Evidence for Genetic Hitchhiking Effect Associated With Insecticide Resistance in Aedes aegypti

Corresponding author: Guiyun Yan, Department of Biological Sciences, State University of New York, 109 Cooke Hall, Buffalo, NY 14260, gyan{at}calshp.cals.wisc.edu (E-mail).

Communicating editor: A. G. CLARK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology. Previous studies with Drosophila suggest that genetic variation of selectively neutral loci in a large fraction of genome may be constrained by fixation of advantageous mutations associated with hitchhiking effect. This study examined restriction fragment length polymorphisms of four natural Aedes aegypti mosquito populations from Trinidad and Tobago, at 16 loci. These populations have been subjected to organophosphate (OP) insecticide treatments for more than two decades, while dichlor-diphenyltrichlor (DDT) was the insecticide of choice prior to this period. We predicted that genes closely linked to the OP target loci would exhibit reduced genetic variation as a result of the hitchhiking effect associated with intensive OP insecticide selection. We also predicted that genetic variability of the genes conferring resistance to DDT and loci near the target site would be similar to other unlinked loci. As predicted, reduced genetic variation was found for loci in the general chromosomal region of a putative OP target site, and these loci generally exhibited larger F_ST values than other random loci. In contrast, the gene conferring resistance to DDT and its linked loci show polymorphisms and genetic differentiation similar to other random loci. The reduced genetic variability and apparent gene deletion in some regions of chromosome 1 likely reflect the hitchhiking effect associated with OP insecticide selection.

MOSQUITOES are important vectors for several human pathogens because of their close association with humans. Mosquito habitats often change rapidly as a result of vector control efforts; therefore, successful adaptation to varying human habitats is essential for mosquito reproduction. Adaptation ability of an organism depends on its genetic variability. Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology (TABACHNICK and BLACK 1996 ). Protein electrophoresis and DNA sequence analyses have revealed remarkable variation in many genes in natural populations of Drosophila and other species, but the genetic variability seems to differ substantially for genes in different genome regions (AQUADRO 1992 ). Distribution patterns of genetic variants in natural populations are the joint effects of various evolutionary forces and demographic factors, including random genetic drift, selection, recombination, mutation, gene flow, mating system and life history (e.g., colonization, range expansions or contractions; SLATKIN 1985 ). Population life history and mating structure influence all loci equally, but selection affects only the target loci (KREITMAN and AKASHI 1995 ). Variation of selectively neutral loci may also be constrained by the hitchhiking effect, particularly in genome regions with low recombination rates and under extensive selection (MAYNARD SMITH and HAIGH 1974 ). Recent studies with D. melanogaster suggest that the hitchhiking effect may have occurred over a large fraction of the insect genome (BEGUN and AQUADRO 1992 ). In this study we analyzed restriction site variation of 16 loci in natural populations of the yellow fever mosquitoes, Aedes aegypti, and provide evidence that the hitchhiking effect may have reduced genetic variation in the genome regions around a putative insecticide resistance locus.

