RESUMO
One of the major obstacles for studies of the biology, ecology, and behavior of Neotropical vectors of human Plasmodium has been the lack of reliable and efficient means of identifying many species. Although the subgenus Nyssorhynchus includes most species responsible for human transmission in South America, there are no polymerase chain reaction (PCR)-based techniques for identifying members of this taxon. We describe the first multiplex PCR for identifying four species in the subgenus Nyssorhynchus that are vectors of human Plasmodium spp. Four species specific primers, together with a universal primer that anneals to the 5.8S rDNA region, produce amplicons of the internal transcribed spacer two with base pair sizes of 131,308,371, and 441 for An. triannulatus, An. trinkae, An. strodei, and An. rangeli, respectively.
Assuntos
Anopheles/parasitologia , Plasmodium/genética , Animais , Sequência de Bases , Primers do DNA , Ecossistema , Geografia , Humanos , New Mexico , Plasmodium/classificação , Plasmodium/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , América do SulRESUMO
Based on similarity of male genitalia, the malaria vector Anopheles trinkae Faran from the eastern Andean piedmont of Colombia, Ecuador, Peru, and Bolivia was determined by Peyton (1993) to be a junior synonym of An. dunhami Causey, then known from a single locality in Amazonian Brazil. Following an appraisal of molecular, chromosomal, and morphological characters, we conclude herein that the 2 taxa are specifically distinct and remove An. trinkae from synonymy with An. dunhami. Eggs of the 2 species are distinguished easily by the anterior crown, long floats, and closed deck that occur only in An. trinkae. The X chromosome of larval polytenes is divisible into R and L arms in An. dunhami, but not in An. trinkae. A phenogram based on banding pattern scores from 18 random amplified polymorphic DNA primers separated with 100% resolution An. dunhami, An. trinkae, Anopheles nuneztovari Gabaldón and Anopheles darlingi Root. In the ITS2 region of rDNA, 25% of base sites distinguished An. trinkae from An. dunhami and 21% from the related An. nuneztovari; males of these 3 species had accessory glands of significantly different sizes. Preliminary isoenzyme screening indicated that 3 of 11 loci were diagnostic for separating An. trinkae from An. dunhami. The results indicate that An. dunhami is related more closely to An. nuneztovari than to An. trinkae and illustrate the merits of a multidisciplinary approach to mosquito systematics.
Assuntos
Anopheles/genética , Filogenia , Cromossomo X , Animais , Anopheles/classificação , Anopheles/parasitologia , Sequência de Bases , Sequência Consenso , Evolução Molecular , Feminino , Genitália Masculina/anatomia & histologia , Geografia , Humanos , Malária/transmissão , Masculino , Dados de Sequência Molecular , Oócitos/ultraestrutura , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , América do SulRESUMO
The rDNA internal transcribed spacer 2 (ITS2) was sequenced for 5 species of mosquitoes that may be important vectors of human malaria in certain regions of South America and are difficult to distinguish by morphology: Anopheles evansae, An. nuneztovari, An. rangeli, An. strodei and An. trinkae. ITS2 sequences from samples collected in Ecuador, Bolivia, Venezuela and Brazil were aligned and compared in order to determine the usefulness of this spacer for the elaboration of species specific primers and DNA probes. The ITS2 was found to be different in size (ranging from 333 to 397 bp) and sequence between all pairs of species. Highly variable regions were found primarily at the 3' end of the spacer and were interspersed with relatively conserved sites. Instraspecific sequence variation was limited to a single transversion between specimens of An. rangeli from distant geographic locations suggesting concerted evolution and homogenization of the ITS2.
Assuntos
Anopheles/genética , DNA Ribossômico/genética , RNA Ribossômico 28S/genética , RNA Ribossômico 5,8S/genética , Animais , Anopheles/classificação , Sequência de Bases , Insetos Vetores/genética , Malária/transmissão , Dados de Sequência Molecular , Análise de Sequência de DNA , América do SulRESUMO
Sequence variation of the ribosomal DNA internal transcribed spacer 2 (ITS2) was examined for populations of the malaria vector Anopheles nuneztovari collected in Colombia, Venezuela, Bolivia, Suriname, and Brazil. Mosquitoes from Colombia and Venezuela had identical ITS2 sequences and were distinguished from sequences in other populations by three insertion/deletion events (indels) and by one transversion. The length of the ITS2 was 363-369 bp, and it had a G+C content of 55.3%-55.7%. Variation in the length of the ITS2 between and within populations was due to indels in simple repeats. ITS2 consensus sequences were similar or identical for samples from the following three groups: (1) Colombia, Bolivia, and Venezuela; (2) Suriname and northern Brazil; and (3) eastern and central Brazil. The presence of two different consensus sequences from a single location near Manaus, Brazil, suggests that populations from eastern Brazil and those from Suriname converge in this region of the Amazon Basin. These data show that putative cryptic species of An. nuneztovari are distinguished by very minor differences in DNA sequence of the ITS2 region.
Assuntos
Anopheles/genética , DNA Ribossômico/genética , Variação Genética , Íntrons , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Feminino , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido Nucleico , América do SulRESUMO
Samples of Anopheles freeborni s.1. were collected from areas of New Mexico where malaria had once been common and sporozoites had been isolated from this species. Specimens were identified by analysis of polytene chromosome banding patterns and by specific rDNA fragments generated through the polymerase chain reaction. All samples collected in New Mexico were identified as An. hermsi, which was the probable vector of malaria in this region during the early 20th century.