Genetic
structure of populations of Ochlerotatus
dorsalis (Meigen, 1830)
(Diptera: Culicidae)
V. Milankov* and Lj. Vapa
Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Yugoslavia
*Email: vesnam@im.ns.ac.yu
Abstract
Allozyme variability at nine loci of Palaearctic and Nearctic populations of Ochlerotatus dorsalis was determined. Estimates of genetic variability showed that the Palaearctic population ranked higher than the Nearctic population in percentage of polymorphic loci (P: 0.77; 0.33), the mean number of alleles per locus (A: 3.3; 1.8) and value of mean heterozygosity (Ho: 0.263; 0.059). In addition to the lack of identical alleles at Est-6 locus, genetic differentiation of Palaearctic and Nearctic populations was due to differences of allele frequency variances at Had, Idh-2 and Gpi loci.
Introduction
Polyacrylamide gel electrophoresis (PAGE) allows precise, efficient and rapid analysis of enzyme variability as a basis for studying biochemical adaptations. Within the genus Ochlerotatus investigation of the dorsalis group has been hindered by disagreement regarding the taxonomic status of its constituent species. Relationships between the Holarctic Oc. dorsalis and the Nearctic Oc. melanimon (Dyar) (Freeborn, 1926; Barr, 1955), and the Palaearctic Oc. caspius (Pallas) (Gutsevich, 1985; Bozicic-Lothrop, 1988) have long been a cause of controversy. Larvae of Oc. dorsalis may be found in inland saline ponds, lakes and marshes, as well as sea shores of California, Canada, Britain, north, central, and southern Europe, Siberia, China and Mongolia (Milankov et al., unpublished). The adults have a medical importance as vectors of arboviruses causing California encephalitis (Wood et al., 1979), of the California serogroup Hart Park and Cache Valley viruses (Clark et al., 1986) and St. Louis encephalitis virus (Emmons et al., 1987).
This paper reports an investigation of the genetic structure of Palaearctic and Nearctic populations of Oc. dorsalis, using allozyme variability at nine isozyme loci.
Sample Collection. Samples of Oc. dorsalis were collected from northern Yugoslavia (Rusanda, Zrenjanin), (AD1; 131 samples) and from California (Salton Sea), (AD2; 66 samples). Third and fourth instar larvae of the Palaearctic population were collected, reared to adults, and then frozen. Species identification was based on characteristic mesonotal and abdominal tergite scale patterns.
Allozyme Analysis. Genetic variation was studied by standard 5% polyacrylamide gel electrophoresis (Munstermann, 1979). Tris-Boric-EDTA (pH 8.9) buffer was used to assay glucose phosphate isomerase (E.C. 5.3.1.9. GPI), esterase (E.C. 3.1.1.1. EST), phosphoglucomutase (E.C. 2.7.5.1. PGM), malic enzyme (E.C. 1.1.1.40. ME), octanol dehydrogenase (E.C. 1.1.1.73. ODH); and Tris-Citric (pH 7.1) buffer was used to assay a-glycerophosphate dehydrogenase (E.C. 1.1.1.8. GPD), 2-hydroxy acid dehydrogenase (E.C. 1.1.99.6. HAD) and isocitrate dehydrogenase (E.C. 1.1.1.42. IDH).
Statistical Analysis. Statistical analysis of electrophoretic variability data was performed using the computer program BIOSYS-2 (original version: BIOSYS-1, Swofford & Selander, 1981; as modified by Black, 1997). The tests included: genotype and allele frequencies, the percentage of polymorphic loci (P), mean observed and expected heterozygosity (Ho, He) for small samples corrected using Levene’s (1949) formula.
Deviation between Ho and He of separate variable loci was evaluated using Wright's inbreeding coefficient (F) (Wright, 1951) with the mean F statistics calculated by a jack-knifing procedure over loci (Weir, 1990) and Selander’s (1970) D statistics. Wright’s F statistics, the difference between the frequency of heterozygotes observed and those expected, three levels of genetic differentiation: Fis (variation within subpopulations), Fit (homozygosity of an individual relative to total population), and Fst (amount of subdivision relative to the limiting amount under complete fixation) were calculated.
Results
The allozyme analysis of Est-1, Est-6, Gpd-2, Gpi, Had, Idh-2, Me, Pgm, and Odh loci revealed the presence of 28 alleles in AD1 population and 16 in AD2. Genotype frequencies were calculated (Table 2). Loci Gpd-2 and Odh in AD1 population were monomorphic, while Me, Pgm and Est-1 of AD2 were polymorphic (based on the 95% criterion).