A. aegypti is an important vector of yellow fever and dengue fever viruses in many tropical countries, including Trinidad and Tobago, West Indies. Control efforts for A. aegypti have focused primarily on habitat reduction and chemical treatment, which is based on the destruction of breeding sites and the use of insecticides, including dichlor-diphenyltrichlor (DDT) in the 1950s and several organophosphates (OP) since the 1960s. The wide use of insecticides has been a powerful selection agent, and rapid development of resistance to DDT and OPs is well documented (GILKES et al. 1956 ; RAWLINS and WAN 1995 ). The genetic mechanisms of insect resistance to various insecticides have been well characterized. For example, a point mutation in the para sodium channel gene confers one form of resistance to DDT (WILLIAMSON et al. 1996 ), and esterase (EST) gene amplification is associated with resistance to OPs in Culex mosquitoes (MOUCHES et al. 1990 ). A genetic linkage map, based largely on random cDNA sequences, has been constructed for A. aegypti (SEVERSON et al. 1993 ), and several insecticide resistance genes have been mapped (SEVERSON et al. 1997 ). In this study, we used cDNA markers distributed across the mosquito genome to examine DNA polymorphism and population genetic differentiation, and to examine mosquito genome structural changes associated with strong selection imposed by insecticides. Several studies have investigated the genetic variation of various genera of mosquito populations with isozyme, RAPD-DNA, microsatellite and mitochondrial DNA markers (POWELL et al. 1980 ; TABACHNICK and WALLIS 1985 ; CONN et al. 1993 ; CHEVILLON et al. 1995 ; APOSTOL et al. 1996 ). Restriction fragment length polymorphism (RFLP) markers are particularly suitable for population genetic studies, because they are presumably neutral, highly polymorphic, segregate as codominant markers, and can be used for studies of other mosquito species (SEVERSON et al. 1994A ). We chose Trinidad and Tobago populations because the population history is known and surveillance programs have been well established there. Population historical information is important for the interpretation of genetic data. Because the mosquito populations have been under selection of OP insecticides, genetic variation of loci closely linked to an esterase locus conferring resistance would be reduced if the hitchhiking effect has occurred. The hitchhiking effect would be prominent in genome regions with strong linkage disequilibrium and intense selection (e.g., insecticides). In contrast, gene diversity at the para locus and other neighboring loci is expected to be similar to unlinked loci in the genome, because selection pressure has been removed at the para locus since DDT was abandoned more than two decades ago.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Natural history of A. aegypti in Trinidad and Tobago:
It is generally believed that domestic A. aegypti originated from an African sylvan ancestor, and was introduced to the New World from West Africa via transoceanic trade during the fifteenth to seventeenth centuries (TABACHNICK 1991 ). Caribbean populations probably represent the initial introduction of the mosquito species into the New World in the course of New World colonization. The first outbreak of yellow fever in Trinidad was recorded in 1796, and in 1820 in Tobago.

In the 1950s, intensive vector control programs aimed toward mosquito eradication were adopted, primarily by the widespread usage of DDT. In the early 1960s, Trinidad was considered free of A. aegypti, but was reinfested in 1962. Overall, mosquito populations in Tobago have been exposed to fewer insecticides than Trinidad populations. A. aegypti is now widely distributed in Trinidad and Tobago, despite continued intensive vector control efforts through the use of OP insecticides. Insecticide applications not only impose strong selection on the target loci, but also lead to recurrent reductions of population sizes.

Collection of samples:
In conjunction with the A. aegypti surveillance program, in April, 1995, we collected three geographically-distinct samples from Trinidad and one sample from Tobago (Figure 1). These four villages share similar climates, including temperature and the annual amount of rainfall. For each village, 100 ovitraps were distributed, and about half of the village's residential area was covered (approximately two traps every five houses). Each ovitrap consisted of a black plastic container roughly half-filled with water into which a rectangular masonite strip was placed in an upright manner. Female A. aegypti mosquitoes will readily oviposit on the masonite strip, near the water interface. After 2–3 days, the masonite strips were removed, transported to the laboratory, where attached eggs were allowed to hatch, and reared into adults. All adults were identified as A. aegypti by microscopic examination, and were frozen for subsequent DNA analysis. Previous studies with A. aegypti in Puerto Rico suggest that the mean number of families represented per ovitrap was 4.7 (e.g., several female mosquitoes frequently oviposit in the same container; APOSTOL et al. 1994 ). Therefore, it is unlikely that siblings within a subpopulation would be sampled.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Map of Trinidad and Tobago. Three samples were collected from Trinidad: Curepe (10°38.62'N, 61°24.23'W), Couva (10°26.12'N, 61°28.19'W), and San Fernando (10°18.11'N, 61°28.21'W). One sample was collected from Tobago (11°11.23'N, 60°44.21'W).

RFLP and probe selection:
We genotyped a total of 870 mosquitoes for four populations (n = 150 for Curepe, 262 for Couva, 258 for San Fernando, and 200 for Tobago). DNA extraction from individual mosquitoes, digestion with EcoRI, Southern blotting and hybridization were as previously described (SEVERSON et al. 1993 ). Fifteen mapped RFLP markers were selected to provide broad coverage of the A. aegypti genome with an average resolution of 10.6 cM (Figure 2). All clones used were random cDNA clones with the exception of the para and Mal I clones. Mal I is a gene specifically expressed in the salivary glands, and its putative function is related to sugar metabolism ( JAMES et al. 1989 ). The mosquito genome consists of single or low-copy DNA sequences and repetitive DNA with short-period interspersion (BLACK and RAI 1988 ). In this study, we focused on allelic variations of single- or low-copy cDNA sequences.

