RNAi-Mediated Silencing of Laccase 2 in Culex pipiens Pupae via Dehydration and Soaking Results in Multiple Defects in Cuticular Development.

Mosquitoes transmit a range of pathogens, causing devastating effects on human health. Population genetic control strategies have been developed and successfully used for several mosquito species. The most important step in identifying potential targets for mosquito control is the understanding of gene function. RNA interference (RNAi) is a powerful tool for gene silencing which has been widely used to study gene function in insects via knockdown of expression. The success of RNAi in insects depends on the efficient delivery of dsRNA into the cells, with microinjections being the most commonly used to study mosquito gene function. However, microinjections in the pupal stage lead to significant mortality in Aedes and Culex species, and few studies have performed microinjections in Culicinae pupae. Advanced techniques, such as CRISPR/Cas9 knockout, require establishing individual mosquito lines for each gene studied, and maintaining such lines may be limited by the insect-rearing capacity of a laboratory. Moreover, at times gene knockout during early development (embryo stage) has a deleterious effect on mosquito development, precluding the analysis of gene function in the pupal and adult stages and its potential for mosquito control. There is a need for a simple procedure that can be used for the fast and reliable examination of adult gene function via RNAi knockdown. Here, we focus on the aquatic stages of the mosquito life cycle and suggest a quick and easy assay for screening the functional role of genes in Culex pipiens mosquitoes without using microinjections. By dehydration of early stage pupae and subsequent rehydration in highly concentrated dsRNA, we achieved a moderate knockdown of laccase 2, a gene that turns on in the pupal stage and is responsible for melanization and sclerotization of the adult cuticle.

The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

Phylogenomics revealed migration routes and adaptive radiation timing of Holarctic malaria mosquito species of the Maculipennis Group.

BACKGROUND: Phylogenetic analyses of closely related species of mosquitoes are important for better understanding the evolution of traits contributing to transmission of vector-borne diseases. Six out of 41 dominant malaria vectors of the genus Anopheles in the world belong to the Maculipennis Group, which is subdivided into two Nearctic subgroups (Freeborni and Quadrimaculatus) and one Palearctic (Maculipennis) subgroup. Although previous studies considered the Nearctic subgroups as ancestral, details about their relationship with the Palearctic subgroup, and their migration times and routes from North America to Eurasia remain controversial. The Palearctic species An. beklemishevi is currently included in the Nearctic Quadrimaculatus subgroup adding to the uncertainties in mosquito systematics. RESULTS: To reconstruct historic relationships in the Maculipennis Group, we conducted a phylogenomic analysis of 11 Palearctic and 2 Nearctic species based on sequences of 1271 orthologous genes. The analysis indicated that the Palearctic species An. beklemishevi clusters together with other Eurasian species and represents a basal lineage among them. Also, An. beklemishevi is related more closely to An. freeborni, which inhabits the Western United States, rather than to An. quadrimaculatus, a species from the Eastern United States. The time-calibrated tree suggests a migration of mosquitoes in the Maculipennis Group from North America to Eurasia about 20-25 million years ago through the Bering Land Bridge. A Hybridcheck analysis demonstrated highly significant signatures of introgression events between allopatric species An. labranchiae and An. beklemishevi. The analysis also identified ancestral introgression events between An. sacharovi and its Nearctic relative An. freeborni despite their current geographic isolation. The reconstructed phylogeny suggests that vector competence and the ability to enter complete diapause during winter evolved independently in different lineages of the Maculipennis Group. CONCLUSIONS: Our phylogenomic analyses reveal migration routes and adaptive radiation timing of Holarctic malaria vectors and strongly support the inclusion of An. beklemishevi into the Maculipennis Subgroup. Detailed knowledge of the evolutionary history of the Maculipennis Subgroup provides a framework for examining the genomic changes related to ecological adaptation and susceptibility to human pathogens. These genomic variations may inform researchers about similar changes in the future providing insights into the patterns of disease transmission in Eurasia.

Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegyptiNix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

Chromosome and Genome Divergence between the Cryptic Eurasian Malaria Vector-Species Anopheles messeae and Anopheles daciae.

Chromosomal inversions are important drivers of genome evolution. The Eurasian malaria vector Anophelesmesseae has five polymorphic inversions. A cryptic species, An. daciae, has been discriminated from An. messeae based on five fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) of ribosomal DNA. However, the inversion polymorphism in An. daciae and the genome divergence between these species remain unexplored. In this study, we sequenced the ITS2 region and analyzed the inversion frequencies of 289 Anopheles larvae specimens collected from three locations in the Moscow region. Five individual genomes for each of the two species were sequenced. We determined that An. messeae and An. daciae differ from each other by the frequency of polymorphic inversions. Inversion X1 was fixed in An. messeae but polymorphic in An. daciae populations. The genome sequence comparison demonstrated genome-wide divergence between the species, especially pronounced on the inversion-rich X chromosome (mean Fst = 0.331). The frequency of polymorphic autosomal inversions was higher in An. messeae than in An. daciae. We conclude that the X chromosome inversions play an important role in the genomic differentiation between the species. Our study determined that An. messeae and An. daciae are closely related species with incomplete reproductive isolation.

Partial-arm translocations in evolution of malaria mosquitoes revealed by high-coverage physical mapping of the Anopheles atroparvus genome.

BACKGROUND: Malaria mosquitoes have had a remarkable stability in the number of chromosomes in their karyotype (2n = 6) during 100 million years of evolution. Moreover, autosomal arms were assumed to maintain their integrity even if their associations with each other changed via whole-arm translocations. Here we use high-coverage comparative physical genome mapping of three Anopheles species to test the extent of evolutionary conservation of chromosomal arms in malaria mosquitoes. RESULTS: In this study, we developed a physical genome map for Anopheles atroparvus, one of the dominant malaria vectors in Europe. Using fluorescence in situ hybridization (FISH) of DNA probes with the ovarian nurse cell polytene chromosomes and synteny comparison, we anchored 56 genomic scaffolds to the An. atroparvus chromosomes. The obtained physical map represents 89.6% of the An. atroparvus genome. This genome has the second highest mapping coverage among Anophelinae assemblies after An. albimanus, which has 98.2% of the genome assigned to its chromosomes. A comparison of the An. atroparvus, An. albimanus, and An. gambiae genomes identified partial-arm translocations between the autosomal arms that break down the integrity of chromosome elements in evolution affecting the structure of the genetic material in the pericentromeric regions. Unlike An. atroparvus and An. albimanus, all chromosome elements of An. gambiae are fully syntenic with chromosome elements of the putative ancestral Anopheles karyotype. We also detected nonrandom distribution of large conserved synteny blocks and confirmed a higher rate of inversion fixation in the X chromosome compared with autosomes. CONCLUSIONS: Our study demonstrates the power of physical mapping for understanding the genome evolution in malaria mosquitoes. The results indicate that syntenic relationships among chromosome elements of Anopheles species have not been fully preserved because of multiple partial-arm translocations.

Mitotic-chromosome-based physical mapping of the Culex quinquefasciatus genome.

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome.