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Abstract Background Nyssorhynchus darlingi (also known as Anopheles darlingi) is the primary malaria vector in the Amazon River Basin. In Brazil, analysis of single nucleotide polymorphisms (SNPs) previously detected three major population clusters, and a common garden experiment in a laboratory setting revealed significant population variation in life history traits. Increasing temperatures and local level variation can affect life history traits, i.e. adult longevity, that alter vectorial capacity with implications for malaria transmission in Ny. darlingi. Methods We investigated the population structure of Ny. darlingi from 7 localities across Brazil utilizing SNPs and compared them to a comprehensive Ny. darlingi catalog. To test the effects of local level variation on life history traits, we reared F1 progeny from the 7 localities at three constant temperatures (20, 24 and 28 °C), measuring key life history traits (larval development, food-starved adult lifespan, adult size and daily survival). Results Using nextRAD genotyping-by-sequencing, 93 of the field-collected Ny. darlingi were genotyped at 33,759 loci. Results revealed three populations (K = 3), congruent with major biomes (Amazonia, Cerrado and Mata Atlântica), with greater F ST values between biomes than within. In the life history experiments, increasing temperature reduced larval development time, adult lifespan, and wing length in all localities. The variation of family responses for all traits within four localities of the Amazonia biome was significant (ANOVA, P

Abstract Background In the Amazon Basin, Nyssorhynchus (Anopheles) darlingi is the most aggressive and effective malaria vector. In endemic areas, behavioral aspects of anopheline vectors such as host preference, biting time and resting location post blood meal have a key impact on malaria transmission dynamics and vector control interventions. Nyssorhynchus darlingi presents a range of feeding and resting behaviors throughout its broad distribution. Methods To investigate the genetic diversity related to biting behavior, we collected host-seeking Ny. darlingi in two settlement types in Acre, Brazil: Granada (~ 20-year-old, more established, better access by road, few malaria cases) and Remansinho (~ 8-year-old, active logging, poor road access, high numbers malaria cases). Mosquitoes were classified by the location of collection (indoors or outdoors) and time (dusk or dawn). Results Genome-wide SNPs, used to assess the degree of genetic divergence and population structure, identified non-random distributions of individuals in the PCA for both location and time analyses. Although genetic diversity related to behavior was confirmed by non-model-based analyses and F ST values, model-based STRUCTURE detected considerable admixture of these populations. Conclusions To our knowledge, this is the first study to detect genetic markers associated with biting behavior in Ny. darlingi. Additional ecological and genomic studies may help to understand the genetic basis of mosquito behavior and address appropriate surveillance and vector control.

Abstract Background In Loreto Department, Peru, a successful 2005–2010 malaria control programme (known as PAMAFRO) included massive distribution of long-lasting insecticidal nets (LLINs). Additional local distribution of LLINs occurred in individual villages, but not between 2012 and 2015. A 2011–2012 study of the primary regional malaria vector Anopheles darlingi detected a trend of increased exophagy compared with pre-PAMAFRO behaviour. For the present study, An. darlingi were collected in three villages in Loreto in 2013–2015 to test two hypotheses: (1) that between LLIN distributions, An. darlingi reverted to pre-intervention biting behaviour; and, (2) that there are separate sub-populations of An. darlingi in Loreto with distinct biting behaviour. Results In 2013–2015 An. darlingi were collected by human landing catch during the rainy and dry seasons in the villages of Lupuna and Cahuide. The abundance of An. darlingi varied substantially across years, villages and time periods, and there was a twofold decrease in the ratio of exophagic:endophagic An. darlingi over the study period. Unexpectedly, there was evidence of a rainy season population decline in An. darlingi. Plasmodium-infected An. darlingi were detected indoors and outdoors throughout the night, and the monthly An. darlingi human biting rate was correlated with the number of malaria cases. Using nextRAD genotyping-by-sequencing, 162 exophagic and endophagic An. darlingi collected at different times during the night were genotyped at 1021 loci. Based on model-based and non-model-based analyses, all genotyped An. darlingi belonged to a homogeneous population, with no evidence for genetic differentiation by biting location or time. Conclusions This study identified a decreasing proportion of exophagic An. darlingi in two villages in the years between LLIN distributions. As there was no evidence for genetic differentiation between endophagic and exophagic An. darlingi, this shift in biting behaviour may be the result of behavioural plasticity in An. darlingi, which shifted towards increased exophagy due to repellence by insecticides used to impregnate LLINs and subsequently reverted to increased endophagy as the nets aged. This study highlights the need to target vector control interventions to the biting behaviour of local vectors, which, like malaria risk, shows high temporal and spatial heterogeneity.