View larger version (25K): In this window In a new window Download PPT slide   Figure 2. Relative map positions of the 16 Aedes aegypti (2N = 6) RFLP loci used in the study. Chromosome numbers are in italics. Map distances are in Kosambi centimorgans. Underlined loci were not used in the study. Esterase gene amplification is involved with resistance to organophosphate insecticides in mosquitoes (MOUCHES et al. 1990 ). Three esterase loci were mapped to the general chromosomal locations as shown in the figure (MUNSTERMANN 1990 ). LF250 represents duplicated loci. Markers in italics are genes with known functions; other markers are random cDNA.

Data analysis:
DNA polymorphism and Hardy-Weinberg equilibrium (HWE) tests: Molecular weights of fragments detected by each clone were estimated by comparing them to lambda-HindIII digest standards included on each gel, using the Eagle Sight image capture and analysis software (Stratagene, La Jolla, CA). DNA polymorphisms may be measured by the proportion of polymorphic loci, number of alleles, and heterozygosity. Conformance with HWE was tested using the probability test for each locus and each population, using the GENEPOP computer program (RAYMOND and ROUSSET 1995 ). Because this test is robust to allele frequencies, rare alleles were not pooled. We further tested whether distortion from HWE resulted from deficient or excessive heterozygosity, using the F_IS statistics (WEIR 1990 ; ROUSSET and RAYMOND 1995 ). F_IS is defined as [1 - (observed heterozygosity/expected heterozygosity from HWE)]. Because F_IS estimates at individual loci may be unduly influenced by rare alleles, we tested the significance of the average F_IS over all loci using the method of ROBERTSON and HILL 1984 . Variations in heterozygosity among the populations were analyzed following the method of WEIR 1990 , using the analysis of variance (ANOVA) with subpopulations, individuals, loci and interactions of loci and individuals as factors. All factors were treated as random effects except loci.

Population genetic structure, gene flow and genetic distance: Population genetic structure was examined with Wright's F-statistics, based on the procedure of WEIR and COCKERHAM 1984 and using the FSTAT computer program (GOUDET 1995 ). Standard deviations (SD) of F-statistics were obtained for each locus by a jackknife procedure over the alleles, and were used to test the significance of the F statistics. We first tested whether the three populations from Trinidad were significantly substructured, then included the Tobago population data in the analysis.

Gene flow (Nm) was estimated from the standardized-among-population genetic variance (F_ST) estimate of each locus using the relationship Nm = (1/F_ST - 1)/4, where N is the effective population size of a deme, and m is the rate of gene flow (WRIGHT 1943 ). This equation assumes the infinite-island model of population structure and gene flow. Few populations probably conform to this assumption, but it provides a useful approximation of the relative magnitude of gene flow. Gene flow was also estimated using the private-alleles method for the appropriate loci (SLATKIN and BARTON 1989 ). Private alleles are the alleles unique to a given deme. Nei's unbaised genetic distance for all pairs of populations was calculated based on population allele frequency for all loci (NEI 1987 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA polymorphisms and HWE tests:
Fifteen cDNA markers examined in this study were all polymorphic. The RFLP patterns of one marker (LF250) indicate that this marker represents a gene duplication (data not shown), and therefore, the 15 markers represented a total of 16 loci. A total of 91 unique alleles were identified, 68 alleles (74.7%) were common to all four populations. The average number of alleles was about five per locus (Table 1). Six loci (LF198, ARC1, LF250a, para, LF168 and Mal I) exhibited private alleles, and five private alleles were in the Tobago population. An excess of rare alleles was found: 16 alleles (18%) had a frequency less than 0.05. Under the infinite alleles model (equation 8.24; KIMURA 1983 ), we expected to find only four alleles in this frequency class with our sample size (n = 870). The mean sizes of restriction fragments detected by the cDNA clones weighted by their frequencies ranged from 0.73 kb at locus LF250b to 12.90 kb at locus LF352, and exhibited an overall mean of 5.13 kb (95% confidence interval: 3.61–6.64 kb).

In general, high heterozygosity was observed in all four mosquito populations, except at the LF90 locus. The LF90 locus showed significantly lower heterozygosity than the other 15 loci examined (Table 1; ANOVA, t = 8.37, d.f. = 1, P < 0.0001). The most heterozygous loci were LF178 on chromosome 1 and LF282 on chromo-some 2. The high heterozygosity at the LF178 locus does not seem to be a result of sex linkage (see Figure 2), because males and females showed similar heterozygosity (data not shown). Population average heterozygosity over all 16 loci varied little among populations (ranged from 0.582 for Couva to 0.627 for Tobago), and such variations were not statistically significant (Table 1; ANOVA, F = 1.08, d.f. = 3, 49, P > 0.05). Heterozygosity is not correlated with the mean size of restriction fragments weighted by frequencies at a locus (r = 0.22, d.f. = 15, P > 0.05), but seems to correlate with the number of observed alleles (r = 0.49, d.f. =15, P = 0.052).

