Mem Inst Oswaldo Cruz, Rio de Janeiro, 101(8) December 2006
Higher genetic variation estimated by microsatellites compared to isoenzyme markers in Aedes aegypti from Rio de Janeiro
ILaboratório de Transmissores de Hematozoários, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brasil
IIInsectes et Maladies Infectieuses, Institut Pasteur, Paris, France
Aedes aegypti populations from five districts in Rio de Janeiro were analyzed using five microsatellites and six isoenzyme markers, to assess the amount of variation and patterns of gene flow at local levels. Microsatellite loci were polymorphic enough to detect genetic differentiation of populations collected at small geographic scales (e.g. within a city). Ae. aegypti populations were highly differentiated as well in the city center as in the outskirt. Thus, dengue virus propagation by mosquitoes could be as efficient in the urban area as in the outskirt of Rio de Janeiro, the main entry point of dengue in Brazil.
Dengue is the most important mosquito-borne disease in Brazil where it is essentially transmitted by Aedes aegypti. Rio de Janeiro is considered to be the main introduction location for dengue viruses which have caused most epidemics in Brazil with increasing incidence of severe cases (Nogueira et al. 2002, Teixeira et al. 2002). The urban mosquito Ae. aegyptiis a major dengue vector in Brazil. It is present in high density at the vicinity of highly populated areas (Lourenço-de-Oliveira et al. 2004). The species shows high level of resistance to insecticides (Luz et al. 2003, Braga et al. 2004, Da-Cunha et al. 2005) and is highly susceptible to dengue viruses (Lourenço-de-Oliveira et al. 2004). Moreover, uncontrolled urbanization and intensive travel and migration are additional factors that favor both vector and virus dis-semination (Gubler 1998, Herrera et al. 2006).
Studies on Ae. aegypti population structure based on allozyme analysis have been reported for more than 40 years (Tabachnick et al. 1979). More recently, micro-satellite markers have been extensively applied to study genetic variations or to detect gene flow (Bruford & Wayne 1993, Jarne & Lagoda 1996). Microsatellites are apparently neutral, codominant and highly polymorphic markers (Tautz 1989).
Field studies on genetic differentiation within vector populations can yield important information regarding to evolution and population biology (Wang et al. 2001). Variations in isoenzymes and microsatellites among vector populations have provided evidence that water storage habits and human densities may affect their differentiation (Huber et al. 2002). Besides, micro-satellite markers provide a sensitive measure of divergence and therefore can potentially distinguish populations that may have recently diverged (Lanzaro et al. 1995). Here, we report a genetic study on Ae. aegypti in Rio de Janeiro using both microsatellite and iso-enzyme markers.
MATERIALS AND METHODS
Mosquito samples - Ae. aegypti was sampled in five districts in Rio de Janeiro in March 2003. Samples were divided into two groups according to the human density in the sampled district: (i) high human density (city center) Pilares (PILR) and Tijuca (TIJU) (15,364.17 inhabitants/km² and 17,431.88 ha/km2, respectively) and (ii) moderate human density (outskirt) - Barra da Tijuca (BART), Campo Grande (CAMG), and Taquara (TAQU) (1,726.46 ha/km2, 2,182.58 ha/km2 and 6,444.13 ha/km2, respectively). To avoid collections of descendents from small number of Ae. aegypti females, 20 ovitraps (Reiter et al. 1991) were set per district during two consecutive weeks (Dibo et al. 2005). Districts are separated by a minimal distance of 8.2 km and a maximal of 32.4 km (see Costa-Ribeiro et al. 2006 for more details).
Mosquitoes were reared to the adult stage (F0 gen-eration) in insectaries under standardized conditions (25 ± 1°C, 80 ± 10% relative humidity and 12 h light/dark cycle) and subsequently, stored at 80°C. Mosquitoes from the same samples were analyzed by both the microsatellite and isoenzyme assays.