Wolbachia pipientis, a bacterial symbiont infecting arthropods and nematodes, is vertically transmitted through the female germline and manipulates its host’s reproduction to favor infected females. Wolbachia also infects somatic tissues where it can cause nonreproductive phenotypes in its host, including resistance to viral pathogens. Wolbachia-mediated phenotypes are strongly associated with the density of Wolbachia in host tissues. Little is known, however, about how Wolbachia density is regulated in native or heterologous hosts. Here, we measure the broad-sense heritability of Wolbachia density among families in field populations of the mosquito Culex pipiens, and show that densities in ovary and nongonadal tissues of females in the same family are not correlated, suggesting that Wolbachia density is determined by distinct mechanisms in the two tissues. Using introgression analysis between two different strains of the closely related species C. quinquefasciatus, we show that Wolbachia densities in ovary tissues are determined primarily by cytoplasmic genotype, while densities in nongonadal tissues are determined by both cytoplasmic and nuclear genotypes and their epistatic interactions. Quantitative-trait-locus mapping identified two major-effect quantitative-trait loci in the C. quinquefasciatus genome explaining a combined 23% of variance in Wolbachia density, specifically in nongonadal tissues. A better understanding of how Wolbachia density is regulated will provide insights into how Wolbachia density can vary spatiotemporally in insect populations, leading to changes in Wolbachia-mediated phenotypes such as viral pathogen resistance.

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.

The major drivers of the extensive biodiversity of the Neotropics are proposed to be geological and tectonic events together with Pliocene and Pleistocene environmental and climatic change. Geographical barriers represented by the rivers Amazonas/Solimões, the Andes and the coastal mountain ranges in eastern Brazil have been hypothesized to lead to diversification within the primary malaria vector, Anopheles (Nyssorhynchus) darlingi Root, which primarily inhabits rainforest. To test this biogeographical hypothesis, we analyzed 786 single nucleotide polymorphisms (SNPs) in 12 populations of An. darlingi from across the complex Brazilian landscape. Both model-based (STRUCTURE) and non-model-based (Principal Components and Discriminant Analysis) analysis of population structure detected three major genetic clusters that correspond with newly described Neotropical biogeographical regions: 1) Atlantic Forest province (= southeast population); 2) Parana Forest province (= West Atlantic forest population, with one Chacoan population - SP); and 3) Brazilian dominion population (= Amazonian population with one Chacoan population - TO). Significant levels of pairwise genetic divergences were found among the three clusters, allele sharing among clusters was negligible, and geographical distance did not contribute to differentiation. We infer that the Atlantic forest coastal mountain range limited dispersal between the Atlantic Forest province and the Parana Forest province populations, and that the large, diagonal open vegetation region of the Chacoan dominion dramatically reduced dispersal between the Parana and Brazilian dominion populations. We hypothesize that the three genetic clusters may represent three putative species.

Herein we tested the repeatability of phylogenetic inference based on high throughput sequencing by increased taxon sampling using our previously published techniques in the pitcher-plant mosquito, Wyeomyia smithii in North America. We sampled 25 natural populations drawn from different localities nearby 21 previous collection localities and used these new data to construct a second, independent phylogeny, expressly to test the reproducibility of phylogenetic patterns. Comparison of trees between the two data sets based on both maximum parsimony and maximum likelihood with Bayesian posterior probabilities showed close correspondence in the grouping of the most southern populations into clear clades. However, discrepancies emerged, particularly in the middle of W. smithii's current range near the previous maximum extent of the Laurentide Ice Sheet, especially concerning the most recent common ancestor to mountain and northern populations. Combining all 46 populations from both studies into a single maximum parsimony tree and taking into account the post-glacial historical biogeography of associated flora provided an improved picture of W. smithii's range expansion in North America. In a more general sense, we propose that extensive taxon sampling, especially in areas of known geological disruption is key to a comprehensive approach to phylogenetics that leads to biologically meaningful phylogenetic inference.

The mosquito Wyeomyia smithii overwinters in a larval diapause that is initiated, maintained and terminated by day length (photoperiod). We use a forward genetic approach to investigate transcriptional events involved in the termination of diapause following exposure to long-days.We incorporate a novel approach that compares two populations that differentially respond to a single day length. We identify 30 transcripts associated with differential response to day length. Most genes with a previously annotated function are consistent with their playing a role in the termination of diapause, in downstream developmental events, or in the transition from potentially oxygen-poor to oxygen-rich environments. One gene emerges from three separate forward genetic screens as a leading candidate for a gene contributing to the photoperiodic timing mechanism itself (photoperiodic switch). We name this gene photoperiodic response gene 1 (ppdrg1). WsPpdrg1 is up-regulated under long-day response conditions, is located under a QTL for critical photoperiod and is associated with critical photoperiod after 25 generations of recombination from a cross between extreme phenotypes.Three independent forward genetic approaches identify WsPpdrg1 as a gene either involved in the photoperiodic switch mechanism or very tightly linked to a gene that is. We conclude that continued forward genetic approaches will be central to understanding not only the molecular basis of photoperiodism and diapause, but also the evolutionary potential of temperate and polar animal populations when confronted with rapid climate change.