The genotype frequencies at several loci did not conform to HWE. Loci on chromosome 2 generally exhibited a heterozygote excess, but loci on chromosome 3 that showed HWE distortion exhibited a heterozygote deficit (Table 1). The F_IS values varied greatly among the loci, suggesting no systematic inbreeding occurred in these populations. The average F_IS over all loci was not significantly different from 0 for each population (Table 1). Departure from HWE probably reflects either the effect of insecticide selection on some loci linked to the resistance loci, or simply sampling error.

Population genetic structure, gene flow and genetic distance:
Analysis of F statistics for the three Trinidad populations found small, but statistically significant F_ST estimates for all loci (Table 2), suggesting that these populations are genetically differentiated. F_ST estimates showed a six-fold difference among loci, with an average F_ST over all loci of 0.043. When the Tobago population was included in the analysis, the basic pattern of F_ST estimation among the loci was not altered (Table 2). As expected, slightly larger F_ST values were obtained for most loci, and the average F_ST over all loci was 0.056. The para locus exhibited similar polymorphism and genetic differentiation as other random loci.

Assuming that the populations are at an equilibrium between migration and random drift, the average number of migrants exchanged per generation can be calculated. Average gene flow (Nm) among the four populations, based on the F_ST method, was 4.2 migrants per generation (95% confidence interval: 3.2–5.7). This estimate was similar to the estimate based on the average frequency of six private alleles present in the populations (Nm = 4.5). Table 3 shows genetic distances and gene flow between each pair of populations calculated from the pair-wise average F_ST . A large gene flow between the Tobago and Trinidad populations was detected. There was no significant correlation between genetic distance and geographic distance (r² = 0.5, d.f. = 5, P > 0.05).

Hitchhiking effect on DNA polymorphisms:
Genetic hitchhiking occurs when a (neutral) mutation changes frequency through genetic linkage to a mutation that is selected, resulting in reduced genetic variation surrounding the target site of selection. Low DNA polymorphism at the LF90 locus suggests that hitchhiking has probably occurred in the genome region of the EST-4 locus. We collected additional evidence to test for this hypothesis by examining genetic polymorphism at the LF230 locus, which also is in the general genomic region of EST-4 (Figure 2). An apparent chromosomal deletion event occurred around this locus in 42–59% of the individuals (Figure 3), and low heterozygosity (0.07–0.29) was observed among those individuals without the apparent deletion (Table 1). However, substantial reduction of heterozygosity for the para locus and other loci in the vicinity of para was not observed (Table 1).

View larger version (105K): In this window In a new window Download PPT slide   Figure 3. Southern blot analysis of natural Aedes aegypti populations probed with cDNA clones LF230 (top) and LF90 (bottom). The mosquito genomic DNA was digested with EcoRI. Each lane is for a single mosquito. (Top) Apparent gene deletion around the LF230 locus in 55% of individuals (11 out of 20). (Bottom) Probe LF90 was used as a control to demonstrate that absence of hybridization of mosquito genomic DNA to LF230 was not due to incomplete DNA digestion, or to poor probe conditions. DNA hybridization was observed for the same individuals with all other markers tested. See Table 1 for heterozygosity and percentage of gene deletion at the LF230 locus.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA polymorphisms of four A. aegypti mosquito populations were examined using RFLP markers. The Trinidad populations have been exposed to OPs every 3–4 months for about two decades. These populations have therefore experienced intense selection by insecticides, that probably resulted in periodic population bottlenecks. A population bottleneck maintains a long-term effect on population heterozygosity, even for species with a large intrinsic rate of growth such as A. aegypti (NEI et al. 1975 ). Thus, genetic polymorphisms are expected to decline rapidly during insecticide use for any locus in the mosquito genome. Loci conferring OP resistance are expected to maintain lower genetic variability than other random loci in the genome, and genetic variation of other closely-linked neutral loci may be reduced through genetic linkage.

