Enhancing Genome Investigations in the Mosquito Culex quinquefasciatus via BAC Library Construction and Characterization

Background Culex 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. Findings We 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. Conclusion The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes

New World screwworm (Cochliomyia hominivorax) myiasis in feral swine of Uruguay: one Health and transboundary disease implications

Background: Feral swine (Sus scrofa) are highly invasive and threaten animal and human health in the Americas. The screwworm (Cochliomyia hominivorax) is listed by the World Organization for Animal Health as a notifiable infestation because myiasis cases affect livestock, wildlife, and humans in endemic areas, and outbreaks can have major socioeconomic consequences in regions where the screwworm has been eradicated. However, a knowledge gap exists on screwworm infestation of feral swine in South America, where the screwworm is endemic. Here, we report screwworm infestation of feral swine harvested in Artigas Department (Uruguay), where the Republic of Uruguay shares borders with Brazil and Argentina. Methods: Myiasis caused by the larvae of screwworm were identified in feral swine with the support and collaboration of members of a local feral swine hunting club over a 3-year period in the Department of Artigas. Harvested feral swine were examined for the presence of lesions where maggots causing the myiasis could be sampled and processed for taxonomic identification. The sites of myiasis on the body of infested feral swine and geospatial data for each case were recorded. The sex and relative size of each feral swine were also recorded. Temperature and precipitation profiles for the region were obtained from public sources. Results: Myiases caused by screwworms were recorded in 27 of 618 the feral swine harvested. Cases detected in males weighing > 40 kg were associated with wounds that, due to their location, were likely caused by aggressive dominance behavior between adult males. The overall prevalence of screwworm infestation in the harvested feral swine was associated with ambient temperature, but not precipitation. Case numbers peaked in the warmer spring and summer months. Conclusions: This is the first report on myiasis in feral swine caused by screwworm in South America. In contrast to myiasis in cattle, which can reach deep into host tissues, screwworms in feral swine tended to cause superficial infestation. The presence of feral swine in screwworm endemic areas represents a challenge to screwworm management in those areas. Screwworm populations maintained by feral swine may contribute to human cases in rural areas of Uruguay, which highlights the importance of the One Health approach to the study of this invasive host species–ectoparasite interaction

Genome-Based Microsatellite Development in the Culex pipiens Complex and Comparative Microsatellite Frequency with Aedes aegypti and Anopheles gambiae

Mosquitoes 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.Six 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 F(ST) 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.We 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%)

Molecular Signatures of Sexual Communication in the Phlebotomine Sand Flies

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

Genome annotation improvements from cross-phyla proteogenomics and time-of-day differences in malaria mosquito proteins using untargeted quantitative proteomics

The malaria mosquito, Anopheles stephensi, and other mosquitoes modulate their biology to match the time-of-day. In the present work, we used a non-hypothesis driven approach (untargeted proteomics) to identify proteins in mosquito tissue, and then quantified the relative abundance of the identified proteins from An. stephensi bodies. Using these quantified protein levels, we then analyzed the data for proteins that were only detectable at certain times-of-the day, highlighting the need to consider time-of-day in experimental design. Further, we extended our time-of-day analysis to look for proteins which cycle in a rhythmic 24-hour ("circadian") manner, identifying 31 rhythmic proteins. Finally, to maximize the utility of our data, we performed a proteogenomic analysis to improve the genome annotation of An. stephensi. We compare peptides that were detected using mass spectrometry but are 'missing' from the An. stephensi predicted proteome, to reference proteomes from 38 other primarily human disease vector species. We found 239 such peptide matches and reveal that genome annotation can be improved using proteogenomic analysis from taxonomically diverse reference proteomes. Examination of 'missing' peptides revealed reading frame errors, errors in gene-calling, overlapping gene models, and suspected gaps in the genome assembly

The role of informal dimensions of safety in high-volume organisational routines:an ethnographic study of test results handling in UK general practice

Abstract Background The handling of laboratory, imaging and other test results in UK general practice is a high-volume organisational routine that is both complex and high risk. Previous research in this area has focused on errors and harm, but a complementary approach is to better understand how safety is achieved in everyday practice. This paper ethnographically examines the role of informal dimensions of test results handling routines in the achievement of safety in UK general practice and how these findings can best be developed for wider application by policymakers and practitioners. Methods Non-participant observation was conducted of high-volume organisational routines across eight UK general practices with diverse organisational characteristics. Sixty-two semi-structured interviews were also conducted with the key practice staff alongside the analysis of relevant documents. Results While formal results handling routines were described similarly across the eight study practices, the everyday structure of how the routine should be enacted in practice was informally understood. Results handling safety took a range of local forms depending on how different aspects of safety were prioritised, with practices varying in terms of how they balanced thoroughness (i.e. ensuring the high-quality management of results by the most appropriate clinician) and efficiency (i.e. timely management of results) depending on a range of factors (e.g. practice history, team composition). Each approach adopted created its own potential risks, with demands for thoroughness reducing productivity and demands for efficiency reducing handling quality. Irrespective of the practice-level approach adopted, staff also regularly varied what they did for individual patients depending on the specific context (e.g. type of result, patient circumstances). Conclusions General practices variably prioritised a legitimate range of results handling safety processes and outcomes, each with differing strengths and trade-offs. Future safety improvement interventions should focus on how to maximise practice-level knowledge and understanding of the range of context-specific approaches available and the safeties and risks inherent in each within the context of wider complex system conditions and interactions. This in turn has the potential to inform new kinds of proactive, contextually appropriate approaches to intervention development and implementation focusing on the enhanced deliberation of the safety of existing high-volume routines

Data from: Composite linkage map and enhanced genome map for Culex pipiens complex mosquitoes

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

Mapping data: two point linkage estimates and standard errors.

Composite two point linkage estimates and standard errors for each linkage group in the Culex pipiens genetic map

Data from: The making of a pest: insights from the evolution of chemosensory receptor families in a pestiferous and invasive fly, Drosophila suzukii

Background: Drosophila 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. Results: Our 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. Conclusion: Our 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