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

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.

Patterns of genetic differentiation imply distinct evolutionary histories of the sibling mosquito speciesAnopheles messeaeandAnopheles daciaein Eurasia

“…3). Because one of the arms in the sex chromosome is absent in a polytene chromosome compliment, we indicated it as X, but not as XL or XR (Artemov et al, 2021). The Y chromosome is heterochromatic and is absent in the polytene chromosome compliment.…”

“…messeae and An. daciae (Artemov et al, 2021; Stegniy, Kabanova, & Novikov, 1976). A total of 11 chromosomal inversions — X1, X2, X3, X4, 2R1, 2R3, 2R4, 3R1, 3R2, 3L1, and 3L4 – were identified and considered in this study (Table 1).…”

“…Inversion X1 is additionally shown by red brackets. Numbered divisions and lettered subdivisions of the chromosomes are shown below the chromosomes.The order of the divisions on chromosome 1 is based on inverted arrangement X11 according to the previous published chromosomal map(Artemov et al 2021). A total of 11 chromosomal inversions are shown.…”

Invasion of malaria mosquitoes natural population by parasitic nematodes Dirofilaria along Ob River basin in Western Siberia

“…Parasites species identification was performed by PCR analysis with primers designed for molecular diagnostics of filariasis in dogs, based on the difference in the sequences of COI gene of the mitochondrial DNA (Rishniw M. et al, 2006). Malaria mosquito species identification was performed using PCR and PCR–RFLP analysis of internal transcribed spacer 2 of ribosomal genes cluster (ITS2, Vaulin, et al, 2018, Artemov et al, 2021).…”

“…The homogenate was incubated at 95 °C for 5 min, immediately frozen at -20 °C, and thawed at room temperature to release the DNA from the tissues after 5 min centrifugation at 10000 g. The supernatant with DNA was transferred into the clean 1.5 ml tube and stored at -20° C to be used for PCR reaction.Parasites species identification was performed by PCR analysis with primers designed for molecular diagnostics of filariasis in dogs, based on the difference in the sequences of COI gene of the mitochondrial DNA(Rishniw M. et al, 2006). Malaria mosquito species identification was performed using PCR and PCR-RFLP analysis of internal transcribed spacer 2 of ribosomal genes cluster (ITS2,Vaulin, et al, 2018, Artemov et al, 2021.…”