Microsatellites - Thirty mosquito adults from each district were analyzed using six microsatellite loci: C2A8, 34/72, T3A7, AED19, 38/38, and 9A89 (see Huber et al. 2001 for more details). DNA was extracted in DNAzol solution (Gibco BRL). PCR reactions containing 1X buffer (Eurobio), 1.2 mM MgCl2, 60 µM of each dNTP, 5 pmol of each primer, 0.25 units of Taq polymerase (Eurobio), and 2 µl of DNA were performed in a 9600 thermal cycler (Perkin-Elmer). The reaction conditions were 5 cycles (2 min at 96°C, 30 s annealing at locus-specific, an-nealing temperature (Ta) and 1 min 15 s extension at 72°C) followed by 25 cycles (30 s at 95°C, 30 s at Ta and 1 min 15 s at 72°C) and a final elongation step (5 min at 72°C). One primer (1.5 pmoles) was end-labeled with one of the three different fluorescent phospho-ramidite dyes (FAM, HEX or NED) appropriate for ABI PRISM instruments. One microliter of each PCR reaction was diluted to 1:20 times with distilled water. Samples were prepared by adding 0.5 µl of internal size standard 400HD ROX (Perkin Elmer) and deionised formamide for a final volume of 20 µl.
Isoenzymes - Forty-eight adults from each district were analyzed for 10 enzyme systems: glucose phosphate isomerase (Gpi, EC 188.8.131.52.), glutamate oxaloacetate transaminases (Got1 and Got2, EC 184.108.40.206.), glycerol phosphate dehydrogenase (Gpd, EC 220.127.116.11.), hexokinases (Hk, EC 18.104.22.168.), malate dehydrogenase (Mdh, EC 22.214.171.124.), malic enzyme (Me, EC 126.96.36.199.), and phosphoglucomutase (Pgm, EC 188.8.131.52.). Each individual mosquito was ground in 25 µl of distilled water and centrifuged (12,000 g for 3 min at +4°C). The supernatant containing soluble proteins was loaded onto a 12.8% starch gel in Tris-Maleate-EDTA (pH 7.4) buffer and run for 4-5 h (Pasteur et al. 1988). A laboratory strain of Ae. aegypti named "Paea" (collected in 1994 in Tahiti, French Polynesia) was used as a mobility control for isoenzyme polymorphism.
Genetic analysis - Hardy-Weinberg (HW) propor-tions were compared using GENEPOP software (version 3.4) (Raymond & Rousset 1995). Deviations from HW were tested using an exact test procedure with the alternative hypothesis H1 corresponding to deficits or excess in heterozygotes (Rousset & Raymond 1995). Linkage disequilibrium was tested between pairs of loci for each sample using Fisher's exact test on rank ' column contingency tables. FIS, the inbreeding coef-ficient, and FST, the fixation index, were estimated as described by Weir and Cockerham (1984). Genetic differentiation across samples was estimated by calculating the P value associated to the FST estimate. Significance levels for multiple testing were corrected using sequential Bonferroni's procedures (Holm 1979). Genetic isolation by geographic distance was tested by estimating rank correlations between FST/(1-FST) calculated between pairs of samples and Ln distances (Slatkin 1993).
Microsatellite polymorphism - The locus 9A89 was monomorphic and 3-4 alleles were scored for the other five loci: three for 34/72, AED19, and 38/38, and four for C2A8 and T3A7 (Table I). The allelic frequencies varied for the loci C2A8 from 0.036 (allele 226) in CAMG to 0.717 (allele 229) in TIJU; for 34/72, from 0.017 (allele 89) in CAMG to 0.833 (allele 87) in PILR; for T3A7, from 0.018 (alleles 216) to 0.821 (allele 224) in BART; for AED19, from 0.054 (allele 178) in TAQU to 0.929 (allele 172) in BART; and for 38/38, from 0 to 1 (Table I).