If recurrent population bottlenecks have occurred in the mosquito populations, low polymorphism for all loci in the genome would be expected. In contrast to the expectation, we observed high polymorphisms for most loci. For the same loci, average heterozygosities of the populations studied here are substantially higher than a laboratory population, which has not been exposed to insecticides for more than 20 years and has not experienced population bottleneck (YAN et al. 1997 ). The highest heterozygosity was observed for loci at two chromosomal regions (LF198-LF178 on chromosome 1, and LF282-LF98 on chromosome 2). Coincidentally, these two chromosomal regions in A. aegypti harbor genes determining vector competence for filarial worms and malaria parasites (SEVERSON et al. 1994B , SEVERSON et al. 1995 ). The high levels of heterozygosity observed may be explained by two mechanisms. The first is that the effective size of population bottlenecks has never been small, because heterogeneous habitats may provide effective shelters for the field populations. The second is that genetic polymorphisms are introduced and maintained by large gene flow among populations. The gene flow estimates seem to support the second hypothesis.

The LF90 locus consistently exhibited lower heterozygosity than other loci in the genome for the four populations used in this study. The heterozygosity of a RFLP locus may be influenced by several factors, including the size of the probes, the size of the regions being probed by the probes, reduced mutation or recombination rates in these genome regions, natural or artificial selection on a particular locus, and hitchhiking (selective sweep) of a selectively neutral locus by selectively favored substitutions at linked loci. We argue that the polymorphism pattern of the LF90 locus likely reflects the result of a hitchhiking effect. First, the putative function of the LF90 clone is coding for ribosomal protein S14 (SEVERSON and ZHANG 1996 ), and thus the RFLP fragments of LF90 themselves are presumably neutral. Second, LF90 is located in the general chromosomal region of EST-4, and gene amplification at an esterase locus is a common mechanism of OP resistance. Our populations have been under selective pressure by OP insecticides for decades. Third, low heterozygosity of the LF90 locus is likely not related to the size of the genome region being probed by the LF90 marker, because we found no significant correlation between heterozygosity and RFLP fragment sizes. Fourth, the observed heterozygosity of the LF90 locus in laboratory colonies of A. aegypti that have not been exposed to insecticide selection was similar to other random loci across the genome (YAN et al. 1997 ).

Our argument for a hitchhiking effect is strengthened by the RFLP data of the LF230 locus, which is linked to LF90 and also is in the general genomic region of EST-4. We found very low DNA polymorphism and an apparent gene deletion for many individuals at this locus, a phenomenon which has not been observed in other laboratory colonies of A. aegypti (YAN et al. 1997 ). Gene deletions may be the result of unequal recombination in this chromosomal region, associated with the esterase gene amplification. For example, OP resistant Culex mosquitoes possess 250–500 copies of a 30-kb esterase B1 gene, compared to a single copy in susceptible individuals (MOUCHES et al. 1990 ). Given the genome size of A. aegypti of about 320 Mb (ZAITLIN and SEVERSON, unpublished results), if the magnitude of esterase gene amplification in the Aedes mosquitoes is similar to Culex mosquitoes, then the hitchhiking effect associated with OP insecticide selection could affect meiotic pairing across a large genome region of chromosome 1, and could lead to chromosomal abnormalities (i.e., deletions or duplications) within this region. In contrast, a hitchhiking effect associated with DDT usage is not evident for the para locus, as indicated by the fact that genetic heterozygosity at the para locus and loci closely linked to para was similar to other random loci in the genome. This result is consistent with the hypothesis that in the years since DDT was abandoned, the populations have had time to re-equilibrate.

Ideally, the hitchhiking effect should be demonstrated at the nucleotide diversity level (KAPLAN et al. 1989 ). Unfortunately, nucleotide diversity cannot be appropriately estimated for the present data, because our RFLP data is based on one restriction enzyme. To statistically rule out the possibility of low heterozygosity caused by reduced mutation rates and/or increased functional constraints in the LF90 gene region, one needs to examine intraspecific variation and interspecific divergence over several gene regions for closely-related species. This method has been elegantly applied to Drosophila studies (BEGUN and AQUADRO 1991 , BEGUN and AQUADRO 1992 ). The rationale is that, if reduced mutation rate in a gene region leads to low intraspecific variation, then interspecific divergence is expected to be smaller than in other gene regions. However, hitchhiking effect only reduces intraspecific polymorphism, but will not affect interspecific divergence (BEGUN and AQUADRO 1991 ). The magnitude of the hitchhiking effect should be inversely proportional to the recombination distance. In addition, laboratory experiments should be conducted to investigate the hypothesis that gene deletions in the genome region containing LF230 result from unequal recombination between susceptible individuals and resistant individuals with an amplified esterase gene.

