New Records of Mosquitoes in Bolivia and Northwestern Argentina.

Two mosquito species, Culex (Culex) hepperi and Culex (Culex) maxi are reported for the first time for Bolivia, in Tarija department. Aedes (Ochlerotatus) stigmaticus and Mansonia (Mansonia) indubitans are the first records in Aniceto Arce province, in Tarija department, southern Bolivia. In addition, Aedes (Protomacleaya) alboapicus and Culex (Phytotelmatomyia) renatoi are reported for the first time in the northwestern region of Argentina. Anopheles (Nyssorrhynchus) nuneztovari s.l. is reported for the first time in Jujuy and Tucumán provinces, and Aedes (Ochlerotatus) raymundi and Anopheles (Anopheles) neomaculipalpus are the first reports in Tucumán province. The 3 species are extending their geographical distribution in these provinces. Data on the collection localities and comments about the medical relevance of some species are also presented.

Ecology and larval population dynamics of the primary malaria vector Nyssorhynchus darlingi in a high transmission setting dominated by fish farming in western Amazonian Brazil.

Vale do Rio Juruá in western Acre, Brazil, is a persistent malaria transmission hotspot partly due to fish farming development that was encouraged to improve local standards of living. Fish ponds can be productive breeding sites for Amazonian malaria vector species, including Nyssorhynchus darlingi, which, combined with high human density and mobility, add to the local malaria burden.This study reports entomological profile of immature and adult Ny. darlingi at three sites in Mâncio Lima, Acre, during the rainy and dry season (February to September, 2017). From 63 fishponds, 10,859 larvae were collected, including 5,512 first-instar Anophelinae larvae and 4,927 second, third and fourth-instars, of which 8.5% (n = 420) were Ny. darlingi. This species was most abundant in not-abandoned fishponds and in the presence of emerging aquatic vegetation. Seasonal analysis of immatures in urban landscapes found no significant difference in the numbers of Ny. darlingi, corresponding to equivalent population density during the rainy to dry transition period. However, in the rural landscape, significantly higher numbers of Ny. darlingi larvae were collected in August (IRR = 5.80, p = 0.037) and September (IRR = 6.62, p = 0.023) (dry season), compared to February (rainy season), suggesting important role of fishponds for vector population maintenance during the seasonal transition in this landscape type. Adult sampling detected mainly Ny. darlingi (~93%), with similar outdoor feeding behavior, but different abundance according to landscape profile: urban site 1 showed higher peaks of human biting rate in May (46 bites/person/hour), than February (4) and September (15), while rural site 3 shows similar HBR during the same sampling period (22, 24 and 21, respectively). This study contributes to a better understanding of the larvae biology of the main malaria vector in the Vale do Rio Juruá region and, ultimately will support vector control efforts.

Complexity of malaria transmission dynamics in the Brazilian Atlantic Forest.

Plasmodium malariae and Plasmodium vivax are protozoan parasites that can cause malaria in humans. They are genetically indistinguishable from, respectively, Plasmodium brasilianum and Plasmodium simium, i.e. parasites infecting New World non-human primates in South America. In the tropical rainforests of the Brazilian Atlantic coast, it has long been hypothesized that P. brasilianum and P. simium in platyrrhine primates originated from P. malariae and P. vivax in humans. A recent hypothesis proposed the inclusion of Plasmodium falciparum into the transmission dynamics between humans and non-human primates in the Brazilian Atlantic tropical rainforest. Herein, we assess the occurrence of human malaria in simians and sylvatic anophelines using field-collected samples in the Capivari-Monos Environmental Protection Area from 2015 to 2017. We first tested simian blood and anopheline samples. Two simian (Aloutta) blood samples (18%, n = 11) showed Plasmodium cytb DNA sequences, one for P. vivax and another for P. malariae. From a total of 9,416 anopheline females, we found 17 pools positive for Plasmodium species with a 18S qPCR assay. Only three showed P. cytb DNA sequence, one for P. vivax and the others for rodent malaria species (similar to Plasmodium chabaudi and Plasmodium berghei). Based on these results, we tested 25 rodent liver samples for the presence of Plasmodium and obtained P. falciparum cytb DNA sequence in a rodent (Oligoryzomys sp.) liver. The findings of this study indicate complex malaria transmission dynamics composed by parallel spillover-spillback of human malaria parasites, i.e. P. malariae, P. vivax, and P. falciparum, in the Brazilian Atlantic forest.

Phylogeny of Anopheles (Kerteszia) (Diptera: Culicidae) Using Mitochondrial Genes.

