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.

In the arboviral vector Aedes aegypti, adaptation to anthropogenic environments has led to a major evolutionary shift separating the domestic Aedes aegypti aegypti (Aaa) ecotype from the wild Aedes aegypti formosus (Aaf) ecotype. Aaa mosquitoes are distributed globally and have higher vectorial capacity than Aaf, which remained in Africa. Despite the evolutionary and epidemiological relevance of this separation, inconsistent morphological data and a complex population structure have hindered the identification of genomic signals distinguishing the two ecotypes. Here we assessed the correspondence between the geographic distribution, population structure and genome-wide selection of 511 Aaf and 123 Aaa specimens and report adaptive signals in 186 genes that we call Aaa molecular signatures. Our results indicate that Aaa molecular signatures arose from standing variation associated with extensive ancestral polymorphisms in Aaf populations and have been co-opted for self-domestication through genomic and functional redundancy and local adaptation. Overall, we show that the behavioural shift of Ae. aegypti mosquitoes to live in association with humans relied on the fine regulation of chemosensory, neuronal and metabolic functions, as seen in the domestication processes of rabbits and silkworms. Our results also provide a foundation for the investigation of new genic targets for the control of Ae. aegypti populations.

Background/Objectives: Chromosomal inversions play an important role in the evolution of insects by forming genetic barriers between closely related species and facilitating local adaptation. Polymorphic inversions in malaria mosquitoes of the Maculipennis subgroup have been studied for over 50 years, yet the evolutionary ancestry of the gene orders remains unknown. In this study, we mapped the genes flanking the breakpoints of two polymorphic X-chromosome inversions in the cryptic species Anopheles messeae and Anopheles daciae of the Maculipennis subgroup. Methods: We used an iterative mapping approach to define the breakpoint regions, selecting flanking markers based on the genome assembly of the reference species, Anopheles atroparvus. To identify the ancestral X chromosomal arrangement in An. messeae and An. daciae, we developed and implemented the genomic inversion calculator (GIC), which uses greedy heuristics to determine the shortest evolutionary scenario of rearrangements. Results: Our knowledge of the relative genomic positions of the inversion breakpoints in An. daciae and An. messeae enabled us to use the An. atroparvus genome as an outgroup and the GIC tool to show that the X0 and X2 arrangements emerged independently along the evolutionary lineages of An. daciae and An. messeae, respectively, based on the X1 arrangement. Conclusions: These results refine the structure and boundaries of the X chromosome rearrangements and reconstruct the sequence of evolutionary events in the cryptic complex An. messeae-An. daciae, demonstrating that the X1 arrangement is ancestral. This study lays the groundwork for analyzing the molecular organization of breakpoints, the mechanisms of inversion formation, and their role in speciation.

In this article we provide a brief overview of research in the main areas of evolutionary cytogenetics of malaria mosquitoes initiated in the 1960s at Tomsk State University. The threat of mass malaria in the Soviet Union was eliminated in the 1950s and 1960s. However, malaria mosquitoes were not destroyed and still remain potential or actual carriers of infectious diseases such as malaria and dirofilariasis. A great contribution to the study of the ecology of malaria mosquitoes in our country was made by the outstanding Russian zoologist and ecologist Vladimir Nikolaevich Beklimishev (1890-1962), who wrote the fundamental work "Ecology of the Malaria Mosquito" in 1944. In the 1950s and 1970s, the American zoologist James B. Kitzmiller (1918-1995) was very actively involved in the cytogenetics of malaria mosquitoes in North America. Using interspecific hybridization, he proved the relationship between the mosquito species of North America and Eurasia and the more ancient origin of the Nearctic group. Kitzmiller's works clearly demonstrated the relevance and timeliness of studying the paleoarctic group of mosquitoes using cytogenetic methods. The ideological inspirer of conducting cytogenetic research at Tomsk University was Tatyana Semenovna Pestryakova (1909-1996), who headed the Department of Invertebrate Zoology at TSU from 1964 to 1974 and had previously studied the ecology of Siberian malaria mosquitoes. Work on the study of the chromosomes of malaria mosquitoes of the Maculipennis group began with the support and participation of the head of the Department of Cytology and Genetics of TSU Nadezhda Nikolaevna Kartashova (19071998). The first published works were devoted to the characterization of the karyotype of Anopheles messeae of the Middle Ob region and the cytogenetic identification of sibling species of malaria mosquitoes An. maculipennis and An. messeae. Vladimir Nikolaevich Stegniy (1946-2023), together with Valentina Matveyevna Kabanova, was the first to use the cytogenetic method to describe a new species of mosquito -An. beklemishevi Stegniy & Kabanova, 1976, named in honor of V. N. Beklemishev. Studying the chromosomes of different mosquito species at different stages of development, V.N. Stegniy discovered that the spatial organization of chromosomes changes significantly during ontogenesis and speciation. It turned out that An. messeae has the largest range among the species of the Maculipennis group, covering almost the entire Boreal subregion of the Paleoarctic, and the greatest inversion polymorphism, which is a classic example of adaptive chromosomal polymorphism. The first works formed three main areas of research on the evolutionary cytogenetics of mosquitoes: 1) species composition and phylogenetic relationships of malaria mosquitoes; 2) reorganization of the spatial architecture of the malaria mosquito genome in ontogenesis and evolution; 3) population and ecological genetics of malaria mosquitoes. Reconstruction of the phylogenetic relationships of mosquito species is important for understanding how and when their genomes changed, and how this contributed to adaptation to new habitats and new pathogens, and also allows researchers to determine the geographical routes of migration and the order of speciation. The study established the geographic distribution, reproductive relationships, and chromosomal phylogenetic relationships in the Anopheles maculipennis species group. It turned out that An. beklemishevi is closest to the Eurasian species and separated from them ~20 million years ago. Thus, the common ancestor of An. beklemishevi and other Eurasian species could have migrated from North America to Eurasia when the Bering Land Bridge existed. The main malaria carriers evolved independently of each other in the Maculipennis group. These data make it possible to approach the study of the molecular genetic basis of the ability of mosquitoes of different phylogenetic lineages to effectively transmit malaria. Studies on the evolutionary cytogenetics of malaria mosquitoes, initiated at Tomsk State University in the 1960s, played an important role in the development of this field of science. They made it possible to significantly advance the understanding of the species composition and phylogenetic relationships of malaria carriers and other diseases. Studies of the spatial organization of chromosomes have provided new insights into the regulation of genomic activity during cell differentiation and the mechanisms of genome architecture reorganization in evolution. These achievements provide a basis for further study of the mechanisms of speciation and adaptation of malaria mosquitoes to changing environmental conditions and interactions with pathogens. The article contains 41 References. The authors dedicate this article to the memory of Vladimir Nikolaevich Stegniy, the founder of the scientific school for the study of cytogenetic mechanisms of evolution and adaptation of natural populations of animals and plants at Tomsk State University. The Authors declare no conflict of interest.

Understanding how life is adapting to urban environments represents an important challenge in evolutionary biology. In this work, we investigate a widely cited example of urban adaptation, Culex pipiens form molestus, also known as the London Underground mosquito. Population genomic analysis of ~350 contemporary and historical samples counters the popular hypothesis that molestus originated belowground in London <200 years ago. Instead, we show that molestus first adapted to human environments aboveground in the Mediterranean or Middle East over the course of more than 1000 years, possibly in association with ancient agricultural civilizations of the Middle East. Our results highlight the role of early human society in priming taxa for contemporary urban evolution. They also provide insight into whether and how molestus contributes to West Nile virus transmission in modern cities.

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 yellow fever mosquito (Aedes aegypti) is an organism of high medical importance because it is the primary vector for diseases such as yellow fever, Zika, dengue, and chikungunya. Its medical importance has made it a subject of numerous efforts to understand their biology. One such effort, was the development of a high-quality reference genome (AaegL5). However, this reference genome was sourced from a highly inbred laboratory strain with unknown geographic origin. Thus, the reference is not representative of a wild mosquito, let alone one from its native range in sub-Saharan Africa. To better understand the genetic architecture of Ae. aegypti and their sister species, we developed two de novo chromosome-scale genomes with sequences sourced from single individuals: one of Ae. aegypti formosus (Aaf) from Burkina Faso and one of Ae. mascarensis (Am) from Mauritius. Both genomes exhibit high contiguity and gene completeness, comparable to AaegL5. While Aaf exhibits high degree of synteny to AaegL5, it also exhibits several large inversions. We also conducted comparative genomic analyses using our genomes and other publicly available culicid reference genomes to find extensive chromosomal rearrangements between major lineages. The expanded gene families common to Aaf, AaegL5, and Am revealed that while the overarching category of genes that have expanded are similar, the specific genes that have expanded differ. Our findings elucidate novel insights into chromosome evolution at both microevolutionary and macroevolutionary scales. The genomic resources we present are additions to a growing arsenal for biologists in understanding mosquito biology and genome evolution.

