ARTIGO ARTICLE 83 Chromosome homogeneity in populations of Triatoma brasiliensis Neiva 1911 (Hemiptera – Reduviidae – Triatominae) Francisco Panzera 1 Homogeneidade cromossômica em populações Ruben Pérez 1 Paula Nicolini 1 de Triatoma brasiliensis Neiva 1911 Sonia Hornos 1 (Hemiptera – Reduviidae – Triatominae) Jane Costa 2 Érika Borges 3 Liléia Diotaiuti 3 Christopher J. Schofield 4 1 Sección Genética Evolutiva, Abstract Triatoma brasiliensis is the most important vector of Chagas disease in the semiarid Facultad de Ciencias. zone of the Northeast of Brazil. Several authors have reported the occurrence of four chromatic Iguá 4225 esq. Mataojo, Casilla de Correos 18110, patterns with morphological, ecological, and genetic differences. In order to determine the exis- 11400 Montevideo, Uruguay. tence of cytogenetic differentiation between these chromatic forms, we analyzed their karyotypes [email protected] and the chromosome behavior during the male meiotic process. Triatoma brasiliensis shows dis- 2 Coleção Entomológica, Departamento de tinct and specific chromosome characteristics, which differ from those observed in all other tri- Entomologia, Instituto atomine species. However, no cytogenetic differences were observed between the four chromatic Oswaldo Cruz, forms of T. brasiliensis. The lack of chromosome differentiation among them could indicate that Fundação Oswaldo Cruz. Av. Brasil 4365, CP 926, the populations of this species are in a process of differentiation that does not involve their chro- Rio de Janeiro, RJ mosomal organization. 21045-900, Brazil. Key words Chagas Disease; Triatominae; Triatoma brasiliensis; Cytogenetics; Karyotyping 3 Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz. Resumo Vários autores têm reportado a ocorrência de quatro padrões cromáticos e diferentes CP 1743, Belo Horizonte, MG 30190-002, Brasil. padrões morfológicos, genéticos e ecológicos de Triatoma brasiliensis no semi-árido nordestino 4 Department of Infectious do Brasil, região onde é o mais importante vetor da doença de Chagas. Para verificar diferenças and Tropical Diseases, citogenéticas nessas populações, foram analisados seus cariótipos e o comportamento cromosso- London School of Hygiene and Tropical Medicine. mial durante o processo meiótico em exemplares machos. Observou-se que o T. brasiliensis mos- London WC1 E7TH, UK. tra características cromossômicas distintas e específicas entre as quatro populações, diferindo daquelas observadas em outras espécies de triatomíneos. No entanto, diferenças citogenéticas não foram observadas entre as quatro populações de T. brasiliensis, o que poderia indicar que as referidas populações ainda se encontram em uma etapa inicial do processo de diferenciação, ainda não envolvendo a organização cromossomial. Palavras-chave Doença de Chagas; Triatominae; Triatoma brasiliensis; Citogenética; Carioti- pagem Cad. Saúde Pública, Rio de Janeiro, 16(Sup. 2):83-88, 2000 84 PANZERA, F. et al. Introduction 5) Scanning electron microscopy and mor- phometric measures of the eggs showed signif- Triatoma brasiliensis is distributed exclusively icant differences (Costa et al., 1997b). in Brazil and is considered the most important 6) Each population showed stability and Chagas disease vector of the northeastern area homogeneity of their chromatic pattern for sev- of this country (Silveira et al., 1984). This species eral years in laboratory colonies (Costa et al., has a wide geographic distribution, including 1996). Intermediate forms reported by Lent & eleven States of Brazil. Three subspecies were Wygodzinsky (1979) were not observed either described on the basis of chromatic characters in the field or in the laboratory. of the pronotum, leg, and hemelytron (Galvão, 7) Reciprocal crosses among all popula- 1956): T. brasiliensis brasiliensis Neiva, 1911 tions showed the F1 fertile. However, in the F2, (from Caicó, Rio Grande do Norte), T. brasilien- a high percentage of sterile eggs, longer devel- sis melanica Neiva & Lent, 1941 (from Espino- opment period, and high mortality rate of fifth sa, Minas Gerais), and T. brasiliensis macrome- instar nymphs were observed for the brasilien- lasoma Galvão, 1956 (from Petrolina, Pernam- sis male x melanica female (Costa, 1999). buco). Lent & Wygodzinsky (1979) considered In order to determine the existence of chro- these subspecies as synonymous based on the mosome differentiation between these chro- fact that intermediate forms seem frequent. Re- matic forms, we analyzed them in terms of cyto- cently, a fourth chromatic pattern was collect- genetic characters previously used to differenti- ed in Juazeiro (Bahia), named as “juazeiro pop- ate triatomine species (Pérez et al., 1992; Pan- ulation” (Costa et al., 1997a). zera et al., 1995, 1997) and