Identification of mosquito species is necessary for determining the entomological components of malaria transmission, but it can be difficult in morphologically similar species. DNA sequences are largely used as an additional tool for species recognition, including those that belong to species complexes. Kerteszia mosquitoes are vectors of human and simian malaria in the Neotropical Region, but there are few DNA sequences of Kerteszia species in public databases. In order to provide relevant information about diversity and improve knowledge in taxonomy of Kerteszia species in Peru, we sequenced part of the mitochondrial genome, including the cytochrome c oxidase I (COI) barcode region. Phylogenetic analyses structured all species of mosquitoes collected in Peru into a single clade, separate from the Brazilian species. The Peruvian clade was composed of two lineages, encompassing sequences from Anopheles Kerteszia boliviensis and Anopheles Kerteszia pholidotus. An. pholidotus sequences were recorded for the first time in Peru, whereas An. boliviensis sequences were for the first time published in the GenBank database. Sequences generated from specimens morphologically identified as Anopheles Kerteszia cruzii clustered into three separate clades according to the collection localities of Serra do Mar, Serra da Mantiqueira, and Serra da Cantareira, confirming An. cruzii as a species complex, composed of at least three putative species.

Molecular phylogeny of Culex subgenus Melanoconion (Diptera: Culicidae) based on nuclear and mitochondrial protein-coding genes.

The subgenus Melanoconion of the mosquito genus Culex is taxonomically diverse and is widely distributed in the Neotropical Region, with 10 species occurring in the Nearctic Region. Species of this subgenus pose a taxonomical challenge because morphological identification is based largely on anatomical characters of the male genitalia. We addressed the monophyly of the Spissipes and Melanoconion Sections of the subgenus Melanoconion and some of the informal groups in each section. Our sample taxa included 97 specimens representing 43 species, from which we analysed fragments of two single-copy nuclear genes (CAD, HB) and one mitochondrial gene (COI). Phylogenetic relationships within the subgenus are presented based on results of maximum-likelihood and Bayesian analyses using a multi-locus matrix of DNA sequences. We show a molecular phylogeny of Melanoconion in which both sections were recovered as monophyletic groups. The monophyly of the Atratus and Pilosus groups was confirmed. Within each section, other monophyletic groups were recovered highlighting the potential need for future nomenclature rearrangement. The phylogenetic signal contained in nuclear genes, when analysed together, was more informative than each gene analysed separately, corroborating monophyly of Melanoconion relative to Culex (Culex) species included in the analyses, the Melanoconion and Spissipes Sections and some species groups. Our results provide new information for the classification of the subgenus and additional data that can be used to improve species identification when a more representative taxon sampling is available.

Larval habitats of Anopheles species in a rural settlement on the malaria frontier of southwest Amazon, Brazil.

Rural settlements are social arrangements expanding in the Amazon region, which generate disturbances in the natural environment, thus affecting the ecology of the species of Anopheles and thus the malaria transmission. Larval habitats are important sources for maintenance of mosquito vector populations, and holding back a natural watercourse is a usual process in the establishment of rural settlements, since the formation of micro-dams represents a water resource for the new settlers. Identifying characteristics of the larval habitats that may be associated with both the presence and abundance of Anopheles vectors species in an environment under ecological transition is background for planning vector control strategies in rural areas in the Amazon. Anopheles larvae collections were performed in two major types of habitats: natural and flow-limited water collections that were constructed by holding back the original watercourse. A total of 3123 Anopheles spp. larvae were captured in three field-sampling collections. The majority of the larvae identified were taken from flow-limited water collections belonged to species of the Nyssorhynchus subgenus (92%), whereas in the natural larval habitats a fewer number of individuals belonged to the Stethomyia (5%) and Anopheles (3%) subgenera. The total of Nyssorhynchus identified (1818), 501 specimens belonged to An. darlingi, 750 to An. triannulatus and 567 for others remaining species. In addition, 1152 could not be identified to subgenus/species level, because they were either in the first-instar or damaged. The primary vector in areas of the Amazon river basin, An. darlingi, was found exclusively in man-made habitats. Statistical analysis display An. triannulatus with specialist behavior for characteristics of man-made habitats. Modifications in the natural environment facilitate the rise of larval habitats for species with epidemiological importance for malaria in the region. This study showed that man-made habitats flow-limited water collections from dry lands could be a factor associated with the increase of An. darlingi and An. triannulatus populations, and other Nyssorhynchus species as well in endemic areas of the Amazon Region.