The Eurasian malaria vector Anopheles messeae is a widely spread and genetically diverse species. Five widespread polymorphic chromosomal inversions were found in natural populations of this mosquito. A cryptic species, Anopheles daciae, was differentiated from An. messeae by the presence of several nucleotide substitutions in the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA. However, because of the absence of a high-quality reference cytogenetic map, the inversion polymorphisms in An. daciae and An. messeae remain poorly understood. Moreover, a recently determined heterogeneity in ITS2 in An. daciae questioned the accuracy of the previously used Restriction Fragment Length Polymorphism (RFLP) assay for species diagnostics. In this study, a standard-universal cytogenetic map was constructed based on orcein stained images of chromosomes from salivary glands for population studies of the chromosomal inversions that can be used for both An. messeae and An. daciae. In addition, a new ITS2-RFLP approach for species diagnostics was developed. Both methods were applied to characterize inversion polymorphism in populations of An. messeae and An. daciae from a single location in Western Siberia in Russia. The analysis demonstrates that cryptic species are remarkably different in their frequencies of chromosomal inversion variants. Our study supports previous observations that An. messeae has higher inversion polymorphism in all autosomes than the cryptic species An. daciae.

Chromosomal inversions are important drivers of genome evolution. The Eurasian malaria vector Anopheles messeae 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.

Chromosomal inversions play a crucial role in evolution and have been found to regulate epidemiologically significant traits in malaria mosquitoes. However, they have not been characterized in Aedes aegypti, the primary vector of arboviruses, due to the poor structure of its polytene chromosomes. The Hi-C proximity ligation approach was used to identify chromosomal inversions in 25 strains of A. aegypti obtained from its worldwide distribution and in one strain of Aedes mascarensis. The study identified 21 multimegabase polymorphic inversions ranging in size from 5 to 55 Mbp. Inversions were more abundant in African than in non-African strains, 15 versus 3 inversions, with the highest number observed in West Africa. All inversions were grouped into two geographic clusters of African or non-African origin, suggesting their association with A. aegypti subspecies. Inversions were unevenly distributed along chromosomal arms, with the highest number found in the 1q and 3p arms homologous to the inversion-rich 2R chromosomal arm in the malaria vector Anopheles gambiae. Direct comparison of inversions between A. aegypti and An. gambiae revealed significant overlap in their genomic locations. This finding may explain the parallel evolution of the two species under similar environmental conditions. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, suggesting their potential role in host preference and disease transmission. Our study revealed the large pool of structural variations in the A. aegypti genome and provides the foundation for future studies of their impact on the biology of this important arboviral vector.

The Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. Here we present AalbF2, a dramatically improved assembly of the Ae. albopictus genome that has revealed widespread viral insertions, novel microRNAs and piRNA clusters, the sex determining locus, new immunity genes, and has enabled genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we built the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. These up-to-date resources of the genome provide a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures. One Sentence Summary: Long-read and Hi-C-based de novo assembly of the arboviral vector Aedes albopictus genome fosters deeper understanding of its biological features.

Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.

The northward expansion of Dirofilaria spp. is a current medical and veterinary concern. However, it is unclear how far north the parasite has spread in Western Siberia and what species of mosquito can carry and transmit it. This study examined Dirofilaria spp. infection in Aedes mosquitoes in the taiga zone of the Irtysh Basin. The mosquito species were identified based on morphology, and Dirofilaria spp. were identified using PCR. Of the 13 habitats surveyed, 24 of 2205 mosquito samples were infected with D. repens and 1 with Dirofilaria sp. The highest infection rate (~7.6) was recorded in Tobolsk, Tyumen region, at 58.4° N. Mosquito infection was recorded as far north as 61° N in Khanty-Mansiysk. The presence of the infective L3 stage of the parasite was recorded up to 60° N in Bobrovsky. Nine species of mosquitoes were found to be infected with D. repens: Aedes rossicus, Aedes behningi, Aedes cantans, Aedes communis, Aedes cyprius, Aedes euedes, Aedes excrucians, Aedes flavescens, and Aedes sticticus. Two of these species, Ae. behningi and Ae. communis, were competent vectors of the parasite. Thus, D. repens has successfully adapted to the Aedes mosquito in the taiga zone of Siberia.