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.

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. 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. 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.

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.

A dominant and hemizygous male-determining locus (M locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegypti . Nix is a male-determining factor (M factor) in the M locus and its transient expression in females (m/m) results in partial masculinization. Here, we show that the Nix transgene alone was sufficient to convert females into fertile males, which continued to produce sex-converted progeny in subsequent generations. However, assisted mating with wild-type females was necessary, as the converted m/m males could not fly. Knockout of myo-sex , a myosin heavy chain gene and the only other protein-coding gene reported in the M locus, rendered wild-type males flightless. Thus, Nix alone converts female Ae. aegypti to fertile males and myo-sex is required for male flight. Only female Ae. aegypti mosquitoes bite and transmit disease-causing viruses. Nix -mediated female-to-male conversion is 100% penetrant and stable over many generations, indicating great potential for mosquito control.

Ecological and genetic studies of malaria mosquitoes of the Black Sea coast have not been recently conducted despite increasing human-caused environmental changes in the area. In this study, we investigated the species composition, geographical distribution, ecological preferences, and chromosomal polymorphism of malaria mosquitoes of the Crimean Peninsula and the Black Sea coast of the Caucasus. Species were diagnosed using a combination of morphological, cytogenetic, and molecular markers. The ecological conditions of the larval habitats, such as dissolved oxygen content in the water, acidity, salinity, and temperature, were measured. Seven species of malaria mosquitoes were identified in the pool of 2229 individual mosquitoes collected at 56 breeding sites including An. atroparvus, An. claviger, An. daciae (formely identified as An. messeae s. l.), An. hyrcanus, An. maculipennis s. s., An. plumbeus and An. melanoon. The previously recorded species of An. algeriensis, An. messeae s. s., An. sacharovi, An. superpictus were not found in this study. Anopheles maculipennis was dominant in typical anophylogenic water bodies. Anopheles plumbeus, which used to breed mainly in tree holes in coastal forests, has spread to urban settlements along the Black Sea coast and breeds in artificial containers. Chromosomal polymorphism was studied and found in An. atroparvus and An. daciae populations. Differences in the chromosomal composition of An. daciae populations in Crimea and on the Black Sea coast of the Caucasus were revealed. The Crimean population had a low level of polymorphism in autosomal inversions. The data obtained in this study can be used to inform a better control of potential malaria vectors in the Black Sea coastal region.

In this study, we sampled malaria mosquito larvae in natural and artificial breeding places to study the geographical distribution, ecological preferences, and chromosomal variability of different species of the genus Anopheles in the territory of the Crimean Peninsula and the Black Sea coast of the Caucasus. Species were diagnosed using a combination of morphological, cytogenetic, and molecular markers. The ecological conditions of the larval habitats, such as dissolved oxygen content in the water, acidity, salinity, and temperature, were measured. Seven species of malaria mosquitoes were identified in the pool of 2229 individual mosquitoes collected at 56 breeding sites, including An. atroparvus, An. claviger, An. daciae (formerly identified as An. messeae s. l.), An. hyrcanus, An. maculipennis s. s., An. plumbeus, and An. melanoon. The previously recorded species of An. algeriensis, An. messeae s. s., An. sacharovi, and An. superpictus were not found in this study. Anopheles maculipennis was dominant in typical anophylogenic water bodies. Anopheles plumbeus, which used to breed mainly in tree holes in coastal forests, has spread to urban settlements along the Black Sea coast and breeds in artificial containers. Chromosomal polymorphism was studied and found in An. atroparvus and An. daciae populations. Differences in the chromosomal composition of An. daciae populations in Crimea and on the Black Sea coast of the Caucasus were revealed. The Crimean population had a low level of polymorphism in autosomal inversions. The data obtained in this study can be used to inform a better control of potential malaria vectors in the Black Sea coastal region.