Heterochromatin variation in chromosomes of Anopheles ( Nyssorhynchus ) darlingi Root and A . ( N . ) nuneztovari Gabaldón ( Diptera : Culicidae )

Pela tecnica do bandamento C detectou-se variacao de marcacao dos blocos heterocromaticos dos cromossomos de A. darlingi e A. nuneztovari de Manaus, Amazonas, e de Macapa, Amapa, Brasil. Os cromossomos sexuais de ambas as especies mostraram duas formas de cromossomos X e o Y foi totalmente heterocromatico. No cromossomo X1 de A. darlingi a marcacao atingiu 1/3 e no cromossomo X2 foi apenas na regiao centromerica. Nos autossomos de ambas as especies as marcacoes foram constantes nas regioes centromericas, e o cromossomo II de A. darlingi mostrou um bloco heterocromatico em um dos bracos. A. nuneztovari mostrou polimorfismo de tamanho para o cromossomo X, tendo o X maior (X1) tres blocos e o menor (X2) dois blocos heterocromaticos. Femeas homozigotas (X2X2) nao foram detectadas nas duas localidades. Em machos de A. darlingi foram encontrados os cromossomos X1 e X2, enquanto que em machos de A. nuneztovari somente o cromossomo X1 foi detectado. Apenas variacao intraespecifica de blocos heterocromaticos nos cromossomos X e nos autossomos foi registrada nas duas populacoes de ambas as especies estudadas em cada localidade.

Chromosomal studies of A. darlingi populations from Minas Gerais, Brazil, and of other South American species showed a karyotype of 2n = 6 (Schreiber and Guedes, 1959), as in other Anopheles species (Coluzzi, 1988).Rafael and Tadei (1998) reported an identical karyotype (2n = 6) for A. darlingi and A. nuneztovari populations from the Amazon region.
C-banding analysis of mitotic chromosomes of Anopheles species from continental Asia (Baimai et al., 1995) revealed species complexes which included Anopheles dirus (Baimai, 1984;Hii, 1985) and Anopheles maculatus in the Neocellia series (Cellia) (Baimai et al., 1993).C-banding was reported to be useful for identifying sibling species based on differences in the morphology, quantity and distribution of heterochromatic blocks, principally in X and Y chromosomes (Baimai et al., 1993).
In spite of the epidemiological importance of A. darlingi and A. nuneztovari in the Amazon region, there are no data on C-banding of the metaphase chromosomes of these species.We studied the variation in heterochromatic block markings in metaphase chromosomes to determine the heterochromatic patterns in the Manaus and Macapá populations of these species.

MATERIAL AND METHODS
Two natural populations of A. darlingi were sampled, with 20 individuals from Manaus (3°08'S, 60°01'W), Amazonas State, and 14 from Macapá (0°02'S, 51°03'W), Amapá State.For A. nuneztovari, 17 individuals from Manaus and 11 from Macapá were analyzed.Slides were prepared from fourth instar larval brain ganglia, treated with a 0.005% colchicine-hypotonic solution, as described by Imai et al. (1988).The slides were washed with distilled water, air dried and stored at room temperature for 72 h.C-banding was done using the method of Sumner (1972), with a reduction in the barium exposure time (3 min).The best preparations were photographed using a phase-contrast microscope fitted with a green filter.