For HW equilibrium, 90 tests were carried out (1 test for each locus in each sample). Significant deviations from Hardy-Weinberg equilibrium associated to deficits of heterozygotes were detected in the locus 34/72 in TAQU (FIS= 0.5591, P = 0.0020) and in the locus T3A7 for all samples: BART (FIS= 0.5355, P = 0.0081), PILR (FIS= 0.8058, P <10-4), TIJU (FIS= 1, P < 10-4), TAQU (FIS= 0.8842, P < 10-4), and CAMG (FIS= 0.7321, P = 0.0001). Tests considering all loci for each sample showed significant deviations from Hardy-Weinberg equilibrium due to heterozygote deficits (Table I). No linkage disequilibrium was detected among 34 comparisons tested. Genetic differentiation evaluated by estimating FST values was high and significant when considering mosquitoes from all five districts (FST = 0.3304, P < 0.05; Table II). When samples were grouped according to human density (city center versus outskirt), a higher genetic differentiation (FST = 0.3792, P < 0.05) was detected among samples from the outskirt (Table II). When testing isolation by geographic distance by estimating rank correlations between FST/(1-FST) calculated between pairs of samples and log geographical distances, no isolation was demonstrated (P = 0.1522) (data not shown).
Isoenzyme polymorphism - Six (Pgm, Pgi, Mdh, Hk2, Got1, and Got2) out of 10 loci investigated were poly-morphic. 2-3 alleles were scored for each locus: 2 for Pgm, Mdh, Got1 and Got2, and 3 for Pgi and Hk2 (Table III). The allelic frequencies varied for the loci Pgm from 0.115 (100) in PILR to 0.958 (80) in BART; for Mdh, from 0.344 (110) in CAMG to 0.656 (100) in CAMG; for Hk2, from 0 to 0.778 (100) in BART; for Got1, from 0.085 (60) in CAMG to 0.915 (100) in BART and CAMG; and for Pgi and Got2, from 0 to 1 (Table III).
For Hardy-Weinberg equilibrium, 150tests were carried out. Only two significant deviations from Hardy-Weinberg equilibrium were observed: a heterozygote deficit in TIJU (FIS= 1, P = 0.0105) and a heterozygote excess in CAMG (FIS= -0.7484, P < 10-4 (Table III). When considering all loci in each sample, a significant heterozygote excess was observed in CAMG (FIS= -0.0856 and P < 10-4) (Table III). No linkage dis-equilibrium was detected among 42 comparisons. Differentiation was significant when considering all samples (FST = 0.0200, P < 0.05), and samples from the outskirt (FST= 0.0375, P < 0.05) (Table III). No correlation was detected when testing genetic isolation by geographic distance (data not shown).
As expected, microsatellite markers displayed higher polymorphism when compared to isoenzyme loci. For microsatellite loci, five among six loci were polymorphic whereas for isoenzyme loci, only six among ten were polymorphic.
Microsatellite loci in Ae. aegypti populations from Rio de Janeiro were less polymorphic when compared with Asian populations (Huber et al. 2002, 2004, Paupy et al. 2004). Low abundance and limited polymorphism in microsatellites have been already described for Ae. aegypti (Fagerberg et al. 2001, Ravel et al. 2001, 2002). In all samples, heterozygote deficits were observed due to the locus T3A7. This may be related to four main factors: (i) the locus is under selection, (ii) null alleles may be present, (iii) inbreeding may be common in populations, or (iv) the presence of population substructure which leads to a Wahlund's effect. The locus T3A7 was probably subjected to mutations in its flanking regions which resulted in inhibition of primer annealing with the complementary DNA sequences. The failure in detection of microsatellite PCR products generates null alleles and thus, a deficit in heterozygotes (Lanzaro et al. 1995). Inbreeding could not explain heterozygote deficits as deficits were not observed in all loci. The Wahlund effect which describes deviations due to the pooling of subpopulations in HW equilibrium could be one explanation. However, the less polymorphic isoenzyme loci only detected two samples with a deviation from HW equilibrium due to a heterozygote excess or deficit.
Isoenzyme markers revealed a larger genetic dif-ferentiation among samples from the outskirt whereas samples from the city center were not differentiated. This pattern contrasted with those observed in Vietnam (Tran Khanh et al. 1999).