In contrast to AD2 population differences in observed (Ho) and expected (He) heterozygosity based on Hardy-Weinberg, values in the Palaearctic (AD1) population of Oc. dorsalis were statistically significant for Est-6, Gpi and Pgm loci. The genotype fixation index, F, indicated excess homozygosity (Fis>0) in both populations at most loci, except at Gpi (AD1), Had (AD2), Idh-2 (AD1), Pgm (AD2) and Odh (AD1). These results were in accordance with values calculated by Selander’s D statistics. D was 0 at Had locus in the AD2 population.
F statistics was used to describe the genetic structure in Palaearctic and Nearctic populations of Oc. dorsalis. The mean fixation index in the total population was high (FIT=0.674). FIT values for Est-1, Est-6, Had and Me loci were considerably higher (from 0.748 to 1.000) followed by Pgm (0.356) and Idh-2 (0.326) loci. Negative FIT values were registered at Odh (FIT= -0.018) and Pgm (FIT= -0.058) loci.
28 alleles at 8 variable loci were registered in populations of the species Oc. dorsalis. No significant differences in the allele frequencies at Me, Pgm and Odh loci were observed. Fst used as a measure of genetic differentiation between populations indicated high genetic subdivision (Fst=0.329). This population subdivision of Oc. dorsalis was due to differences of allele frequency variance (Fst) at Had, Est-6, Gpi and Idh-2. Value of F parameters by jack-knife estimates over loci were in concurrence with previous results.
Analysis of population genetic structure parameters showed many differences between the two populations. All parameters indicated higher genetic variability in the Palaearctic population (Table 1).
Discussion
Protein-enzyme polymorphism can serve as an indicator of changes in genes occurring during evolution. These processes can be complex since they involve co-adaptive changes in systems of genes.
Analyses of isozyme variability have shown that various loci have different levels of variability. Thus, locus Gpd-2 was monomorphic in four Nearctic populations (Shultz at al., 1986) and in a Palaearctic population (Lambert et al., 1990) which agrees with the results in this study. Furthermore, the Idh-2 locus in all Nearctic populations was monomorphic (Shultz et al., 1986) as we found to be the case in the Nearctic AD2 population, but not in the Palaearctic AD1 population (Milankov et al., 2000).
Unlike populations from New Mexico, Michigan, Utah and Wyoming where the Me locus was monomorphic (Shultz et al., 1986), we found 2 and 3 alleles, respectively, in the Salton Sea (AD2) and Rusanda (AD1) populations.
Shultz et al., (1986) and Lambert et al., (1990) found the Pgm locus to be polymorphic in all analysed populations of Oc. dorsalis. No heterozygotes have previously been registered at locus Pgm in Nearctic populations (Shultz et al., 1986), but we found one homozygote and one heterozygote in the Salton Sea (AD2) population. One homozygote and three heterozygotes were found at the Pgm locus in a Palaearctic population of Oc. dorsalis from France (Lambert et al., 1990), and we observed three homozygotes and two heterozygotes in the population from Rusanda (AD1).
Lambert et al. (1990) reported two homozygotes and one heterozygote at the Gpi locus in a population from France; we observed one homozygote and one heterozygote in the Rusanda (AD1) population. The Gpi locus is monomorphic in the Salton Sea (AD2) population (Milankov et al., 2000). In contrast to the Palaearctic population, the Idh-2, Had and Est-6 loci were monomorphic in the Nearctic population (AD2). Locus Odh is monomorphic in both populations (Milankov et al., 2000), except that the rare allele Odh108 occurs in the Nearctic population.
The Est-6 locus is diagnostic for separating the conspecific AD1 and AD2 populations. At this locus the largest number of genotypes (5 homozygotes and 7 heterozygotes) was found in the Rusanda population, and the allele Est-684/84 was also found. In sympatric populations of Oc. dorsalis and Oc. caspius from Rusanda identical alleles were observed at the Est-6 locus (Milankov & Vapa, unpublished).
