Abstract. Aedes aegypti is an invasive, highly anthropophilic mosquito and a major vector for dengue and chikungunya.

BackgroundDrosophila suzukii differs from other melanogaster group members in their proclivity for laying eggs in fresh fruit rather than in fermenting fruits. Olfaction and gustation play a critical role during insect niche formation, and these senses are largely mediated by two important receptor families: olfactory and gustatory receptors (Ors and Grs). Earlier work from our laboratory has revealed how the olfactory landscape of D. suzukii is dominated by volatiles derived from its unique niche. Signaling and reception evolve in synchrony, since the interaction of ligands and receptors together mediate the chemosensory behavior. Here, we manually annotated the Ors and Grs in D. suzukii and two close relatives, D. biarmipes and D. takahashii, and compared these repertoires to those in other melanogaster group drosophilids to identify candidate chemoreceptors associated with D. suzukii’s unusual niche utilization.ResultsOur comprehensive annotations of the chemosensory genomes in three species, and comparative analysis with other melanogaster group members provide insights into the evolution of chemosensation in the pestiferous D. suzukii. We annotated a total of 71 Or genes in D. suzukii, with nine of those being pseudogenes (12.7 %). Alternative splicing of two genes brings the total to 62 genes encoding 66 Ors. Duplications of Or23a and Or67a expanded D. suzukii’s Or repertoire, while pseudogenization of Or74a, Or85a, and Or98b reduced the number of functional Ors to roughly the same as other annotated species in the melanogaster group. Seventy-one intact Gr genes and three pseudogenes were annotated in D. suzukii. Alternative splicing in three genes brings the total number of Grs to 81. We identified signatures of positive selection in two Ors and three Grs at nodes leading to D. suzukii, while three copies in the largest expanded Or lineage, Or67a, also showed signs of positive selection at the external nodes.ConclusionOur analysis of D. suzukii’s chemoreceptor repertoires in the context of nine melanogaster group drosophilids, including two of its closest relatives (D. biarmipes and D. takahashii), revealed several candidate receptors associated with the adaptation of D. suzukii to its unique ecological niche.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2983-9) contains supplementary material, which is available to authorized users.

We report here the development of 65 novel microsatellite loci and construction of a composite genetic linkage map for Culex pipiens complex mosquitoes. Microsatellites were identified by in silico screening of the Culex quinquefasciatus genome assembly. Cross-species utility of 73 microsatellites for population studies in C. pipiens sensu stricto and C. quinquefasciatus was evaluated by genotyping a subset of samples collected in Indiana, United States, and Point Fortin, Trinidad. Allele frequencies of 67 microsatellites were within Hardy-Weinberg expectations in both population subsets. A composite linkage map was constructed based on restriction fragment length polymorphism and microsatellite polymorphisms in 12 independent F1 intercross mapping populations. The composite map consists of 61 marker loci totaling 183.9 cM distributed across the 3 linkage groups. These loci cover 29.5, 88.8, and 65.6 cM on chromosomes I-III, respectively, and allow for assignment of 10.4% of the genome assembly and 13.5% of the protein coding genes to chromosome position. Our results suggest that these microsatellites will be useful for mapping and population studies of 2 pervasive species in the C. pipiens complex. Moreover, the composite map presented here will serve as a basis for the construction of high-resolution genetic and physical maps, as well as detection of quantitative trait loci to aid in the investigation of complex genetic traits influencing phenotypes of interest.

The New World Screwworm fly, Cochliomyia hominivorax, is a major pest of livestock in South America and Caribbean. However, few genomic resources have been available for this species. A genome of 534 Mb was assembled from long read PacBio DNA sequencing of DNA from a highly inbred strain. Analysis of molecular evolution identified 40 genes that are likely under positive selection. Developmental RNA-seq analysis identified specific genes associated with each stage. We identify and analyze the expression of genes that are likely important for host-seeking behavior (chemosensory), development of larvae in open wounds in warm-blooded animals (heat shock protein, immune response) and for building transgenic strains for genetic control programs including gene drive (sex determination, germline). This study will underpin future experiments aimed at understanding the parasitic lifestyle of the screwworm fly and greatly facilitate future development of strains for efficient systems for genetic control of screwworm.

Phlebotomine sand flies employ an elaborate system of pheromone communication wherein males produce pheromones that attract other males to leks (thus acting as an aggregation pheromone) and females to the lekking males (sex pheromone). In addition, the type of pheromone produced varies among populations. Despite the numerous studies on sand fly chemical communication, little is known of their chemosensory genome. Chemoreceptors interact with chemicals in an organism’s environment to elicit essential behaviors such as the identification of suitable mates and food sources. Thus, they play important roles during adaptation and speciation. Major chemoreceptor gene families, odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) together detect and discriminate the chemical landscape. Here, we annotated the chemoreceptor repertoire in the genomes of Lutzomyia longipalpis and Phlebotomus papatasi, major phlebotomine vectors in the New World and Old World, respectively. Comparison with other sequenced Diptera revealed a large and unique expansion where over 80% of the ~140 ORs belong to a single, taxonomically restricted clade. We next conducted a comprehensive analysis of the chemoreceptors in 63 L. longipalpis individuals from four different locations in Brazil representing allopatric and sympatric populations and three sex-aggregation pheromone types (chemotypes). Population structure based on single nucleotide polymorphisms (SNPs) and gene copy number in the chemoreceptors corresponded with their putative chemotypes, and corroborate previous studies that identified multiple populations. Our work provides genomic insights into the underlying behavioral evolution of sexual communication in the L. longipalpis species complex in Brazil, and highlights the importance of accounting for the ongoing speciation in central and South American Lutzomyia that could have important implications for vectorial capacity.

BackgroundMosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence.Methodology/Principal FindingsSix lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3–5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise FST indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively.Conclusions/SignificanceWe demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).

Culex pipiens mosquitoes are important disease vectors inhabiting temperate zones, worldwide. The seasonal reduction in temperature and photoperiod accompanying late summer and early fall prompts female mosquitoes to enter diapause, a stage of developmental arrest and physiological conditioning that enhances survival during the winter months. To investigate the molecular mechanisms underlying diapause induction, we used custom whole-transcriptome microarrays to identify differences in gene expression following exposure to nondiapause (long days, 25°C) and diapause-inducing (short days, 18°C) environmental conditions. Using a two-way ANOVA we identified 1130 genes that were differentially expressed. We used the expression of these genes across three time points to construct a gene co-expression network comprising five modules. Genes in modules 1, 2 and 3 were largely up-regulated, while genes in modules 4 and 5 were down-regulated when compared to nondiapause conditions. Pathway enrichment analysis of the network modules revealed some potential regulatory mechanisms driving diapause induction. Module 1 was enriched for genes in the TGF-β and Wnt signaling pathways; module 2 was enriched for genes involved in insect hormone biosynthesis, specifically, ecdysone synthesis; module 3 was enriched for genes involved in chromatin modification, and module 5 was enriched for genes in the circadian rhythm pathway. Our results suggest that TGF-β signaling and chromatin modification are key drivers for the integration of environmental signals into the diapause induction phase in C. pipiens mosquitoes.

BackgroundCulex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly.FindingsWe constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus.ConclusionThe NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes.