Mem Inst Oswaldo Cruz, Rio de Janeiro, 100(6) October 2005
Variability and genetic differentiation among Anopheles (Ano.) intermedius Chagas, 1908 and Anopheles (Ano.) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) from the Brazilian Amazon
Instituto Nacional de Pesquisas da Amazônia, Coordenação de Pesquisas em Ciências da Saúde, Av. André Araújo 2936, 69011-970 Manaus, AM, Brasil
*Dirección Regional de Salud de Loreto, Dirección Ejecutiva de Salud Ambiental, Iquitos, Peru
Anopheles (Anopheles) intermediusu00a0andu00a0Anopheles (Ano.) mattogrossensisu00a0are Brazilian anopheline species belonging to the scarcely studiedu00a0Anophelesu00a0subgenus. Few studies have been done on the genetic differentiation of these species. Both species have been found infected byu00a0Plasmodiumu00a0and are sympatric with other anopheline species from theu00a0Nyssorhynchusu00a0subgenus. Eighteen enzymatic loci were analyzed in larval specimens ofu00a0An. intermediusu00a0andu00a0An. mattogrossensisu00a0aiming to estimate the variability and genetic differentiation between these species.u00a0An. mattogrossensisu00a0population showed higher genetic variability (P = 44.4 and Ho = 0.081 ± 0.031) than that ofu00a0An. intermediusu00a0(P = 33.3 and Ho = 0.048 ± 0.021). Most analyzed loci showed genotypic frequencies according to Hardy-Weinberg equilibrium, except foru00a0LAP1u00a0andu00a0LAP2u00a0inu00a0An. intermedius, andu00a0EST1u00a0andu00a0PGMu00a0loci inu00a0An. mattogrossensis. The genetic distance between these species (D = 0.683) was consistent with the inter-specific values reported foru00a0Anophelesu00a0subgenus. We verified that the polymorphism and heterozygosity percentile values found in both species and compared to those in the literature, showed no relation between the level of isozyme variability and geographical distribution. The low variability found in these two species is probably more related to the niche they occupy than to their geographic distribution.
Information concerning variability and genetic differentiation in species of the subgenus Anopheles is very scanty in Brazil, even thought they include malaria vector species in other American neighboring countries (Ro-driguez et al. 2000, Santos et al. 2003). An. intermedius and An. mattogrossensis show a wide geographical distribution in South America (Bolivia, Brazil, Peru, Colombia, Venezuela, Trinidad, and Guiana), being most abundant in forested areas like the Brazilian Amazon Region (Forattini 1962). Larvae are mostly found in shaded lagoons, marshes, floodplains, residual puddles in the forest, and river stretches in the middle of the bushes. Females are exophilic and zoophilic, and there have been no evidence of being able to transmit malaria (Forattini 1962, Belkin et al. 1971). However, dissection and ELISA analyses revealed that these species were infected by Plasmodium(Arruda et al. 1986, Tadei & Dutary-Thatcher 2000). In an area where anthropic modified environments are rapidly spreading like the Brazilian Amazon, this species maybe under vigilance as able to transmit malaria.
This work aims to analyze the isoenzimatic genetic variability of An. (Ano.) intermedius and An. (Ano.)mattogrossensis and to compare to results found for species of the subgenus Nyssorhynchus.
MATERIALS AND METHODS
Specimens and collection sites - Wild-caught An. mattogrossensis adult females were collected on cattle-bait in Lake Janauari (right margin of the Negro River, near Manaus), Amazonas (03º08'00.5"S, 60º00'34.7"W). An. intermedius adult females were captured on human-bait in forest border areas close to domiciles (30 m approximately) of Highway Pacoval, km 4, Macapá, Amapá (00º02'19.8"N, 51º03'40.7"W), between 18:00 and 22:00 h. Females mosquitoes were isolated for individual oviposition. After hatching, larvae from individual progenies were maintained at 26º ± 1ºC until 4th instar when they were frozen at _70ºC and underwent isoenzymatic analyses, according to Santos et al. (1981). Fourth instar larvae were employed in all the systems, except for a-GPDH, in which adults were used. Morphologic identifications were done on egg and adults based on Cova-Garcia and Sutil (1977) and Consoli and Lourenço-de-Oliveira (1994).