to detect intraspecif- Besides the variation of the cuticle color ic variation (Panzera et al., 1992). This group of patterns, other differences were reported be- heteropteran insects exhibits holocentric chro- tween the four forms of T. brasiliensis: mosomes, which do not have a morphologically 1) The brasiliensis populations have the differentiated centromere. Previous cytogenetic most widespread distribution (Maranhão, Piauí, information concerning T. brasiliensis is mainly Ceará, Rio Grande do Norte, and Paraíba) and restricted to its diploid chromosome number occupy a great variety of ecotopes (both domicil- (2n = 20 autosomes plus XY in the males and XX iary and wild). The macromelasoma and juazeiro in the females)(Schreiber & Pellegrino, 1950; populations were reported only from Pernambu- Schreiber et al., 1967). co and Bahia, respectively, occupying peridomi- ciliary and sylvatic ecotopes. The melanica popu- lation, from the North of Minas Gerais, was only Material and methods captured in sylvatic ecotopes (Costa et al., 1998). 2) Two populations, macromelasoma and Material analyzed juazeiro, were not found to be infected with Try- panosoma cruzi, whereas the brasiliensis and The origins of the populations studied in this melanica populations had infection rates of 15% report are shown in Table 1. At least three indi- and 6.6%, respectively (Costa et al., 1998). viduals per population were analyzed. 3) Isoenzymatic data, based on nine loci, suggest a clear genetic differentiation between Cytogenetic studies these populations. Nei’s genetic distance reach- es values corresponding to those found at the • Chromosome preparations interspecific level for other species of Triatomi- and banding procedures nae (Costa et al., 1997a). The greatest distance was obtained between the brasiliensis and me- Gonads (testes and ovaries) were fixed in lanica populations. ethanol-acetic acid (3:1) and softened in a 45% 4) Recent DNA analysis of a region of the aqueous solution of acetic acid. Part of the ma- mitochondrial cytochrome B gene shows a large terial was used in squashes and stained with lac- degree of sequence divergence, ranging from to-acetic orcein for meiotic descriptions. The re- 2.7 (for the brasiliensis-macromelasoma pair) maining material was studied using the C-band- to 11.2% (for the brasiliensis-melanica pair) ing technique according to Pérez et al. (1997), to (Monteiro et al., 1999). The differences in DNA observe the distribution and behavior of C-het- composition observed between some popula- erochromatin during mitosis and meiosis. tions of T. brasiliensis is even higher than those found between other closely related triatomine species, such as Triatoma infestans/Triatoma platensis (7.7%). Cad. Saúde Pública, Rio de Janeiro, 16(Sup. 2):83-88, 2000 CHROMOSOME HOMOGENEITY IN T. brasiliensis POPULATIONS 85 Table 1 Collection sites of the different populations of Triatoma brasiliensis studied in this report. Population State of Brazil Locality Habitat brasiliensis Ceará Independência Intradomestic/Peridomestic/Sylvatic Novo Oriente Intradomestic brasiliensis Piauí Simplício Mendes Intradomestic/Peridomestic brasiliensis Pernambuco Terra Nova Intradomestic/Peridomestic brasiliensis Rio Grande do Norte Caicó Laboratory, 3rd generation. macromelasoma Pernambuco Petrolina Laboratory, 3rd generation. melanica Minas Gerais Espinosa Laboratory, 3rd generation. juazeiro Bahia Juazeiro Laboratory, 3rd generation. • Cytogenetic markers During early male meiotic prophase, one heteropycnotic chromocenter (arrows) and We used several cytogenetic markers according several heteropycnotic dots scattered through- to Pérez et al. (1992). Those traits based on rel- out the nucleus are observed (Figures 1a and ative chromosome size, such as autosomal size 1b). The pachytene stage is very specific and al- and autosomes versus sex chromosomes, were lows a clear identification of this species. In assessed by observing first and second meiotic this stage, all bivalents are rather separated metaphases. and present a terminal C-heterochromatic dot The amount of autosomal C-heterochro- in one or both chromosomal ends (Figure 1a). matin was estimated using specific software The diffuse stage is characterized by a signifi- (Image Pro-Plus – Media Cybernetics, Mary- cant increase in nuclear size (Figure 1b). Dur- land, USA). For each individual at least three ing diplotene, associated XY sex chromosomes metaphase plates were analyzed. remain heteropycnotic, and they are often at- tached to the terminal heteropycnotic regions of one or two autosomal bivalents (arrows Fig- Results ure 1c). The first and second meiotic metaphases No cytogenetic differences were observed be- present the typical ring chromosomal disposi- tween the four