Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera:Culicidae)

INTRODUCTION

Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga, 1878, which presents a wide-ranging geographical distribution throughout South America, has been cited as an important malaria vector locally in Brazil, especially in the Amazon region (Segura, 1998; Tadei and Dutary, 2000), the origin of 99.7% of these reports (Fundação Nacional de Saúde - FNS/FUNASA, 2000).

Cytogenetic studies for A. albitarsis population differentiation were made by Kreutzer et al. (1976). They analyzed samples from Brazil, Venezuela and Colombia and identified three populations based on an inversion on the X polytene chromosomes, which were named B1, B2 and C. Later analyses using isoenzymes (Steiner et al., op. cit., 1982; Narang and Seawright, 1993), hybridization (Klein et al., 1991), morphology and behavior (Rosa-Freitas et al., op. cit., 1990) and RAPD-PCR (Wilkerson et al., 1995a,b) have contributed to the identification of a fourth species; thus, this group is designated as A, B, C, and D. The A species has been described as A. albitarsis sensu stricto, the B species has not been described, the C species was named A. marajoara Galvão and Damasceno and a fourth species was named A. deaneorum Rosa-Freitas. It is known that A. marajoara is a significant malaria vector at least in Northeastern Brazil (Conn et al., 2002), but the status of the other species is poorly understood (Cong Li and Wilkerson, 2005). A complication for accurate taxonomy and geographic distribution of A. albitarsis samples is the fact that adult females are difficult to distinguish morphologically, and most collections of this group are treated as species complexes (Kreutzer et al., op .cit., 1976; Steiner et al., 1982; Rosa-Freitas et al., 1990).

Cytogenetic studies of mitotic chromosomes are important tools for comprehension of the taxonomy and evolution of these mosquitoes of the Anopheles complex, such as Anopheles dirus from continental Asia (Baimai, 1984). Chromosomal studies of anophelines have shown a karyotype of 2n = 6 chromosomes (Baimai et al., 1996), which includes two pairs of sex chromosomes, a pair of metacentric and a pair of submetacentric autosomes; this seems to be a conservative characteristic of these mosquitoes (Rao and Rai, 1987; Coluzzi, 1988). This chromosome number has been found in the Brazilian Anopheles species, including A. darlingi, A. noroestensis, A. argyritarsis, A. aquasalis, A. nuneztovari (Schreiber and Guedes, 1959, 1961; Rafael and Tadei, 1998), A. cruzii and A. bellator (Ramírez, 1989).

We examined metaphase chromosomes of A. albitarsis populations, because of the lack of information on karyotypes. This type of data is important for understanding population differentiation and for the development of strategies for human malaria vector control in the Brazilian Amazon.

MATERIAL AND METHODS

Mosquito samples and chromosome preparations

Adult female A. albitarsis mosquitoes were collected from Iranduba and Coari in the State of Amazonas, and from Ilha Comprida (24°42.75’S, 47°31.6’W), in the State of São Paulo (Figure 1A,B). Adult females of A. albitarsis were collected while they were rest on the houses’ wall in Ilha Comprida, São Paulo State, and rest on the houses’ wall and feeding on cattle in Iranduba and Coari, Amazonas State. The females were transported to the Insectary of the Laboratory of Malaria Vectors at the Instituto Nacional de Pesquisas da Amazônia, in Manaus, Amazonas. The specimens were identified morphologically (Forattini, 1962; Gorham et al., 1967; Faran and Linthicum, 1981; Consoli and Lourenço de Oliveira, 1994).

Slides of brain ganglia of fourth-instar larvae were prepared, 15 from Ilha Comprida, 18 from Iranduba and 14 from Coari. Brain ganglia were treated with 0.005% colchicine-hypotonic solution, stained with Giemsa (Imai et al., 1988). The chromosomes were photographed and then analyzed under phase contrast, with an optovar 1.25X lens on a Zeiss-Axioplan microscope, photographed with Kodak Imagelink HQ ISO 25 film. The chromosomes were numbered according to the nomenclature proposed by Rai (1963). Arm ratios (AR) and relative size (RS%) of all chromosomes were calculated by the Beçak method (1967) and classified according to Levan et al. (1964).