Chromosomal inversions play an important role in the genomic evolution and adaptation of mosquito species. Here, we present a protocol for detecting chromosomal inversions in mosquitoes using Hi-C technology and chromatin contact heatmaps. We describe the steps for Hi-C library preparation using the Hi-C Arima+ kit, along with the Arima Library Prep Module, and provide several adaptations for mosquito samples. We then outline procedures for Hi-C data analysis, Hi-C heatmap generation, and the identification of polymorphic and fixed inversions. For additional details on the use and execution of this protocol, please refer to Lukyanchikova et al.1.

Chromosomal rearrangements are believed to play a critical role in shaping the eco-evolutionary dynamics of diverse organisms, including mosquitoes. The availability of high-quality genome assemblies of multiple mosquito species has revolutionized the study of these rearrangements. Researchers can now employ genomic approaches to identify chromosomal rearrangements and gain new insights into their role in both microevolutionary and macroevolutionary processes. Autosomal inversions are emerging as possible key drivers of adaptation in mosquitoes. By maintaining standing genetic variation, inversions enable mosquito populations to rapidly adapt to environmental pressures, including insecticide-based vector control methods. X-chromosomal inversions may also play a role in speciation by reducing gene flow between diverging populations. Since mosquitoes are primary vectors of many human diseases, understanding the genomic basis of their adaptation and evolution is important from an epidemiological perspective. Studying chromosomal rearrangements can help identify chromosomal regions associated with adaptation to environmental stresses or insecticide resistance, thereby improving the efficacy of disease management programs.

The Asian tiger mosquito, Aedes albopictus, is currently the most widespread invasive mosquito species in the world. It poses a significant threat to human health, as it is a vector for several arboviruses. We used a SNP chip to genotype 748 Ae. albopictus mosquitoes from 41 localities across Europe, 28 localities in the native range in Asia, and 4 in the Americas. Using multiple algorithms, we examined population genetic structure and differentiation within Europe and across our global dataset to gain insight into the origin of the invasive European populations. We also compared results from our SNP data to those obtained using genotypes from 11 microsatellite loci (N = 637 mosquitoes from 25 European localities) to explore how sampling effort and the type of genetic marker used may influence conclusions about Ae. albopictus population structure. While some analyses detected more than 20 clusters worldwide, we found mosquitoes could be grouped into 7 distinct genetic clusters, with most European populations originating in East Asia (Japan or China). Interestingly, some populations in Eastern Europe did not share genetic ancestry with any populations from the native range or Americas, indicating that these populations originated from areas not sampled in this study. The SNP and microsatellite datasets found similar patterns of genetic differentiation in Europe, but the microsatellite dataset could not detect the more subtle genetic structure revealed using SNPs. Overall, data from the SNP chip offered a higher resolution for detecting the genetic structure and the potential origins of invasions.

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.

Chromosomal inversions play a fundamental role in evolution and have been shown to regulate epidemiologically important traits in malaria mosquitoes. However, they have never been characterized in Aedes aegypti, the major vector of arboviruses, because of the poor structure of its polytene chromosomes. In this study, we applied a Hi-C proximity ligation approach to identify chromosomal inversions in 25 strains of Ae. aegypti, acquired from its worldwide distribution, as well as in one strain of Ae. mascarensis. The study identified 21 multi-megabase inversions with uneven distributions along the three chromosomes. All chromosomal inversions, including one specific for Ae. mascarensis, were polymorphic. Nevertheless, geographic origin separated the strains into two clusters carrying African and non-African inversions suggesting their potential association with Ae. aegypti subspecies. Some of the inversions colocalized with chemoreceptor genes and quantitative trait loci associated with pathogen infection, implicating the potential role of inversions in host choice and disease transmission.

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.

