Insects play an important role in biocenoses due to their abundance and wide (cosmopolitan) distribution. Many insects are crop pests. An effective pest control could be realized in case of proper species identification, which is usually managed by morphological analysis. Molecular methods allow to deep study of many issues of insect biology. In particular, traditional approach can not ordinary identify a species at all stages of their life cycle, whereas molecular methods can it. This review covers a wide range of issues related to the molecular genetic analysis of insects. In the first section we consider the methods of fixation and storage of insect specimens, as well as their impact on DNA quality. Further, we provide general information on population study design. Various schemes of DNA extraction, examples of both express techniques and more thorough protocols for DNA extraction and their purification are provided. In addition, methods of DNA isolation that allow to preserve a specimen integrity for further morphological studies are considered. The methods of DNA quality control are described in detail, that is important for PCR analysis. The last section provides various methods of PCR analysis, that we exemplify by studies aimed to elucidate both fundamental issues and practical problems.
This study investigated the relationship between two subspecies of the Citellophilus tesquorum flea, C. t. altaicus and C. t. sungaris, which are vectors of the bacterium Yersinia pestis that causes human plague across the vast territories of the Palearctic. Adult fleas were collected from 16 localities and 11 populations in 2019 and 2020. Specimens were morphologically verified for subspecies status and analysed for mitochondrial cytochrome c oxidase subunit I (COI) DNA, nuclear ribosomal cluster internal transcribed spacer 1 (ITS1) and ITS2, and Wolbachia-infection status. Our results demonstrated a genetic difference between C. t. altaicus and C. t. sungaris. According to mitochondrial data, the genetic distance between clades of C. t. altaicus and C. t. sungaris was comparable with the species divergence of the genus Callopsylla, which is closely related to Citellophilus. All studied populations of C. t. altaicus were Wolbachia-infected, whereas all studied populations of C. t. sungaris were symbiont-free. Data for ITS1 and ITS2 had much lower phylogenetic signals than mitochondrial data; however, diagnostic substitutions for C. t. altaicus and C. t. sungaris delimitation were also revealed. Analysis of a hardly accessible report on cross experiments allowed us to conclude the partial postzygotic isolation between these subspecies. Taken together, the molecular-genetic, geographical, morphological, and reproductive isolation findings verified that C. t. altaicus and C. t. sungaris subspecies could be considered as different species.
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