Isoenzymatic analyses - Eighteen loci were analyzed: esterase (EST1, EST3 - E.C.18.104.22.168), leucine aminopeptidase (LAP1, LAP2, LAP3 - E.C.22.214.171.124), a-glycerophosphate dehydrogenase (a-GPD - E.C.126.96.36.199), phos-phoglucose isomerase (PGI - E.C.188.8.131.52), alcohol dehydrogenase (ADH - E.C.184.108.40.206), aldehyde oxidase (AO - E.C.220.127.116.11), isocitrate dehydrogenase (IDH - E.C.18.104.22.168), 6-phosphogluconate dehydrogenase (6-PGD - E.C.22.214.171.124), malate dehydrogenase (MDH - E.C.126.96.36.199), malic enzyme (ME - E.C.188.8.131.52), hexokinase (HK1, HK2, HK3, HK4 - E.C.184.108.40.206) and phosphoglucomutase (PGM - E.C.220.127.116.11). Electrophoresis was performed in starch gel (12.5%) and starch-agarose (2 and 1%, respectively) following Steiner and Joslyn (1979), Lima and Contel (1990) and Santos et al. (1996). For each locus, the most frequent electromorph was designated the 100 allele and all other alleles were measured relative to it.
Statistic analyses - Approximately three individuals from each of 30 progenies were used for each enzymatic system, in a total of 100 individuals. Genetic variability for each species was estimated using the mean number of alleles per locus, proportion of polymorphic loci (P), mean observed heterozygosity (Ho), and expected hete-rozygosity (He) using the Biosys-1 program (Swofford & Selander 1981). Conformance tests for Hardy-Weinberg equilibrium was done by Chi-square test using the Biosys-1 program and by G-square test (Monjeló 2005). The no-criteria option from the Biosys-1 program was used to estimate the percentage of polymorphic loci. Thus, a locus was considered polymorphic if any variation was observed, independent of the frequency of alleles detected. T-student test was used to assess the significance level of the mean observed and expected heterozygosities between An. intermedius and An. mattogrossensis. Genetic distance was calculated according to Nei (1978).
Five out of the 18 loci analyzed presented polymor-phism for both species: EST1, LAP1, LAP2, LAP3, and PGM. PGM locus was the one presenting the largest number of alleles when considering both species (Table I). EST3, 6PGD, and MDH loci were polymorphic only for An. mattogrossensis, whereas IDH locus was polymorphic only for An. intermedius. IDH presented three alleles _ IDH106, IDH100 and IDH92. In An. mattogrossensis only IDH92 was detected (Fig. 1). IDH92 was considered a diagnostic locus, as well as HK1, HK2, HK3, and HK4 loci, which were monomorphic for the HK1100, HK2100, HK3100 and HK4100alleles in An.intermedius and for theHK194, HK295, HK394 and HK498alleles in An. mattogrossensis (Fig. 2). For the PGMlocus, the PGM100 allele was common in the two species (0.929 for An. intermedius). The PGM105allele wasonly detected in An. intermedius while PGM97 was detected only in An. mattogrossensis (Table I).
Both species showed most loci in Hardy-Weinberg equilibrium as demonstrated by Chi-square and G-square results (Table I). For most loci c2 and G2 were not significant except for loci LAP1 (c2 = 56.998, G2 = 7.953, D.F. = 1, P < 0.01) and LAP2 (c2 = 11.692, G2 = 6.961, D.F. = 1, P < 0.01) for An. intermedius population, and EST1 (c2 = 10.120, G2 = 7.732, D.F. = 1, P < 0.01) and PGM (c2 = 5.071, G2 = 5.756, D.F. = 1, P < 0.05) loci for An. mattogrossensis (Table I). Most of the Hardy-Weinberg equilibrium deviations were probably due to excess of observed homozygous individuals, in comparison with the expected numbers.
Two-banded heterozygotes were stained in EST, LAP, and PGM indicating a monomeric structure for these enzymes, and IDH, 6PGD, and MDH showed three bands in the heterozygotes, a pattern typical of dimeric enzymes. The enzymes ME, HK, PGI, ADH, AO, and a-GPD were monomorphic for both species (i.e. no heterozygote individual was detected).
