BackgroundAnopheles 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.MethodsSpecimens 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.ResultsThe 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. nuneztovaris.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.ConclusionsThe two markers revealed three genetic lineages for A. nuneztovaris.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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1217-6) contains supplementary material, which is available to authorized users.

Background: Saliva of mosquitoes contains anti-platelet, anti-clotting, vasodilatory, anti-complement and antiinflammatory substances that help the blood feeding process. The salivary polypeptides are at a fast pace of evolution possibly due to their relative lack of structural constraint and possibly also by positive selection on their genes leading to evasion of host immune pressure. Results: In this study, we used deep mRNA sequence to uncover for the first time the sialomes of four Amazonian anophelines species (Anopheles braziliensis, A. marajorara, A. nuneztovari and A. triannulatus) and extend the knowledge of the A. darlingi sialome. Two libraries were generated from A. darlingi mosquitoes, sampled from two localities separated~1100 km apart. A total of 60,016 sequences were submitted to GenBank, which will help discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure. Additionally, in these analyses we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of A. triannulatus, A. marajorara and A. darlingi species. Conclusions: Besides their pharmacological properties, which may be exploited for the development of new drugs (e.g. anti-thrombotics), salivary proteins of blood feeding arthropods may be turned into tools to prevent and/or better control vector borne diseases; for example, through the development of vaccines or biomarkers to evaluate human exposure to vector bites. The sialotranscriptome study reported here provided novel data on four New World anopheline species and allowed to extend our knowledge on the salivary repertoire of A. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard within future RNAseq studies.

Recent studies have shown that Anopheles oswaldoi sensu lato comprises a cryptic species complex in South America. Anopheles konderi, which was previously raised to synonymy with An. oswaldoi, has also been suggested to form a species complex. An. oswaldoi has been incriminated as a malaria vector in some areas of the Brazilian Amazon, Colombia, Peru and Venezuela, but was not recognized as a vector in the remaining regions in its geographic distribution. The role of An. konderi as a malaria vector is unknown or has been misattributed to An. oswaldoi. The focus of this study was molecular identification to infer the evolutionary relationships and preliminarily delimit the geographic distribution of the members of these complexes in the Brazilian Amazon region. The specimens were sampled from 18 localities belonging to five states in the Brazilian Amazon and sequenced for two molecular markers: the DNA barcode region (COI gene of mitochondrial DNA) and Internal Transcribed Spacer 2 (ITS2 ribosomal DNA). COI (83 sequences) and ITS2 (27 sequences) datasets generated 43 and 10 haplotypes, respectively. Haplotype networks and phylogenetic analyses generated with the barcode region (COI gene) recovered five groups corresponding to An. oswaldoi s.s., An. oswaldoi B, An. oswaldoi A, An. konderi and An. sp. nr. konderi; all pairwise genetic distances were greater than 3%. The group represented by An. oswaldoi A exhibited three strongly supported lineages. The molecular dating indicated that the diversification process in these complexes started approximately 2.8 Mya, in the Pliocene. These findings confirm five very closely related species and present new records for these species in the Brazilian Amazon region. The paraphyly observed for the An. oswaldoi complex suggests that An. oswaldoi and An. konderi complexes may comprise a unique species complex named Oswaldoi-Konderi. Anopheles oswaldoi B may be a potential malaria vector in the extreme north of the Brazilian Amazon, whereas evidence of sympatry for the remaining species in other parts of the Brazilian Amazon (Acre, Amazonas, Pará and Rondônia) precluded identification of probable vectors in those areas.

Mansonia (Diptera: Culicidae) are known to cause discomfort to the local populations of Amazon. Considering the fact that the effective control of these mosquitoes can only be obtained by understanding their ecology and behavior, entomological monitoring becomes essential. In view of this, mosquitoes of the genus Mansonia were collected by human landing catches (HLC) from 2015 to 2019, in four locations of Porto Velho, Rondônia, Brazil. The collections were performed inside and outside the homes, once in every four months, uninterrupted for 24 hr. Human bite indices/hour was used to analyze the hourly activity of the species for different seasons and environment (indoor and outdoor). Moreover, nonparametric Mann–Whitney tests were conducted to indicate if there were differences between exophagic and endophagic behavior. The seasonality of Mansonia species was also analyzed. Overall, 96,766 specimens were collected over five years of sampling. Mansonia titillans (Walker) was found to be the most abundant species (76.9%). The highest percentage of mosquitoes was collected in February (48.4%), followed by October (39.6%) and June (12.0%). The biting activity of the two most abundant species showed peak host seeking activity/behavior during twilight and night, more perceptible in the outdoor environment (peridomiciliary). In general, seasonality showed a tendency towards a reduction in the abundance of Mansonia in the years after 2015. Our results will be essential in the formulation of effective control methodology for Mansonia in the studied area.

No abstract