Ae. aegypti females tend to lay eggs in artificial containers at the vicinity of human habitations. So, dispersal varies upon the distribution of breeding sites and human density for blood feeding (Tsuda et al. 2001). Gene flow contributes to homogenize populations and thus reduces genetic differences among them. In Rio de Janeiro, samples were collected at the end of the rainy season, in March, when a large number of unintentional containers such as cans, tires and bottles were productive in larvae and pupae (Honório et al 2006). This type of breeding sites more abundant in highly populated areas predominates in the city center where flow low genetic differentiation or deficits among mosquito population was detected using isoenzyme markers. Conversely, microsatellite markers were polymorphic enough to differentiate populations within the city center whereas isoenzymes failed to detect any differentiation.
Dengue is endemic in Rio de Janeiro since the in-troduction of dengue virus in 1986 (Lourenço-de-Oliveira et al. 2004). Variations in dengue incidence are observed within the city depending on vector distribution and vector control implementation (Luz et al. 2003). Our results demonstrated microsatellite markers to be more adapted to evaluate genetic structure of samples collected within a small geographic scale like a city. The same pattern of genetic differentiation of Ae. aegypti populations in the city center and the outskirt suggested that the dengue virus propagation by mosquitoes could be as efficient in urban area as in the outskirt of Rio de Janeiro.
To Mauro Blanco Brandolini, Fábio Castelo, Fernando da Costa Alves, and the staff of Coordenação de Controle de Vetores in Rio de Janeiro for providing mosquito samples; to Nadia Ayad for technical assistance; to Sara Moutailler and Marcia Castro for their assistance in the laboratory.
Braga IA, Lima JBP, Soares SS, Valle D 2004. Aedes aegypti resistance to temephos during 2001 in several municipalities in the states of Rio de Janeiro, Sergipe and Alagoas, Brazil. Mem Inst Oswaldo Cruz 99: 199-203.
Bruford MW, Wayne RK 1993. Microsatellites and their application to population genetic studies. Curr Opin Genet Dev 3: 939-943.
Costa-Ribeiro MCV, Lourenço-de-Oliveira R, Failloux AB 2006. Geographic and temporal genetic patterns of Aedes aegypti populations in Rio de Janeiro, Brazil. Trop Med Int Hlth 11: 1276-1285.
Da-Cunha MP, Lima JBP, Brogdon WG, Moya GE, Valle D 2005. Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003. Mem Inst Oswaldo Cruz 100: 441-444.
Dibo MR, Chiaravalloti-Neto F, Battigaglia M, Mondini A, Favaro EA, Barbosa AAC, Glasser CM 2005. Identification of the best ovitrap installation sites for gravid Aedes (Stegomyia) aegypti in residences in Mirassol, state of São Paulo, Brazil. Mem Inst Oswaldo Cruz 100: 339-343.
Fagerberg AJ, Fulton RE, Black IV WC 2001. Microsatellite loci are not abundant in all arthropod genomes: analyses in the hard tick, Ixodes scapularis and the yellow fever mosquito, Aedes aegypti. Insect Mol Biol 10: 225-236.
Gubler DJ 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480-496.
Herrera F, Urdaneta L, Rivero J, Zoghbi N, Ruiz J, Carrasquel G, Martinez JA, Pernalete M, Villegas P, Montoya A, Rubio-Palis Y, Rojas E 2006. Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela. Mem Inst Oswaldo Cruz 101: 625-633.
Holm S 1979. A simple sequentially rejective multiple test procedure. Scand J Stat 6: 65-70.
Honório NA, Cabello PH, Codeço CT, Lourenço-de-Oliveira R 2006. Preliminary data on the performance of Aedes aegypti and Aedes albopictus immatures developing in water-filled tires in Rio de Janeiro. Mem Inst Oswaldo Cruz 101: 225-228.
Huber K, Luu Le L, Chanta N, Failloux AB 2004. Human transportation influences Aedes aegypti gene flow in Southeast Asia. Acta Trop 90: 23-29.
Huber K, Luu Le L, Tran Huu H, Tran Khanh T, Rodhain F, Failloux AB 2002. Temporal genetic variation in Aedes aegypti populations in Ho Chi Minh City (Vietnam). Heredity 89: 7-14.