Gene flow estimates among the four mosquito populations were very high compared to other animal species (SLATKIN 1985 ). Our gene flow estimates are, however, consistent with other studies of natural A. aegypti (APOSTOL et al. 1996 ) and C. pipiens (CHEVILLON et al. 1995 ) populations. Extinction and recolonization may constitute an important and powerful form of gene flow for the mosquito populations in small geographic areas. Suitable niches may often be vacated by insecticide applications, and then subsequently recolonized by mosquitoes. Direct estimates of A. aegypti's natural dispersal ability, however, found limited flight ranges in urban areas, usually within 1 km when there are no geographic barriers (REITER et al. 1995 ). Therefore, gene flow between the Trinidad and Tobago populations must be assisted by human activities, because a 35-km strait is far beyond A. aegypti's flight ability. A. aegypti-infested water containers transported from Trinidad to Tobago have occasionally been detected since 1983 (CHADEE 1990 ).

SLATKIN and BARTON 1989 showed that, in a subdivided population that is at a demographic equilibrium, both F_ST and private-alleles methods can provide reasonably accurate estimates of Nm under a variety of conditions. Our results indicate that Nm estimates based on F_ST were more consistent among loci and are probably more reliable than the private-alleles method. However, two factors may lead to biased estimates of gene flow for both methods. First, strong selection by insecticides may have significant effects on population allele frequencies, and produce local differentiation. Thus, gene flow based on F_ST would be underestimated. Second, the assumption that the populations are in genetic equilibrium may not be true for mosquitoes. Assuming that mutation is small relative to migration, the half time to equilibrium (t_1/2) between gene flow and genetic drift is calculated as t_1/2 = Ln2/(2m + 1/N ) (CROW and AOKI 1984 ). If the generation time of mosquitoes is 1 month, and Nm = 4.2 (from the present study), t_1/2 is about 3 years for a population with N = 500. The interval between insecticide sprayings is typically only 3–4 months, far less than the time required to reach genetic equilibrium.

In this study, we applied classical population genetic theory and molecular techniques to study the evolutionary consequences of insecticide utilization in medically-important field pest populations. We made specific predictions concerning gene polymorphisms and spatial variations based on the history of insecticide application. These predictions were then tested by RFLP analysis of loci representative of the mosquito genome. Our data were generally consistent with the predictions. We observed evidence for a hitchhiking effect in the general chromosomal region containing genes presumed to confer resistance to OPs. The hitchhiking effect was reflected by low DNA polymorphisms and gene deletions for loci surrounding the EST-4 locus gene region. Gene deletions and reduced genetic variability in genome regions of chromosome 1 may be the result of the hitchhiking effect associated with the spread of the amplified EST-4 gene, which increases the fitness of the mosquitoes in the OP environment (WOOD and BISHOP 1981 ). Large gene flow among the mosquito populations likely resulted from human-assisted migration, and may explain the rapid spread of insecticide resistant genes (RAYMOND et al. 1991 ).

	FOOTNOTES

¹ Present address: Department of Biological Sciences, State University of Notre Dame, Notre Dame, IN 46556.

	ACKNOWLEDGMENTS

We thank M. FERO, M. KASSNER, V. KASSNER, L. SMITH and J. WALERAK for technical assistance. M. RAYMOND, W. J. TABACHNICK and D. ZAITLIN provided valuable discussions. We are grateful to J. F. CROW, C. DENNISTON, K. F. GOODNIGHT and two anonymous reviewers for critical review. This work was funded by a National Institutes of Health (NIH) National Research Service Award No. T32 (NIH grant AI-07414) to G.Y., and NIH grant AI-33127 to D.W.S.

Manuscript received April 3, 1997; Accepted for publication October 29, 1997.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

APOSTOL, B. L., W. C. BLACK, P. REITER, and B. R. MILLER, 1994  Use of randomly amplified polymorphic DNA amplified by polymerase chain reaction markers to estimate the number of Aedes aegypti families at oviposition sites in San Juan, Puerto Rico. Am. J. Trop. Med. Hyg. 51:89-97.

APOSTOL, B. L., W. C. BLACK, P. REITER, and B. R. MILLER, 1996  Population genetics with RAPD-PCR markers: the breeding structure of Aedes aegypti in Puerto Rico. Heredity 76:325-334.

