ALLOZYME VARIABILITY IN OCHLEROTATUS CASP / US ( PALLAS , 1771 ) POPULATION ( DIPTERA : CULICIDAE )

The biological characters of approximately 3200 mosquito species (W 0 I'd 1992) and their vectorial capacity point to the importance of an extensive investigation of this group of organisms. The subgenus Ochlerotatus with 181 species recorded in Palacarctic represents one of 38 subgenera of the genus Aedes (K n i g h t and S ton e 1977). This subgenus includes mainly salt marsh. inland or littoral mosquitoes. Particular attention has been devoted to the investigations of Aedes caspius. subgenus Ochlerauuus. as a potential vector of human pathogens: Spiroplasma sabaudiensc (L eGo f f et at. 1990) and Crisiulospora aedis (K hod z h a e v a and Iss i 1989). The most important vectors of Tahyna virus in Europe are Ae. \'C.HIIlS and Ac. caspius (P i I ask i 1987). The species Ae. caspills is a vector of Tahyna (TAH) virus in the Mediterranean region and also a potential reservoir of "Riff Valley Fever Virus" (RVF) during interepizootic periods (M i t c hell 1995). Palaearctic species Oe. caspius inhabits saline lakes and pools of the Mediterranean region, shores oi' Great Britain, and fresh-water and lower salt marshes in the continental parts of Europe, Russia, Mongolia, northern China, Pakistan, north and north-eastern Africa, Asia Minor, and Persian Gulf (C I' a n s ton ct al. 1987). Recently, the subgenus Ochlcrotatus has been elevated to generic level. The need for a more comprehensive understanding of the role of De. casipiusas a disease vector, as an integral part of vector control and suppression programs, induced us to analyze genetic structure of its Palacarctic population using allozyme variability at 9 isozyme loci. Specimens of Ochlerotatus caspius were collected from northern Yugoslavia Rusanda. Zrenjanin (160 samples). Third and fourth instal'S of the population were collected and reared to adults, and then frozen. The genetic variation was studied by standard 5% polyacrylamide gel electrophoresis (M un s term ann 1979). Tris-l3oric-EDTA (pH 8.9) buffer was used to assay glucose phosphate isomerase (E. C. 5.3.1.9; Gpi locus), esterase (3.1.1.1; Est-I, ESI-6), phosphoglucomutase (2.7.5.1; Pgm), malic enzyme (1.1.1.40; Me), octanol dehydrogenase (1.1.1.73; D(Y1); and Tris-Citric (pH 7.1) buffer was used to assay a-glycerophosphate dehydrogenase (1.1.1.8; Gpd-Ti, 2-hydrm:y acid dehydrogenase (1.1.99.6; Had) and isocitrate dehydrogenase (1.1.1.42; Mh-2). Statistical analysis of electrophoretic variability data was performed using the computer program BIOSYS-l (S w 0 1'1' 0 I' d and S e I and e I' 1981). The tests included: genotype and allele frequencies, the percentage of polymorphic loci (P), mean observed and expected heterozygosity (H()o He) for small samples corrected .using Leven e's (1949) formula. Deviation between lio and He of separate variable loci was evaluated using Wright's inbreeding coefficient (F; W I' i g h t 1951) with the mean F statistics calculated by a Jackknifing procedure over loci (Wei I' 1990) and S e I and e r's (1970) D statistics.

