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Simple Summary The most dangerous vectors of malaria in the northern regions of the world belong to the Maculipennis group. Among the 22 species in this group, six are considered dominant vectors of malaria. Of these six, Anopheles messeae represents the most widely spread and genetically diverse species in Eurasia and Anopheles daciae, a cryptic species whose taxonomic status is still under debate, has been differentiated from An. messeae based on differences in their ribosomal DNA. However, genetic studies of these species are scarce. The availability of well-developed polytene chromosomes in malaria mosquitoes provides an opportunity to construct high-resolution cytogenetic photomaps that can be used to investigate the genetic divergence between these species. In this study, we created a standard universal cytogenetic map for the salivary gland polytene chromosomes of An. messeae and An. daciae and developed a simple and robust molecular approach for species diagnostics. The quality of the cytogenetic map was validated by studying inversion polymorphisms in populations of An. messeae and An. daciae from a location in the Asian part of Russia. The map will facilitate further investigation of the genetic diversity of these cryptic species. Abstract 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.

Simple Summary Mosquitoes from the family Culicidae are vectors of numerous human diseases such as dengue, Zika, and West Nile fevers, malaria, and lymphatic filariasis. The availability of high-quality genome assemblies for mosquitoes assists in identifying genes responsible for important epidemiological traits such as vector competence, insecticide resistance, and mosquito behavior and stimulates a better understanding of the genetic structure of mosquito populations. Thus, the development of genomic resources and tools is a necessary step for designing and implementing novel genome-based approaches to control mosquitoes. The emphasis of this study is the further development and optimization of a physical genome mapping approach. Physical mapping is placing genomic scaffolds to the chromosomes based on hybridization of the specific probes. This task is difficult for mosquitoes with large genome sizes which are enriched with repetitive DNA sequences and can be potentially misassembled. Here, we describe in detail a simple and robust technique for the physical mapping of such mosquito genomes. This method can be further used for physical genome mapping in other mosquitoes and insects. Abstract 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.

Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

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. Published version

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.

Background A dominant malaria vector, Anopheles messeae, is a highly polymorphic species with wide distribution throughout Eurasia. Five highly polymorphic inversions associated with the geographical distribution of the species have been reported. A sister species, An. daciae, was described and discriminated from An. messeae based on five fixed nucleotide substitutions in the internal transcribed spacer 2 (ITS2) of ribosomal DNA. However, the levels of genomic divergence, chromosomal variation, and ecological differentiation between these two cryptic species remain unexplored. Results In this study, we sequenced ITS2 and analyzed the inversion frequencies of 289 Anopheles larvae specimens collected from three locations in the Moscow region. We identified a high abundance of both An. messeae and An. daciae in all three locations. Five individual genomes for each species of An. messeae and An. daciae from one location were sequenced. Our study confirmed five previously described nucleotide substitutions in the ITS2 of An. messeae. However, we found that the ITS2 sequence in An. daciae was heterogenic in three of the five positions. Fixed nucleotide differences between An. messeae and An. daciae were found only in the last two positions. One mosquito was identified as a hybrid between An. messeae and An. daciae based on heterogeneous substitutions in all five positions. Although, the genomic sequence comparison demonstrated genome-wide divergence between the two species, which is especially pronounced on the X chromosome, an ADMIXTURE cluster analysis demonstrated the presence of two admixed individuals suggesting ongoing hybridization. Cytogenetic analysis demonstrated that An. messeae and An. daciae significantly differ from each other by their frequency of polymorphic inversions. Inversion X1 was fixed in An. messeae but was polymorphic in all An. daciae populations. The frequency of polymorphic autosomal inversions was higher in An. messeae than in An. daciae. The species composition was different among the studied locations suggesting species-specific ecological preferences. Conclusions Our study demonstrated that An. messeae and An. daciae represent closely related cryptic species with incomplete reproductive isolation that are able to maintain genomic differentiation in sympatry despite ongoing genetic introgression. The X chromosome plays an important role in the reproductive isolation between the species.