AQUADRO, C. F., 1992  Why is the genome variable? Insights from Drosophila.. Trends Genet. 8:355-361[Medline].

BEGUN, D. J. and C. F. AQUADRO, 1991  Molecular population genetics of the distal portion of the X chromosome in Drosophila: evidence for genetic hitchhiking of the yellow-achaete region. Genetics 129:1147-1158[Abstract].

BEGUN, D. J. and C. F. AQUADRO, 1992  Levels of naturally occurring DNA polymorphism correlate with recombination rates in D. melanogaster.. Nature 356:519-520[Medline].

BLACK, W. C. and K. S. RAI, 1988  Genome evolution in mosquitoes: intraspecific and interspecific variation in repetitive DNA amounts and organization. Genet. Res. 51:185-196[Medline].

CHADEE, D. D., 1990  Aedes aegypti surveillance in Tobago, West Indies (1983-88). J. Am. Mosq. Control Assoc. 6:148-150[Medline].

CHEVILLON, C., N. PASTEUR, M. MARQUINE, D. HEYSE, and M. RAYMOND, 1995  Population structure and dynamics of selected genes in the mosquito Culex pipiens.. Evolution 49:997-1007.

CONN, J., A. F. COCKBURN, and S. E. MITCHELL, 1993  Population differentiation of the malaria vector Anopheles aquasalis using mitochondrial DNA. J. Hered. 84:248-253[Abstract/Free Full Text].

CROW, J. F. and K. AOKI, 1984  Group selection for a polygenic behavioral trait: estimating the degree of population subdivision. Proc. Natl. Acad. Sci. USA 81:6073-6077[Abstract/Free Full Text].

GILKES, C. D., F. R. S. KELLETT, and H. P. S. GILLETTE, 1956  Yellow fever in Trinidad and the development of resistance in Aedes aegypti Linn, to D.D.T. formulations. W. I. Med. J. 5:73-89.

GOUDET, J., 1995  FSTAT version 1.2: a computer program to calculate F-statistics. J. Hered. 86:485-486[Free Full Text].

JAMES, A. A., K. BLACKMER, and J. V. RACIOPPI, 1989  A salivary gland-specific maltase-like gene of the vector mosquito, Aedes aegypti.. Gene 75:73-83[Medline].

KAPLAN, N. L., R. R. HUDSON, and C. H. LANGLEY, 1989  The "hitchhiking effect" revisited. Genetics 123:887-899[Abstract/Free Full Text].

KIMURA, M., 1983 The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge.

KREITMAN, M. and H. AKASHI, 1995  Molecular evidence for natural selection. Ann. Rev. Ecol. Syst. 26:403-422.

MAYNARD SMITH, J. and J. HAIGH, 1974  The hitch-hiking effect of a favorable gene. Genet. Res. 23:23-35[Medline].

MOUCHÈS, C., Y. PAUPLIN, M. AGARWAL, L. LEMIEUX, and M. HERZOG et al., 1990  Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.. Proc. Natl. Acad. Sci. USA 87:2574-2578[Abstract/Free Full Text].

MUNSTERMANN, L. E., 1990 Linkage map of the yellow fever mosquito, Aedes aegypti, pp. 179–183 in Genetic Maps, Vol. 3, edited by S. J. O'BRIEN. Cold Spring Harbor Press, Cold Spring Harbor, NY.

NEI, M., 1987 Molecular Evolutionary Genetics. Columbia University Press, New York.

NEI, M., T. MARUYAMA, and R. CHAKRABORTY, 1975  The bottleneck effect and genetic variability in populations. Evolution 29:1-10.

POWELL, J. R., W. J. TABACHNICK, and J. ARNOLD, 1980  Genetics and the origin of a vector population: Aedes aegypti, a case study. Science 208:1385-1387[Abstract/Free Full Text].

RAWLINS, S. C. and J. H. WAN, 1995  Resistance in some Caribbean populations of Aedes aegypti to several insecticides. J. Am. Mosq. Control Assoc. 11:59-65[Medline].

RAYMOND, M. and F. ROUSSET, 1995  GENEPOP (version 1.2): a population genetics software for exact tests and ecumenicism. J. Hered. 86:248-249[Free Full Text].

RAYMOND, M., A. CALLAGHAN, P. FORT, and N. PASTEUR, 1991  Worldwide migration of amplified insecticide resistance genes in mosquitoes. Nature 350:151-153[Medline].