The results of the average number of samples and of alleles per locus were similar between An. intermedius andAn. mattogrossensis species (95.7 ± 4.8 and 98.4 ± 5.5; 1.4 ± 0.1 and 1.4 ± 0.1, respectively). Higher polymorphism and heterozygosity were found in An. mattogrossensis (P = 44.4; He = 0.081 ± 0.031). An. intermedius presented lower polymorphism (P = 33.3) and heterozygosity (He = 0.048 ± 0.021) values (Table II). However, there is not a statistically significant difference between the observed and expected mean heterozygosities of the two species (Student's t = _0.876, _0.956, and P = 0.389, 0.348, respectively).
A comparative analysis of genetic variability parameters between species of the subgenera Nyssorhynchus andAnopheles, based on the present work and published data, is shown in Table III. Genetic distance index betweenAn. intermedius and An. mattogrossensis was 0.683, in conformity with previous values found for interspecific variation in the genus Anopheles.
An. intermedius and An. mattogrossensis are species belonging to the subgenus Anopheles. Because these species do not present epidemiological importance and are of restrict geographical distribution in Brazil, little is known about their population genetics. Isoenzymatic analyses found about 30% polymorphic loci for both species out of 18 loci. Five monomorphic loci were considered diagnostic, i.e. could separate An. intermedius from An. mattogrossensis. These were IDH1, HK1, HK2, HK3, and HK4. Fritz et al. (1995) identified three diagnostic locifor AO, IDH2, and ME, which separate An. nuneztovari, An. rangeli, and An. trinkae, species of the Nys-sorhynchus subgenus, with a closer phylogenetic relationship between the latter two. IDH1andIDH2 also separated the A and B forms of An. quadrimaculatus complex (Lanzaro et al. 1990). According to these authors, the analyses of the genotypic frequencies in these two loci revealed a significant deficiency of heterozygotes, consistent with the Wahlund effect, observed when sympatric populations with limited gene flow are considered as a unique population. However, the lack of heterozygotes found in three of the four loci for An. intermedius andAn. mattogrossensis cannot be accounted for the Wahlund effect, since populations come from different sites apart 2000 km approximately.
Polymorphism values observed for An. intermedius (P = 33.3)and An. mattogrossensis (P = 44.4) are similar to those found for other species of the subgenus Anopheles, such as An. franciscanus (P = 33.3) and An. crucians (P = 39.4) (Manguin et al. 1995). However, in the subgenus Nyssorhynchus, high levels of polymorphism (P > 55.0) were reported for one Brazilian population of An. albitarsis (Narang 1980), three populationsof An. albimanusfrom the Northern Colombia (Narang et al. 1991) and four populations of An. darlingi from the Brazilian Amazon (Santos et al. 1999). Carvalho-Pinto and Lourenço-de-Oliveira (2004) found higher polymorphism levels on An. cruzii Brazilian populations of the subgenus Kerteszia (P = 72.7 - 81.8). Extensive genetic variability in mosquitoes is thought to have implications on malaria control programs, since it could represent plasticity to respond to selection pressure of insecticides (Tadei 1993). Small polymorphism values were registered for Southern Colombian populations of An. albimanus (16 £ P £ 40). An. albimanus low polymorphism values could be due to the relation between the habitat and the reduced population size in the estuaries, which can promote inbreeding, resulting in genetic drift or marginal effect and selection. Low polymorphism values were also detected in the An. triannulatus population from Lake Janauari (25 £ P £ 56.3, Brazilian Amazon, Santos et al. 2004), in An. nuneztovari from Highway BR-174-AM, km 204 (P = 31.3, Brazilian Amazon, Scarpassa et al. 1999), and An. aquasalis population from Rio de Janeiro (P = 34, Narang 1980), of restricted distribution in brackish water on estuarine habitats. For the An. aquasalis population, polymorphism value was similar to that found for An. intermedius (Table III). The low polymorphism in An. triannulatus and An. nuneztovari populations (Scarpassa et al. 1999, Santos et al. 2004) could be related to the fact that collection was performed during dry season, when breeding sites are drastically reduced, leading to mosquitoes population density decrease and favoring inbreeding, that could result in genetic drift or marginal effect and, selection. Low genetic variation was also found in An. mattogrossensis based on mitochondrial DNA-sequencing, when compared with six anopheline species of the subgenus Nyssorhynchus. The same study also showed lower adenine and thymine rates on An. mattogrossensis specimens than on those from subgenus Nyssorhynchus, possibly suggesting a lower mutation rate (Borges R and Santos JMM, unpublished data).
