New Cytogenetic Photomap and Molecular Diagnostics for the Cryptic Species of the Malaria Mosquitoes Anopheles messeae and Anopheles daciae from Eurasia

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.