Species composition of the mosquitoAnopheles hyrcanus(Diptera, Culicidae) Group in the Russian Far East

Abstract

A specific feature of the genus Anopheles (Diptera, Culicidae) is the presence of numerous sibling species displaying high morphological similarity (Stegniy 1991, Collins and Paskewitz 1996, Jeong et al. 2010). These species play different roles in transmitting the pathogens of malaria and other dangerous diseases, thus attracting constant attention of biologists, health professionals, and epidemiological surveillance services. An important component of antimalarial activities is identification of potential vectors, study of their distribution, and determination of their role in pathogen transmission. The mosquitoes of the Anopheles hyrcanus group are known as active vectors of the agents of transmissible diseases (Rueda et al. 2006). This group comprises about 30 species mainly inhabiting the Oriental Zoogeographic Region; however, the distribution range of some species enter the southwestern and southeastern Palearctic. Within the former USSR territory, the distribution range of the mosquitoes belonging to this group consists of two disconnected areas. The western area covers the main part of Ukraine, Moldavia, Northern Caucasus, Transcaucasia, the coast of the Caspian Sea, Middle Asia, and southern Kazakhstan. The eastern area comprises the Primorsk and Khabarovsk regions and several areas of the Amur region (Gutsevich et al. 1970, Gutsevich 1976). Currently, the questions are still open regarding the species diversity and modern boundaries of the distribution ranges of the mosquitoes belonging to the “hyrcanus” group inhabiting the territory of Russia and Commonwealth of Independent States (CIS) countries. The goal of this work was to study the species composition of the malaria mosquito Anopheles hyrcanus group in the Far East. The 3rd and 4th instar larvae were sampled during August 22 to September 5, 2011, in eight typical anopheline habitats in the Primorsk and Khabarovsk regions (Figure 1). The sampling was performed according to a conventional protocol (Gutsevich et al. 1970). The larvae were fixed with 90% ethanol for subsequent molecular genetic analysis. A total of 264 individuals was examined, 24 to 66 larvae per population. DNA was extracted using an Invisorb® Spin Tissue Mini Kit (Invitek, Germany) according to the manufacturer’s protocol with minor modifications. Molecular genetic identification of five species of the hyrcanus group was conducted as proposed by Li et al. (2005). The reaction mixture for multiprimer PCR contained single PCR buffer (60 mM Tris–HCl, 25 mM KCl, 10 mM 2-mercaptoethanol, and 0.1% Triton X-100), 1.5 mM MgCl2, 200 μM of each dNTP, 1 U of Taq DNA polymerase (SibEnzim, Novosibirsk, Russia), 4 pmol of each primer, 10 ng of genome DNA, and deionized water to a total volume of 15 μl. Amplification was conducted in an MJ MiniTM Personal Thermal Cycler (Bio-Rad, USA) using the following mode: initial DNA denaturation for 2 min at 94° C; 35 cycles of 30 s at 94° C, 30 s at 60° C, and 1 min at 72° C; and final extension for 7 min at 72° C. The amplification products were visualized on 1.5% agarose gel containing ethidium bromide. The used molecular genetic assay allowed five species of “hyrcanus” group to be identified: An. sinensis Weidemann, 1828, An. pullus Yamada, 1937, An. lesteri Baisas, Hu, 1936, An. belenrae Rueda, 2005, and An. kleini Rueda, 2005. Until 1997, it was considered that only one species of the hyrcanus group—Anopheles hyrcanus Pallas, 1771—was present on the territory of the former USSR (Gutsevich et al. 1970). Within this species, two spatially separated forms, western and eastern, were distinguished based on morphological characteristics (Gutsevich 1976). However, morphological criteria did not allow the problem of