In contrast to the polymorphic loci Est-6, Gpi, Had, Idh-2, Pgm and Odh (99% criterion) in the Palaearctic population, and Had and Pgm (99% criterion) in the Nearctic population, no heterozygotes were registered in the Me and Est-1 loci. Malic enzyme (NADP-Malate dehydrogenase), coded by alleles of two (Richardson et al., 1986) or three loci (Munstermann, 1979), is a tetrameric protein of the glycolytic pathway. In some aedine mosquitoes two Me loci are visible (Matthews & Munstermann, 1983; Foley & Bryan, 1991), probably corresponding to the mitochondrial (Me-1 or slow locus) and the cytoplasmic forms (Me-2 or fast locus). Absence of heterozygotes in the polymorphic Me locus could be the result of a number of factors, such as selection against heterozygotes (i.e. disruptive selection), the presence of null alleles, assortative mating, population substructuring and presence of cryptic species in sympatry, or some non-genetic, post-translational modification and electrophoretic insufficiency. The presence of heterozygotes in other polymorphic loci showed that inbreeding, non-random mating and genetic drift (which affect all polymorphic loci in a similar fashion) could not be the cause of the deficiency of heterozygotes in the Me and Est-1 loci. Also, null alleles are unlikely since they are very rare for the enzymes of intermediary metabolism (such as glycolytic enzymes). It is possible that the analysed Me locus was not mitochondrialy located (which could also cause the absence of heterozygous genotypes) since ME allozymes in Oc. dorsalis populations showed similar electrophoretic mobility as the allozyme coded by Me-2 in the Aedes aegypti laboratory strain used as a standard. The lack of heterozygosity could be the result of sub-sampling and non-random mating (a sample set consists of individuals from different subpopulations or genetically distinct groups such as cryptic species) since Oc. dorsalis is well known for its capacity to migrate long distances. Additionally, it has been proven that the lack of heterozygosity in the Me-1 and Acon-1 loci, and the Odh locus in the populations of Anopheles quadrimaculatus species C (Narang et al., 1990) and An. minimus (Green et al., 1990), respectively, indicated genetic substructuring - division of analysed species into subpopulations or cryptic species.
Although the comparison of enzyme
maps revealed that numerous inversions (mostly paracentric) and translocations
caused subgeneric genetic divergence, it was also found that certain linkage
associations were conserved in Aedes evolution
(Matthews & Munstermann, 1994). Since linkage mapping results showed that
the changes in Aedes (recently the
sub-genus Ochlerotatus has been
elevated to generic level) occurred at a much lower rate than in other examined
organisms (Rai et al., 1982), it
could be assumed that the registered pattern is present in Oc. dorsalis although the linkage map has not yet been constructed
for this species. Therefore, it could be assumed that the cytoplasmic Me locus in Oc. dorsalis is associated with the sex-determining locus on
chromosome 1, as in Aedes aegypti (Munstermann,
1990), Ae. triseriatus (Matthews
& Munstermann, 1990) and Ae.
scutellaris group (Pashley & Rai, 1983). In this study, however, no
difference between males and females in the alleles of the Me locus was observed that would support this hypothesis.
Esterases (EST) exist as molecular forms possessing a large number of isoenzymes. Two types of esterases have been identified in mosquito species, preferentially hydrolysing either a- or b- naphthyl acetate. Two esterase loci (Est-1, Est-6) were analysed in this study using the substrate a- naphthyl acetate. At the Est-1 locus no heterozygotes were registered. The absence of heterozygotes might be due to the presence of null alleles (quite common for multiple-locus enzyme complexes, e.g. esterases). Null alleles, causing the excess of phenotypic homozygotes, were registered in the Est-1, Est-2 (Stordeur, 1976) and Est-3 loci (Urbanelli et al., 1980) in populations of Culex pipiens; and Est-4 and Est-5 in Aedes albopictus (Tadano, 1987). Esterase loci are mostly a part of the linkage group II, as in the case of the Est-6 locus in Aedes aegypti (Munstermann & Craig, 1979) and in the Ae. scutellaris group (Pashley & Rai, 1983) as well as Est-5 in the Ae. triseriatus group (Munstermann et al., 1982).
The presence of major and rare alleles indicates genetic divergence among populations of Oc. dorsalis. The same major alleles (>0.5) at Gpi, Pgm and Odh were registered in both populations. On the other hand, those at Gpd-2 (which was monomorphic), alleles at monomorphic Est-6, Idh-2 and Had loci, as well as a major allele at the Me locus in the AD2 population, were not major alleles in the AD1 population. In accordance with these results, no statistically significant differences in allele frequencies at Est-1, Pgm and Odh loci were observed. Apart from the lack of identical alleles at the Est-6 locus, genetic differentiation of Palaearctic and Nearctic populations was due to differences of allele frequency variances at Had (Fst=0.711), Est-6 (Fst=0.536) and Idh-2 (Fst=0.344).
Genetic divergence among populations was, to a lesser extent, affected by allelic frequency variance at Gpi (Fst=0.243), Est-1 (Fst=0.055) and Odh (Fst=0.095) loci.
Estimates of genetic variability showed that the Palaearctic population ranked higher than the Nearctic population in percentage of polymorphic loci, the mean number of alleles per locus, and the value of mean heterozygosity. Observed differences in values of genetic structure parameters were probably due to association of genetic variability, level of ecological heterogeneity, effective size of populations and historical events, as well as independent evolution of geographically distant populations. However, analysis of allozyme variability at
17 isozyme loci in Nearctic populations showed that polymorphism, heterozygosity and mean number of alleles per locus were uniform (5.88; 0.035-0.059; 1.06 respectively) (Shultz et al., 1986) and similar to corresponding values calculated for the AD2 population (A: 1.8; Ho: 0.059), except for frequency of polymorphic loci (P: 33.3). The observed differences can be attributed to different numbers of analysed loci and the fact that not the same enzymes were assayed for both populations. Of 17 isozyme loci analysed by Shultz et al. (1986), only 4 loci (Gpd-2, Idh-2, Me and Pgm) were included in our study. Higher values of all genetic structure parameters were registered in the Palaearctic population than in the Nearctic population, and also when the Rusanda population was compared with a population from France (Lambert et al., 1990). Frequency of polymorphic loci and heterozygosity were slightly higher in the AD1 population than in the population from France (P: 0.714; Ho: 0.178), which was probably due to there being only three common loci (Gpd-2, Gpi and Pgm) analysed.