Huber K, Mousson L, Rodhain F, Failloux AB 2001. Isolation and variability of polymorphic microsatellite loci in Aedesaegypti, the vector of dengue viruses. Mol Ecol Notes 1: 219-222.
Jarne P, Lagoda PJL 1996. Microsatellites, from molecules to populations and back. Trends Ecol Evol 11: 424-429.
Lanzaro GC, Zheng L, Toure YT, Traore SF, Kafatos FC, Vernick KD 1995. Microsatellite DNA and isozyme variability in a West African population of Anopheles gambiae. Insect Mol Biol 4: 105-112.
Lourenço-de-Oliveira R, Vazeille M, Filippis AMB, Failloux AB 2004. Aedes aegypti in Brazil: genetically differentiated populations with high susceptibility to dengue and yellow fever viruses. Trans R Soc Trop Med Hyg 98: 43-54.
Luz PM, Codeco CT, Massad E, Struchiner CJ 2003. Uncertainties regarding dengue modeling in Rio de Janeiro, Brazil. Mems Inst Oswaldo Cruz 98: 871-878.
Nogueira RMR, Miagostovich MP, Schatzmayr HG 2002. Dengue viruses in Brazil. Dengue Bull26: 77-83.
Pasteur N, Pasteur G, Bonhomme F, Catalan J, Britton-Davidian J 1988. Practical Isozyme Genetics, John Wiley and Sons/Ellis Horwood Ltd., England.
Paupy C, Chanta N, Huber K, Lecoz N, Reynes J-M, Rodhain F, Failloux AB 2004. Influence of breeding sites features on genetic differentiation of Aedes aegypti populations analyzed on a local scale in Phnom Penh municipality of Cambodia. Am J Trop Med Hyg 71: 73-81.
Ravel S, Hervé JP, Diarrassouba S, Kone A, Cuny G 2002. Microsatellite markers for population genetic studies in Aedes aegypti (Diptera: Culicidae) from Côte d'Ivoire: evidence for a microgeographic genetic differentiation of mosquitoes from Bouaké. Acta Trop 82: 39-49.
Ravel S, Monteny N, Velasco Olmos D, Escalante Verdugo J, Cuny G 2001. A preliminary study of the population genetics of Aedes aegypti (Diptera: Culicidae) from Mexico using microsatellite and AFLP markers. Acta Trop 78: 241-250.
Raymond M, Rousset F 1995. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86: 248-249.
Reiter P, Amador MA, Anderson RA, Colon N 1991. Enhancement of the CDC ovitrap with hay infusions for daily monitoring of Aedes aegypti populations. J Am Mosq Control Assoc 7: 52-55.
Rousset F, Raymond M 1995. Testing heterozygote excess and deficiency. Genetics 140: 1413-1419.
Slatkin M 1993. Isolation by distance in equilibrium and non-equilibrium populations. Evolution 47: 264-279.
Tabachnick WJ, Munstermann LE, Powell JR 1979. Genetic distinctness of sympatric joins of Aedes aegypti in East Africa. Evolution 33: 287-295.
Tautz D 1989. Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 17: 6463-6471.
Teixeira MG, Costa MCN, Guerra Z, Barreto ML 2002. Dengue in Brazil: Situation-2001 and trends. Dengue Bull26: 70-76.
Tran Khanh T, Vazeille-Falcoz M, Mousson L, Tran Huu H, Rodhain F, Nguyen Thi H, Failloux AB 1999. Aedes aegypti in Ho Chi Minh city (Vietnam): Susceptibility to dengue 2 Virus and genetic differentiation. Trans R Soc Trop Med Hyg 93: 581-586.
Tsuda Y, Takagi M, Wang S, Wang Z, Tang L 2001. Movement of Aedes aegypti (Diptera:Culicidae) released in a small isolated village on Hainan island, China. J Med Entomol 38: 93-98.
Wang R, Zheng L, Touré YT, Dandekar R, Kafatos F 2001. When genetic distance matters: Measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species. Proc Nat Acad Sci USA 98: 10769-10774.
Weir BS, Cockerham CC 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370.