Insects can adapt quickly and effectively to rapid environmental change and maintain long-term adaptations, but the genetic mechanisms underlying this response are not fully understood. In this review, we summarize studies on the potential impact of chromosomal inversion polymorphisms on insect evolution at different spatial and temporal scales, ranging from long-term evolutionary stability to rapid emergence in response to emerging biotic and abiotic factors. The study of inversions has recently been advanced by comparative, population, and 3D genomics methods. The impact of inversions on insect genome evolution can be profound, including increased gene order rearrangements on sex chromosomes, accumulation of transposable elements, and facilitation of genome divergence. Understanding these processes provides critical insights into the evolutionary mechanisms shaping insect diversity.

Detailed knowledge of phylogeography is important for control of mosquito species involved in the transmission of human infectious diseases. Anopheles messeae is a geographically widespread and genetically diverse dominant vector of malaria in Eurasia. A closely related species, An. daciae, was originally distinguished from An. messeae based on five nucleotide substitutions in its ribosomal DNA (rDNA). However, the patterns of phylogeographic history of these species in Eurasia remain poorly understood. Here, using internal transcribed spacer 2 (ITS2) of rDNA and karyotyping for the species identification we determined the composition of five Anopheles species in 28 locations in Eurasia. Based on the frequencies of 11 polymorphic chromosomal inversions used as genetic markers, a large-scale population genetics analysis was performed of 1932 mosquitoes identified as An. messeae, An. daciae and their hybrids. The largest genetic differences between the species were detected in the X sex chromosome suggesting a potential involvement of this chromosome in speciation. The frequencies of autosomal inversions in the same locations differed by 13%-45% between the species demonstrating a restricted gene flow between the species. Overall, An. messeae was identified as a diverse species with a more complex population structure than An. daciae. The clinal gradients in frequencies of chromosomal inversions were determined in both species implicating their possible involvement in climate adaptations. The frequencies of hybrids were low ~1% in northern Europe but high up to 50% in south-eastern populations. Thus, our study revealed critical differences in patterns of phylogeographic history between An. messeae and An. daciae in Eurasia. This knowledge will help to predict the potential of the malaria transmission in the northern territories of the continent.

Abstract 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.

Background Domestication is a complex, multi-stage and species-specific process that results in organisms living close to humans. In the arboviral vector Aedes aegypti adaptation to living in proximity with anthropogenic environments has been recognized as a major evolutionary shift, separating a generalist form, Aedes aegypti formosus (Aaf), from the domestic form Aedes aegypti aegypti (Aaa), which tends to deposit eggs artificial containers and bite humans for a blood meal. These behaviors enhance the mosquito vectorial capacity. The extent to which domestication has impacted the Ae. aegypti genome has not been thoroughly investigated yet. Results Taking advantage of two forms’ distinct and historically documented geographic distributions, we analyzed the genomes of 634 worldwide Ae. aegypti mosquitoes. Using more than 300 million high-confidence SNPs, we found a unique origin for all out-of-Africa Ae. aegypti mosquitoes, with no evidence of admixture events in Africa, apart from Kenya. A group of genes were under positive selection only in out-of-Africa mosquitoes and 236 genes had nonsynonymous mutations, occurring at statistically different frequencies in Aaa and Aaf mosquitoes. Conclusion We identified a clear signal of genetic differentiation between Aaa and Aaf, circumscribed to a catalogue of candidate genes. These “ Aaa molecular signature ” genes extend beyond chemosensory genes to genes linked to neuronal and hormonal functions. This suggests that the behavioral shift to domestication may rely on the fine regulation of metabolic and neuronal functions, more than the role of a few significant genes. Our results also provide the foundation to investigate new targets for the control of Ae. aegypti populations.

Chromosomes are intricately folded within the cell nucleus and interact with peripheral nuclear proteins. The chromatin architecture has a profound effect on how the genome is organized. 3D-FISH is a powerful technique that can reveal the structural and functional organization of chromosomes in the nuclear space. Here, we present a protocol for visualizing specific genomic regions in whole-mount paraformaldehyde-fixed cell nuclei of Anopheles mosquitoes. This protocol was tested in our laboratories and has been showed to be effective and reliable for visualizing genomic regions of various lengths-from 1-kb gene-scale fragments to chromosome-scale segments of DNA.