RESULTS

Fifty-seven metaphase preparations from Ilha Comprida, 59 from Iranduba and 38 from Coari were photographed and analyzed. The A. albitarsis preparations showed a chromosome number of 2n = 6, with acrocentric X and Y punctiform chromosomes, metacentric (pair II) and submetacentric (pair III) autosomes (Figures 2 and 3). Only heteromorphic (XY) and homomorphic (XX) chromosomes in the males and females were observed in the sex pair in the three A. albitarsis populations. The chromosomes were named sex pair (I) and autosome pairs (II and III), according to Rai (1963). Another characteristic detected in the chromosomes of the three populations was somatic pairing during metaphase (Figure 2C).

Morphometric sex chromosome and autosome measurements were obtained from brain ganglium cells from 10 metaphase samples of progeny of A. albitarsis from the three sites; the mean autosome anopheline sizes differed (Tables 1, 2 and 3). The mean lengths and mean arm ratios were higher for the three chromosome pairs in the Coari population (Table 3) than in the Ilha Comprida and Iranduba populations (Tables 1 and 2). The mean size of the X chromosome varied, the smallest being observed in Ilha Comprida (Table 1). The relative size of the Y chromosome was calculated as a proportion of the mean size of the X chromosome. The relative sizes for autosomes and X chromosomes were calculated according to the haploid genome size.

The mean lengths (in micrometers) of the autosomal and sex chromosomes of the A. albitarsis samples from Ilha Comprida, Iranduba and Coari were compared by analysis of variance (ANOVA) and the t-test. There were no significant differences among the three populations.

DISCUSSION

Chromosomal karyotypes have been reported for some 20 genera and more than 300 species in the Culicidae (Rai 1963; White 1980; Rai and Black, 1999). The diploid chromosome number of all species examined has remained at six, despite the ancient origin of the Culicidae family and the incorporation of extensive chromosomal repatterning (Kumar and Rai, 1990; Mori et al., 1998). The only exception is Chagasia bathana (2n = 8), subfamily Anophelinae, which possesses three autosome pairs and a heteromorphic pair of sex chromosomes (Kreutzer, 1978); all other anopheline species possess two pairs of generally metacentric chromosomes of unequal size and one pair of heteromorphic sex chromosomes, X and Y (Kitzmiller, 1963; White, 1980).

Morphological data were obtained on the karyotypes (2n = 6); they had acrocentric X and Y punctiform chromosomes, metacentric (pair II) and submetacentric (pair III) autosomes. These characteristics have also been reported for A. darlingi, A. aquasalis (Frizzi and Ricciardi, 1955), A. noroestensis, A. argyritarsis (Schreiber and Guedes, 1959, 1961) from Minas Gerais, Brazil, A. (Kerstezia) cruzii from southeastern Brazilian populations (São Paulo) by Ramírez (1989) and A. darlingi from Macapá (Amapá) and Manaus (Amazonas) by Rafael and Tadei (1998).

As found in the males of A. albitarsis, punctiform sex (Y) chromosomes have been reported from other Brazilian Anopheles species: A. quadrimaculatus, A. aquasalis (Frizzi and Ricciardi, 1955), A. noroestensis, A. argyritarsis (Schreiber and Guedes, 1959, 1961), A. cruzii (Ramírez, 1989), A. darlingi and A. nuneztovari (Rafael and Tadei, 1998). The prometaphase and metaphase of the A. albitarsis samples that we studied showed somatic pairing. Traut et al. (1990) also reported somatic pairing to be a common phenomenon in Diptera. The same pattern is known for Culicidae chromosomes, such as A. cruzii (Ramírez and Dessen, 1994), A. darlingi and A. nuneztovari (Rafael and Tadei, 1998).

The size of Anopheles mitotic chromosomes varies (Rai and Craig, 1961; Kitzmiller, 1963; Rafael and Tadei, 1998). We found no significant size differences between the X chromosomes of the A. albitarsis populations compared to those of A. darlingi (Rafael and Tadei, 1998) and A. cruzii (Ramírez, 1989). The largest mean size of the three chromosome pairs of A. albitarsis, was found in the Coari population. The Ilha Comprida population had the smallest X chromosomes. However, the differences in chromosome size among these populations were not significant.

Significant chromosome size variation has not occurred during the evolution of the A. albitarsis complex, in contrast to Chagasia bathana species, an Anopheline subfamily with 2n = 8 chromosomes. Based on the variation we found in the mean chromosome size in the three populations of the albitarsis complex, selective pressure in these populations is apparently occurring only at a genetic level.

ACKNOWLEDGMENTS

The authors thank the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM)/PIPT, and the Ministério da Ciência e Tecnologia (MCT) (grant PPI 3680 and the project PIATAM/CTPETRO) for financial support.

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