REITER, P., M. A. AMADOR, R. A. ANDERSON, and G. G. CLARK, 1995  Dispersal of Aedes aegypti in an urban area after blood feeding as demonstrated by rubidium-marked eggs. Am. J. Trop. Med. Hyg. 52:177-179.

ROBERTSON, A. and W. G. HILL, 1984  Deviations from Hardy-Weinberg proportions: sampling variances and use in estimation of inbreeding coefficients. Genetics 107:703-718[Abstract/Free Full Text].

ROUSSET, F. and M. RAYMOND, 1995  Testing heterozygote excess and deficiency. Genetics 140:1413-1419[Abstract].

SEVERSON, D. W. and Y. ZHANG, 1996  Generation of expressed sequence tags and sequence-tagged sites as physical landmarks in the mosquito, Aedes aegypti, genome. Genome 39:224-229[Medline].

SEVERSON, D. W., A. MORI, Y. ZHANG, and B. M. CHRISTENSEN, 1993  Linkage map for Aedes aegypti using restriction fragment length polymorphisms. J. Hered. 84:241-247[Abstract/Free Full Text].

SEVERSON, D. W., A. MORI, Y. ZHANG, and B. M. CHRISTENSEN, 1994a  The suitability of RFLP markers for evaluating genetic diversity among and synteny between mosquito species. Am. J. Trop. Med. Hyg. 50:425-432.

SEVERSON, D. W., A. MORI, Y. ZHANG, and B. M. CHRISTENSEN, 1994b  Chromosomal mapping of two loci affecting filarial worm susceptibility in Aedes aegypti.. Insect Biochem. Mol. Biol. 3:67-72.

SEVERSON, D. W., V. THATHY, A. MORI, Y. ZHANG, and B. M. CHRISTENSEN, 1995  Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti.. Genetics 139:1711-1717[Abstract].

SEVERSON, D. W., N. M. ANTHONY, O. ANDREEV, and R. H. FFRENCH-CONSTANT, 1997  Molecular mapping of insecticide resistance genes in the yellow fever mosquito Aedes aegypti.. J. Hered. 88:520-524[Abstract/Free Full Text].

SLATKIN, M., 1985  Gene flow in natural populations. Ann. Rev. Ecol. Syst. 16:393-430.

SLATKIN, M. and N. H. BARTON, 1989  A comparison of three indirect methods for estimating average levels of gene flow. Evolution 43:1349-1368.

TABACHNICK, W. J., 1991  Evolutionary genetics and arthropod-borne disease: the yellow fever mosquito. Am. Entomol. 37:14-24.

TABACHNICK, W. J., and W. C. BLACK, 1996 Population genetics in vector biology, pp. 417–437 in The Biology of Disease Vectors, edited by B. J. BEATY and W. C. MARQUARDT. University Press of Colorado, Niwot, CO.

TABACHNICK, W. J., and G. P. WALLIS, 1985 Population genetic structure of the yellow fever mosquito Aedes aegypti in the Caribbean: ecological considerations, pp. 371–382 in Ecology of Mosquitoes: Proceedings of a Workshop, edited by L. P. LOUNIBOS, J. R. REY and J. H. FRANK. Florida Medical Entomology Laboratory, Vero Beach, FL.

WEIR, B. S., 1990 Genetic Data Analysis. Sinauer Associates, Sunderland, MA.

WEIR, B. S. and C. C. COCKERHAM, 1984  Estimating F-statistics for the analysis of population structure. Evolution 38:1358-1370.

WILLIAMSON, M. S., D. MARTINEZ-TORRES, C. A. HICK, and A. L. DEVONSHIRE, 1996  Identification of mutations in the housefly para-type sodium channel gene associated with knockdown resistance (kdr) to pyrethroid insecticides. Mol. Gen. Genet. 251:51-60.

WOOD, R. A., and J. A. BISHOP, 1981 Insecticide resistance: genes and mechanisms, pp. 97–127 in Genetic Consequences of Man-Made Change, edited by J. A. BISHOP and L. M. COOK. Academic Press, London.

WRIGHT, S., 1943  Isolation by distance. Genetics 28:114-138[Free Full Text].

YAN, G., B. M. CHRISTENSEN, and D. W. SEVERSON, 1997  Com-parisons of genetic variability and genome structure among mosquito strains selected for refractoriness to a malaria parasite. J. Hered. 88:187-194[Abstract/Free Full Text].

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