The authors would like to acknowledge their gratitude to Dr Branka Bozicic-Lothrop for providing mosquito samples from Salton Sea, California, USA. The authors express their appreciation to J. Stamenkovic for assistance in preparing this manuscript.
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|
Table 1. Estimates
of genetic structure in populations of Ochlerotatus
dorsalis species |
||||||
|
Population |
n (SE) |
A |
He (SE) |
Ho (SE) |
Private allele |
P(0.95) |
|
AD1 |
39.0 (3.6) |
3.3 (0.7) |
0.463 (0.092) |
0.263 (0.086) |
14 |
0.778 |
|
AD2 |
24.2 (3.2) |
1.8 (0.4) |
0.162 (0.085) |
0.059 (0.055) |
1 |
0.333 |
n = Mean sample size per locus, SE = standard error
A = Mean number of alleles per locus
He = Expected heterozygosity averaged over all loci, SE = standard error
Ho = Average frequency of observed heterozygosity, SE = standard error
P(0.95) = Frequency of polymorphic
Table 2. Genotype frequency at variable
loci in populations of Ochlerotatus
dorsalis species |
|||
|
Locus |
Genotype |
AD1 |
AD2 |
|
Est-1 |
92/92 |
0.023 |
0.056 |
|
|
96/96 |
0.139 |
0.389 |
|
|
98/98 |
0.442 |
0.333 |
|
|
100/100 |
0.349 |
0.111 |
|
|
102/102 |
0.047 |
0.111 |
|
|
Ho |
0.000 |
0.000 |
|
|
He |
0.501 |
0.351 |
|
Est-6 |
84/84 |
- |
1.000 |
|
|
88/88 |
0.133 |
- |
|
|
93/93 |
0.133 |
- |
|
|
96/96 |
0.100 |
- |
|
|
98/98 |
0.167 |
- |
|
|
100/100 |
0.033 |
- |
|
|
86/93 |
0.033 |
- |
|
|
88/98 |
0.033 |
- |
|
|
88/93 |
0.067 |
- |
|
|
88/96 |
0.167 |
- |
|
|
96/100 |
0.067 |
- |
|
|
96/102 |
0.033 |
- |
|
|
102/104 |
0.033 |
- |
|
|
Ho |
0.433 |
0.000 |
|
|
He |
0.801 |
0.000 |
|
Gpi* |
88/88 |
0.419 |
1.000 |
|
|
88/100 |
0.581 |
- |
|
|
Ho |
0.581 |
0.000 |
|
|
He |
0.412 |
0.000 |
|
Had* |
90/90 |
0.077 |
0.970 |
|
|
100/100 |
0.564 |
- |
|
|
90/100 |
0.205 |
0.030 |
|
|
100/110 |
0.154 |
- |
|
|
Ho |
0.359 |
0.000 |
|
|
He |
0.409 |
0.000 |
|
Idh-2* |
83/83 |
0.127 |
- |
|
|
90/90 |
0.237 |
1.000 |
|
|
83/90 |
0.309 |
- |
|
|
83/97 |
0.127 |
- |
|
|
90/97 |
0.200 |
- |
|
|
Ho |
0.636 |
0.000 |
|
|
He |
0.613 |
0.000 |
|
Me |
102/102 |
0.625 |
0.773 |
|
|
104/104 |
0.325 |
0.227 |
|
|
106/106 |
0.050 |
- |
|
|
Ho |
0.000 |
0.000 |
|
|
He |
0.501 |
0.351 |
|
Odh* |
100/100 |
0.952 |
1.000 |
|
|
100/108 |
0.048 |
- |
|
|
Ho |
0.048 |
0.000 |
|
|
He |
0.046 |
0.000 |
|
Pgm |
100/100 |
0.187 |
- |
|
|
140/140 |
0.375 |
0.500 |
|
|
160/160 |
0.125 |
- |
|
|
100/140 |
0.250 |
0.500 |
|
|
140/160 |
0.062 |
- |
|
|
Ho |
0.313 |
0.500 |
|
|
He |
0.596 |
0.375 |
*Genotype frequency at Idh-2, Had, Odh and Gpi loci were presented in Milankov et al., 2000