. This permitted the isolation of pure homozygous stocks for various electrophoretic variants (allozymes). When necessary, the parents were stored at 25-60°C below zero and were assayed simultaneously with the F I offspring on the same gel. Electrophoresis in acrylamide gel was performed as described earlier (Narang & Kitzmiller 1972). The zymograms were developed first by incubating the gel in a solution containing 12 mg B-nicotinamide adenine dinucleotide, 8 mgp-nitro blue tetrazolium, I ml isopropanol, 1 ml octanol and 50 ml of 0.1 M tris-Hel buffer pH 8.5 and then adding 2 mg phenazine methosulphate. For the substrate test, various substrates were substituted in the incubation mixture. The natural populations sampled for gene
FIG. 2. Electrophoretic pattern of an alcohol dehydrogenase locus (Adh-l) in Cule.~ Pipiens quinquejasciatus. Refer to the schematic drawing for explanation.
Downloaded from http://jme.oxfordjournals.org/ by guest on June 7, 2016
ADH II
EntomoI.
Narang et al.:
1977
Cul~x p. quinqUl!fasciatus
genetics
481
Inheritance of Adh-l electrophoretic variants in Cx. p.fatigans. PROGENY PHENOTYPE PARENTAL·
1977
\Ve thank
J. Med. Entomol.
Schmid, Sherwood Evans, Charles Hansen and Hilda Lynn for technical assistance. LITERATURE CITED
Bidlingumyer, W. L. 1967. A comparison of trapping methods for adult mosquitoes: speciesresponse and environmental influence. J. Med. Entornol. 4: 200-20. Edman, J. D. 1971. Host-feeding patterns of Florida mosquitoes. I. Aedes, Arzopheles, Coquillettidia, Mansonia and PsoroPhora. J. Med. Entornol. 8: 687-95. 1974. Host-feeding patterns of Florida mosquitoes. IV. Deinocerites. J. Med. Entomot. 11: 105-07. Edman, J. D. & W. L. Bidlingmayer. 1969. Flight capacity of blood engorged mosqutoes. Mosq. News 29: 386-92. Lumsden, W. H. R. 1958. A trap for insects biting small vertebrates. Nature, London 181: 819-20. O'Meara, G. F. & J. D. Edman. 1975. Autogenous egg production in the salt-march mosquito, Aedes taeniorhynchus. Bioi. Bull. 149: 384-96. Seabrook, E. L. & T. E. Duffey. 1946. The occurrence of Megarhinus rutelus Coq. in S.E. Florida. Mosq. News 6: 193-94. Srihongse, S. & C. M. Johnson. 1965. Wyeomyia subgroup of arbovirus: isolation from man. SciCllce 149: 863-64. 1967. The isolation of Ilheus virus from man in Panama. Am. J. Trop. Med. Hyg. 16: 516-18.
Lawrence \Vebber, Amelia
24 December
Vol. 14, no. 4: 479-483
1977
ISOZYMES OF CULEX
II.
PIPIENS QUINQUEFASCIATUS (DIPTERA: CULICIDAE) Genetics of an alcohol dehydrogenase I
By Sudhir Narang2,
Satish C. Bhalla3, Neelam. Narang2 and Miriam. Tavares-Souza4
Abstract: Three zones of ADH activity were observed in the mosquito Culex PiPims quinquefasciatus. Variants were found only in zone I. Genetic studies on these variants suggest that the codominant alleles Adh-l S and Adh-l F are responsible for the 3 phenotypes observed (slow band, fast band and the 3banded pattern). The Adh-Ilocus is located in linkage group II. The gene order is Adh-I-y-ru. The average recombination in the Adh-I-y segment is 9.4 ± 1.01. Gene frequency studies showed that Adh-l S was the more abundant allele in all natural populations tested. Most of the laboratory populations reared for over 3 years showed fixation of this allele. ADH isozymes
'This work was supported by grants from the National Council of Scientific and Technological Development-CNPQ, Brazil and the National Institutes of Health, U.S.A. (Grant No. AI-I 1725). "Institute of Biological Sciences, University of Brasilia, Brasilia, Brazil. 'University of Maryland School of Medicine, International Health Program, 660 West Redwood Street, Baltimore, Maryland 21201, U.S.A. 'University of Bahia, Salvador, Brazil.
show relatively low substrate specificityto various alcohols tested.
