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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.

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 genome assembly of Anopheles darlingi consists of 2221 scaffolds (N50 = 115,072 bp) and has a size spanning 136.94 Mbp. This assembly represents one of the smallest genomes among Anopheles species. Anopheles darlingi genomic DNA fragments of ~37 Kb were cloned, end-sequenced, and used as probes for fluorescence in situ hybridization (FISH) with salivary gland polytene chromosomes. In total, we mapped nine DNA probes to scaffolds and autosomal arms. Comparative analysis of the An. darlingi scaffolds with homologous sequences of the Anopheles albimanus and Anopheles gambiae genomes identified chromosomal rearrangements among these species. Our results confirmed that physical mapping is a useful tool for anchoring genome assemblies to mosquito chromosomes.

Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a Plasmodium transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for Anopheles beklemishevi.