Of the six polymorphic loci found in An. intermedius, only LAP1and LAP2 were significant (Table I). Deviation from Hardy-Weinberg equilibrium was due in part to the large number of homozygotes found. In An. mat-togrossensis, two loci (EST 1 and PGM) also presented significant deviations from their expected allelic frequencies. For EST1 was due to heterozygote deficiency. For PGM, deviation was related to heterozygote excess. Heterozygote deficiency was seen in an An. albitarsis population from Macapá (Amapá) for EST4 and LAP1 loci (Maia 1997). When studying An. nuneztovari populations from Brazil and from Colombia, Hardy-Weinberg disequilibrium was found for some loci, favoring homozygotes excess for a rare allele of EST5 and PGM loci, or heterozygotes individuals constituted by two rare alleles of IDH1 and MDH loci (Scarpassa et al. 1999). A large number of homozygotes was verified for some loci in An. darlingi populations from the Amazon Region, except for theEST2 locus in the Manaus population, in which the heterozygotes were more frequent (Santos et al. 1999).
Lower heterozygosity values were obtained for An. intermedius and An. mattogrossensis when compared to species belonging to Nyssorhynchus and Anopheles subgenera (Table III). However, difference in the mean heterozygosity values for An. intermedius and An. mattogrossensis is not great enough to reject the possibility that the difference is due to random sampling variability.
Species or populations distributed in a large variety of environmental conditions would probably be more genetically heterozygous and/or polymorphic. Contrarily, populations of limited or restricted distribution in special habitats would be less polymorphic (Narang 1980). Low heterozygosity (0.084) found in An. aquasalissuggested this to be an strategy of the species, because larvae breed in brackish water, mainly restricted to the coastal region (Narang 1980).
High heterozygosity values were found for An. crucians (Steiner et al. 1975) and species A, B, and C of the An.quadrimaculatus complex (Narang et al. 1989), that are vectors species of the subgenus Anopheles. However, theAn. pseudopunctipennis population from Granada Island differed from those in the continent on account of lacking heterozygotes and low polymorphism, due to their geographical isolation, suggesting this low level of genetic variability to be brought about by "founder effect" (Manguin et al. 1995).
Data on genetic distance values (D = 0.683) of An. intermedius and An. mattogrossensis are consistent with those found in the literature for interespecific variation in Anopheles subgenus (Manguin et al. 1995). These authors identified lower distance values (D = 0.335) between the closely related species, An. pseudopunctipenis and An.franciscanus, when compared with An. crucians (D = 0.997). Higher value (D = 2.355) was observed between An. pseudopunctipenis and An. (Nys.) albimanus. However, Santos et al. (2003) found lower values (D = 0.373 - 0.989) among five Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera from the Amazon Region. In An. rangeli and An. trinkae, the genetic distance ranged from 0.149 £ D £ 0.197 and between these two species and An. nuneztovari it ranged from 0.286 £ D £ 0.440 (Fritz et al. 1995), both of the Nyssorhynchussubgenus.
The genetic identity (I = 0.382) found between An. intermedius and An. mattogrossensis agrees with the values proposed by Avise (1974) for cogeneric populations (0.26 £ I £ 0.67).
Low heterozygosity and isoenzyme polymorphism found in An. intermedius and An. mattogrossensis populations agree with data found in literature for species of restricted distribution of the subgenus Anopheles and some of the subgenus Nyssorhynchus, and it differs from most malaria vector species. Nonetheless, An. intermedius and An. mattogrossensis can be considered highly specialized species since they can be found only in restricted spotted niches in spite of showing a wide geographical distribution. An. intermedius is found mainly in the border between the primary forest and cattle pastures, while An. mattogrossensis is foundin swamps or shallow flooded areas. The low variability found in these two species is probably related to the specialized ability to thrive on the niches they occupy rather than to their large geographical distribution. The low variability can also be a result ofAn. intermedius and An. mattogrossensis' genetic structures, where selection pressures were not enough to lead to higher variability.
To the technicians of the Malaria and Dengue Vectors Laboratory for their technical support in the collections and identification of the mosquitoes. To Jorge Antunes for helping draft the original English version of the text.
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