RESULTS
The C-banding patterns of 76 out of 103 A. darlingi metaphases from Manaus and 57 out of 74 from Macapá, as well as 63 A. nuneztovari metaphases out of 86 from Manaus and 46 out of 53 from Macapá were photographed and analyzed.A. darlingi and A. nuneztovari populations from both localities showed two types of X chromosomes (X 1 and X 2 ), which differed in the content and distribution of heterochromatic blocks (Figure 1).In A. darlingi from Manaus, the sex chromosomes had centromeric markings that extended to 1/3 of X 1 while the Y chromosome was entirely heterochromatic (Figure 2).The X 2 chromosomes of samples from Macapá (Figure 2B) showed fewer markings, which extended only to the centromeric region.These marking patterns were the same as that of A. darlingi from Manaus.Chromosomes with a longer barium exposure (4 min) were more discolored than other preparations, although centromeric markings were seen in autosomes II and III and in the X 1 X 1 sex pairs (Figure 2C).
The C-banding pattern in autosomes of the A. darlingi population from Macapá was the same as that of A. darlingi from Manaus (Figure 1).In these populations, the II and III chromosomes had well-marked centromeric regions (Figure 2B and C).All of the II chromosomes had a band which extended from the centromere along half the length of one arm of the chromatid in each population (Figure 2B).
The variations in heterochromatic block markings in X 1 , X 2 and autosomal chromosomes of the A. nuneztovari from Manaus were the same as that of A. nuneztovari from Macapá (Figure 1).The X 1 chromosome (longer) consisted of three heterochromatic blocks (two telomeric and one centromeric) and the X 2 chromosome (shorter) contained two heterochromatic blocks, one of which was telomeric and the other centromeric (Figure 3A, B and C).The X 2 chromosomes of female A. nuneztovari had two heterochromatic blocks (Figure 3).The centromeric heterochromatin markings of the autosomes were found in this species (Figure 3D and E).
X 2 X 2 A. darlingi and A. nuneztovari females were not found (Table I).X 1 and X 2 males were found in A. darlingi while A. nuneztovari males had only the X 1 chromosome.

DISCUSSION
C-banding studies of mitotic and meiotic chromosomes have provided important information on inter-and intraspecific population variation in Anopheles species and the technique has proven to be an excellent tool for identifying species complexes (Baimai et al., 1993).In this study the analysis of mitotic chromosomes of A. darlingi and A. nuneztovari described above revealed intraspecific variation in the quantity and distribution of heterochromatic blocks in sex chromosomes and in the centromeric regions of autosomes (Figure 1).Kitzmiller (1977) and Tadei (1985) suggested that in Anopheles genus the X chromosome was more sensitive to rearrangements than the autosomes.Intraspecific variation in sex chromosomes through the acquisition of constitutive heterochromatin is a common phenomenon in Southeast Asian anophelines.Baimai et al. (1996) reported two types of X chromosomes with floating frequencies in natural populations of Anopheles willmori.The X chromosomes in Amazonian populations of A. darlingi and A. nuneztovari most likely have similar mechanisms of adaptation in order to survive in these populations.
The difference in size between the X 1 and X 2 chromosomes of A. nuneztovari may have resulted from the addition to or loss of part of one of these chromosomes.The addition or loss of chromosomal heterochromatin in   Anopheles has played an important role in chromosomal evolution in Anopheles species (Vasantha et. al., 1982;Baimai et al., 1993Baimai et al., , 1996)).The X 2 chromosome in Amazonian populations of A. nuneztovari could have been derived from the presumed X 1 through the loss of an extra heterochromatic block in the distal end of the chromosome arm.The heterochromatic blocks of A. darlingi and A. nuneztovari are similar to those of Anopheles (Kerteszia) cruzii, according to Ramírez (1989) and Ramírez andDessen (1994, 1996).The inversions in the latter species probably arose from differences in the homolog chromosomes of the same specimen.However, the inversion polymorphism detected in A. darlingi (Kreutzer et al., 1972;Tadei et al., 1982;Tadei, 1985) and A. nuneztovari (Kitzmiller et al., 1973;Conn et al., 1993) does not necessarily mean that inversions alone positioned the heterochromatic blocks in the chromosomes of these spe- cies.Rather, these blocks may have originated from differences accumulated during evolution, as proposed by Gatti et al. (1982) to account for differences in the heterochromatic patterns of Anopheles gambiae and Anopheles arabiensis.
The C-banding in the present study in A. darlingi and A. nuneztovari populations exhibited only intraspecific variation of the heterochromatic blocks in X chromosomes and autosomes.The X chromosomes presented greater variation in the content and distribution of heterochromatic blocks than did the autosomes.

Figure 1 -
Figure 1 -Diagrammatic comparison of metaphase karyotypes of Anopheles darlingi and Anopheles nuneztovari from Manaus and Macapá.Only one set of autosomes (II and III) is shown.Variable heterochromatic portions are indicated in black.Chromosomes and heterochromatic portions are shown as a percentage of the total length.c = Centromeric region; sc = secondary constriction.
in A. darlingi and A. nuneztovari chromosomes

Table I -
X 1 and X 2 chromosomes in females and males of Anopheles darlingi and Anopheles nuneztovari populations from Manaus (MAO) and Macapá (MC).