Identification and validation of the first EST-SSR markers based on transcriptome of Anopheles darlingi, the primary transmitter of malaria in Brazil.

BACKGROUND: Anopheles darlingi is a monotypic species in terms of its morphological, genetic, and behavioral aspects and is the primary transmitter of human malaria (99%) in Brazil, especially in the Brazilian Amazon. In this pioneering study, 15 expressed sequence tag (EST)-simple sequence repeat (SSR) markers were obtained and characterized in samples from the municipality of São Gabriel da Cachoeira, Amazonas state, Brazil, with polymorphisms that can be used for further genetic research. METHODS AND RESULTS: The specimens (from egg to larval stage) collected were bred in the insectary at INPA (National Institute for Amazonian Research). The SSR repeats within the contigs of the A. darlingi EST banks were confirmed on the Vector Base site. DNA was extracted and amplified using polymerase chain reaction and then genotyped. Fifteen polymorphic SSR loci were identified and characterized. The number of alleles totaled 76 and ranged from 2 to 9. The observed heterozygosity varied between 0.026 and 0.769, the expected heterozygosity between 0.025 and 0.776, and the mean polymorphism information content was 0.468. Eight loci showed Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (P: (5%) ≤ 0.0033). No linkage disequilibrium was found among the loci. CONCLUSIONS: The polymorphic SSRs of the loci have been shown to be efficient for investigation of the variability and genetic population structure of A. darlingi.

Multiple evolutionary lineages for the main vector of Leishmania guyanensis, Lutzomyia umbratilis (Diptera: Psychodidae), in the Brazilian Amazon.

Lutzomyia umbratilis is the main vector of Leishmania guyanensis in the Brazilian Amazon and in neighboring countries. Previous biological and molecular investigations have revealed significant differences between L. umbratilis populations from the central Brazilian Amazon region. Here, a phylogeographic survey of L. umbratilis populations collected from nine localities in the Brazilian Amazon was conducted using two mitochondrial genes. Statistical analyses focused on population genetics, phylogenetic relationships and species delimitations. COI genetic diversity was very high, whereas Cytb diversity was moderate. COI genealogical haplotypes, population structure and phylogenetic analyses identified a deep genetic differentiation and three main genetic groups. Cytb showed a shallower genetic structure, two main haplogroups and poorly resolved phylogenetic trees. These findings, allied to absence of isolation by distance, support the hypothesis that the Amazon and Negro Rivers and interfluves are the main evolutionary forces driving L. umbratilis diversification. The main three genetic groups observed represent three evolutionary lineages, possibly species. The first lineage occurs north of the Amazon River and east of Negro River, where Le. guyanensis transmission is intense, implying that L. umbratilis is an important vector there. The second lineage is in the interfluve between north of Amazon River and west of Negro River, an area reported to be free of Le. guyanensis transmission. The third lineage, first recorded in this study, is in the interfluve between south of Amazonas River and west of Madeira River, and its involvement in the transmission of this parasite remains to be elucidated.

Exploring deeper genetic structures: Aedes aegypti in Brazil.

Aedes aegypti, being the principal vector of dengue (DENV1 to 4), chikungunya and Zika viruses, is considered as one of the most important mosquito vectors. In Brazil, despite regular vector control programs, Ae. aegypti still persists with high urban density in all the states. This study aimed to estimate the intra and inter population genetic diversity and genetic structure among 15 Brazilian populations of Ae. aegypti based on 12 microsatellite loci. A total of 510 specimens were analyzed comprising eight locations from northern (Itacoatiara, Manaus, Novo Airão, Boa Vista, Rio Branco, Porto Velho, Guajará-Mirim and Macapá), three from southeastern (Araçatuba, São José de Rio Preto and Taubaté), one from southern (Foz do Iguaçu), one from central west (Cuiabá) and two from northeastern (Campina Grande and Teresina) regions of Brazil. Genetic distances (pairwise values of FST and Nm) and the analysis of molecular variance (AMOVA) were statistically significant, independent of geographic distances among the sites analyzed, indicating that them are under a complex dynamic process that influence the levels of gene flow within and among regions of the country. Bayesian analysis in STRUCTURE revealed the existence of two major genetic clusters, as well as there was genetic substructure within them; these results were confirmed by AMOVA, BAPS and DAPC analyses. This differentiation is the cumulative result of several factors combined as events of multiple introduction, passive dispersal, environmental and climatic conditions, use of insecticides, cycles of extinction and re-colonization followed by microevolutionary processes throughout the country. Isolation by distance also contributed to this differentiation, especially among geographically closer localities. These genetic differences may affect its vector competence to transmit dengue, chikungunya, Zika and the response to vector control programs.

Evidence of new species for malaria vector Anopheles nuneztovari sensu lato in the Brazilian Amazon region.

BACKGROUND: Anopheles nuneztovari sensu lato comprises cryptic species in northern South America, and the Brazilian populations encompass distinct genetic lineages within the Brazilian Amazon region. This study investigated, based on two molecular markers, whether these lineages might actually deserve species status. METHODS: Specimens were collected in five localities of the Brazilian Amazon, including Manaus, Careiro Castanho and Autazes, in the State of Amazonas; Tucuruí, in the State of Pará; and Abacate da Pedreira, in the State of Amapá, and analysed for the COI gene (Barcode region) and 12 microsatellite loci. Phylogenetic analyses were performed using the maximum likelihood (ML) approach. Intra and inter samples genetic diversity were estimated using population genetics analyses, and the genetic groups were identified by means of the ML, Bayesian and factorial correspondence analyses and the Bayesian analysis of population structure. RESULTS: The Barcode region dataset (N = 103) generated 27 haplotypes. The haplotype network suggested three lineages. The ML tree retrieved five monophyletic groups. Group I clustered all specimens from Manaus and Careiro Castanho, the majority of Autazes and a few from Abacate da Pedreira. Group II clustered most of the specimens from Abacate da Pedreira and a few from Autazes and Tucuruí. Group III clustered only specimens from Tucuruí (lineage III), strongly supported (97 %). Groups IV and V clustered specimens of A. nuneztovari s.s. and A. dunhami, strongly (98 %) and weakly (70 %) supported, respectively. In the second phylogenetic analysis, the sequences from GenBank, identified as A. goeldii, clustered to groups I and II, but not to group III. Genetic distances (Kimura-2 parameters) among the groups ranged from 1.60 % (between I and II) to 2.32 % (between I and III). Microsatellite data revealed very high intra-population genetic variability. Genetic distances showed the highest and significant values (P = 0.005) between Tucuruí and all the other samples, and between Abacate da Pedreira and all the other samples. Genetic distances, Bayesian (Structure and BAPS) analyses and FCA suggested three distinct biological groups, supporting the barcode region results. CONCLUSIONS: The two markers revealed three genetic lineages for A. nuneztovari s.l. in the Brazilian Amazon region. Lineages I and II may represent genetically distinct groups or species within A. goeldii. Lineage III may represent a new species, distinct from the A. goeldii group, and may be the most ancestral in the Brazilian Amazon. They may have differences in Plasmodium susceptibility and should therefore be investigated further.