The mosquito Culex pipiens quinquefasciatus Say (=Cx. p.fatigans Wiedemann) is an important vector of filariasis in the tropics and the subtropics. In recent years a fair amount of genetic information has become available for this species. However, genetics of the polymorphic enzyme systems in this mosquito have been neglected. Outside of a few general studies on enzyme polymorphism (Miles 1974) and specific studies on esterases, malate dehydrogenases and phosphoglucomutases (Bullini & Colluzi 1974, Cheng & Hacker 1976, Garnett & French 1971, Narang & Narang 1975, Narang et al. 1977, Pasteur & Sinegre 1975, Simon 1969, Stordeur 1976), genetic studies of isozymes are practically nonexistant. In view of the lack of
Downloaded from http://jme.oxfordjournals.org/ by guest on June 7, 2016
species had consistently low engorgement rates, even lower than those observed among other autogenous forms, i.e., Aedes taeniorhynchus and Deinocerites cancer, in the same forest (Edman 1974). However, it should be noted that Wyeomyia were captured in suction traps while actively flying, rather than with aspirators while at rest, as in the case of Ae. taeniorhynchus and De. cancer. The engorgement rate of Wy. vanduzeei was similar to that for Wy. mitchellii. Thus, these field data are not indicative of the different blood requirements observed in the laboratory. Since Wy. mitchellii appears to be more abundant in inland rather than coastal forests, thc question arises as to whether its poorer success in coastal areas might be related to lower host populations (O'Meara & Edman 1975), coupled with its apparent inability to develop eggs without a blood meal. Extensive investigations on the bionomics of Wyeonryia in Florida, which should answer these and other questions, are currently in progress at the FMEL. Ackllowledgmm/s:
479
Florida Wyeornyia host-feeding patterns
J. Moo.
480
+
Adh-I F Hybrid
ADHI [
Adh-IS
-----
ADHm
----
FIG. I. Schematic drawing showing isozyme pattern in Culex pipims quinquifasciatus larvae. The 3 zones of ADH activity are shown on the left and the 3 bands (fast, hybrid and slow) of Adh-l locus are shown on the right. Solid lines=intensely staining, dashed lines = faintly staining.
sufficient genetic markers with high penetrance and uniform expressivity in this species, a need for more biochemical genetic markers can hardly be overemphasized. The zymogram techniques, in addition to providing new genetic markers, provide a vary handy tool to uncover the hidden (cryptic) variability in natural populations. This, in turn, can provide important baseline data of great potential value in any future genetic control strategy of the vector species (Narang et al. 1977, Sakai et al. 1973). Efficient use of the zymogram technique involves the scoring of alleles at loci encoding particular enzymes, rather than mere comparison of numbers and positions of bands of enzyme activity on gels. This approach requires formal progeny studies to establish the number of loci and to support genetic interpretation of zymogram patterns (Wagner & Selander 1974). These studies were undertaken to investigate the following: (1) various isozyme patterns of alcohol
Vol. 14, no. 4
dehydrogenase, (2) the genetic basis of various electrophoretic forms, and (3) the isozyme variability in natural populations of Cx. p. quillqueJasciatus. MATERIALS
AND METHODS
The following strains of Culex p. quinqueJasciatus were used for genetic studies. (I) URUGUAI: This strain was established in 1972 from a locality of the same name in Salvador, Brazil. A subs train homozygous for Adh-l F (fast) allele was isolated from this strain; (2) Y TU: This strain was isolated by inbreeding mosquitoes collected in Salvador, Brazil (Bhalla & Tavares-Souza 1975). A substrain homozygous for Adh-l S (slow) allele was isolated from this strain. Single pair matings were set up and both parents of each cross were subjected to electrophoresis after the female had oviposited. This permitted the isolation of pure homozygous stocks for various electrophoretic variants (allozymes). When necessary, the parents were stored at 25-60°C below zero and were assayed simultaneously with the F I offspring on the same gel. Electrophoresis in acrylamide gel was performed as described earlier (Narang & Kitzmiller 1972). The zymograms were developed first by incubating the gel in a solution containing 12 mg B-nicotinamide adenine dinucleotide, 8 mgp-nitro blue tetrazolium, I ml isopropanol, 1 ml octanol and 50 ml of 0.1 M tris-Hel buffer pH 8.5 and then adding 2 mg phenazine methosulphate. For the substrate test, various substrates were substituted in the incubation mixture. The natural populations sampled for gene
FIG. 2. Electrophoretic pattern of an alcohol dehydrogenase locus (Adh-l) in Cule.~ Pipiens quinquejasciatus. Refer to the schematic drawing for explanation.
Downloaded from http://jme.oxfordjournals.org/ by guest on June 7, 2016
ADH II
EntomoI.
Narang et al.:
1977
Cul~x p. quinqUl!fasciatus
genetics
481
Inheritance of Adh-l electrophoretic variants in Cx. p.fatigans. PROGENY PHENOTYPE PARENTAL·