used to assay a-glycerophosphate dehydrogenase (1.1.1.8;Gpd-Ti, 2-hydrm:y acid dehydrogenase (1.1.99.6;Had) and isocitrate dehydrogenase (1.1.1.42;.Statistical analysis of electrophoretic variability data was performed using the computer program BIOSYS-l (S w 0 1'1' 0 I' d and S e I and e I' 1981).The tests included: genotype and allele frequencies, the percentage of polymorphic loci (P), mean observed and expected heterozygosity (H()o He) for small samples corrected .usingLeven e's (1949) formula.Deviation between li o and He of separate variable loci was evaluated using Wright's inbreeding coefficient (F; W I' i g h t 1951) with the mean F statistics calculated by a Jackknifing procedure over loci (Wei I' 1990) and S e I and e r's (1970) D statistics.The allozyme analysis of Est-L, ESI-6, Gpd-Z, Gpi, Had, Idh-Z.Me.Pgm, and Odli loci revealed the presence of 33 alleles in the population of De. caspius.Out of I) isozyme loci.only Gpd-Z locus was monomorphic (Table 1; Mil a n k 0 v  1'1 al. 2000).Analyses of isozyme variability showed that various loci had different levels of variability.Thus, Gpd-Z locus was monomorphic in two populations from France and population from Egypt (S h u I t z 1'1 al. 1986), as well as the population from Rusanda.Only in the population from Ravenne Gpd-2 was polymorphic, while it was monomorphic (criterion 0.95) in the other two populations from France with two registered heterozygous genotypes (1. a m bel' t 1 '1 al, 1990).Also, Pgm locus was polymorphic in all analyzed populations from France.Egypt (S h u I t z 1'1 al. 1986; 1. a m bel' t 1'1 al, 11)1)0) and North Yugoslavia.According to Shu I t z 1'1 al. (11)86)Pgm locus was diagnostic for separating a population from Egypt and populations from Europe.Similarly to populations from France, heterozygote was found in the population from Rusanda (Tab.1). The Gpi locus had diagnostic value for delineating populations from France and from Cairo (1. a mbel' t 1'1 al. 11)90).In one population from France and a population from Cairo Gpi was monomorphic, while in two French populations two homozygotes and two heterozygotes were found (1. a m her t 1'1 al. 1990).One homozygote (GpilOO/l0~and one heterozygote (Gpi 1(J(}/112) were observed in the population from Rusanda at the Gpi locus (M i I a n k 0 v 1'1 al. 2(00).Contrary to populations from France where Idh-2 and Me loci were monomorphic (S h u I t z 1 '1 al. 1986).in the population from North Yugoslavia three (genotype: Idh_210:U02.Idh_2 10.//10 -l.Idh_298i10.) and four (Me 9,1: 1\1(~lIL'.Me 104.MeJ(J6) alleles.respectively.were registered.The largest number of genotypes in the population of De. caspius from Rusanda was found at the ESI-6 locus (6 homozygotes and 17 heterozygotes).In sympatric populations of the species De. caspills and De.dorsalis (Milankov and Vapa,2(02)    were observed at the ESI-6 locus.The difference in variability.comparing to other analyzed isozyme loci.could be explained by the metabolic function of allozymes coded by alleles at the ESI-6 locus and their association with organophosphate resistance.
As opposed to the Gpi and Odh loci. it has been determined that genotype frequencies at the Est-I, ESI-6.Had.ldh-Z, Me and Pgm loci significantly differed from the expected values.Heterozygous genotypes were registered at all variable •A locus is considered polymorphic on the basis of 0.95 criterion loci.except at Ate. Differences in observed (H0) and expected (H e) heterozygosity based on Hardy-Weinberg values were statistically significant at all loci except for Gpi.Genotype fixation index.J~: indicated excess homozygosity (F,s>O) at all loci.except atl'gm.Had and Gpi.These results were in accordance with the values calculated by Selander's D statistics.
Estimates of genetic variability showed that the population from Rusanda (Table 2) was more variable than populations from France (S h u I t Z 1'1 al. 11)86; 1. a m bel' t 1'1 al. 1990) and Egypt (S h u It Z 1 '1 al. 1986; 1. a m bel' t 1'1 al. 1990).The analysis of allozyme variability at 17 isozyme loci in populations from France and Egypt (S h u It z 1 '1 al, 1986) showed that percentage of polymorphic loci (P). the mean number of alleles per locus (A) and value of mean heterozygosity (H o )  were uniform (17.65; 1.18-1.24;0.035-0.106)and lower than the corresponding values (57.1-60.0;1.8-2.71;0.113-0.148)according to Lam bel' t 1'1 al. (1990).The highest values of genetic structure parameters were registered in the population from Rusanda (P: 88.9; A: 3.8; flo: 0.267).while the lowest ones were recorded in populations from Egypt (P = 0.0, 5.88; A = 1.0.1.06; fl o = 0.0,0.024)(1. a m bel' t ei al. 1990; Shultz 1 '1 al. 1986).The observed differences can be attributed to environmental differences, effective size of populations, historical events.as well as different number of analyzed loci and the fact that there were the differences in the enzymes assayed in different populations.Namely.out of 17 isozyme loci analyzed by Shu I t z-a 1'1 al, (1986).only 4 loci (Gpd-2, Idh-Z.Me and l'gm) were included in our study.Also, out of 7 analyzed loci in populations from France and Egypt (1. a m bel ' t et al, 1990) only three loci (Gpd-2.Gpi and Pgm) were studied.

Table 1 .
Genotype frequency at different loci in the population of He 0.491 * Genotype frequency at Idh-L Had.Odh and Gpiloci were presented in Milankov et al. 2000

Table 2 .
Estimates of genetic variability of the Ochlerototus caspius population (standard errors in parenthesis)