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

2012; Wiley; Volume: 37; Issue: 2 Linguagem: Inglês

10.1111/j.1948-7134.2012.00249.x

1948-7134

Natalia V. Khrabrova, В. П. Перевозкин, Yulia V. Andreeva, Anuarbek K. Sibataev, V. N. Stegniy,

Viral Infections and Vectors

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. Sampling sites for malaria mosquitoes in the Primorsk and Khabarovsk regions. 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 Mini™ 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 taxonomic status for these forms to be resolved because of a high variation in systematic characters. Later, examination of the egg's exochorion and cytogenetic analysis of karyotypes indentified the eastern form of An. hyrcanus as An. sinensis Weidemann, 1828. Moreover, it was indicated that this was the only representative of the genus Anopheles in the Russian Far East (Gordeev and Klein 1997), whereas the presence of a larger number of species belonging to this complex had been demonstrated for the adjacent countries of the Oriental Zoogeographic Region. In particular, the list of hyrcanus species in China comprises 21 species (Rueda et al. 2007) and in the Republic of Korea, six species (Rueda et al. 2006), five of which (An. sinensis, An. pullus, An. lesteri, An. kleini, and An. belenrae) it is extremely difficult to identify by means of morphological criteria (Jeong et al. 2010, Li et al. 2005). From 264 specimens studied by molecular-genetic analysis, three species of Anopheles belonging to "hyrcanus" complex were identified at first time: An. kleini, An. pullus, and An. lesteri. It has been shown that the species composition varies in different biotopes. Table 1 lists the data on distribution and ratios of the "hyrcanus" species in the studied regions of the Primorsk and Khabarovsk regions. An. kleini is the dominant species in all locations; it has been detected in all samples at a frequency of 75 to 100%; An. pullus is subdominant and has been identified in five studied habitats at a frequency of 3.33–20.83%; and An. lesteri has been found at a relatively low frequency only in the south of the Primorskii krai, in the city of Slavyanka. Thus, the maximal species diversity has been recorded in the southernmost site of the studied region. Probably, the revealed specific structure is not constant and subject to seasonal dynamics, as is suggested by the data for the Republic of Korea (Jeong et al. 2010, Rueda et al. 2010, Kim et al. 2011). Of special importance is that An. sinensis has not been found in the examined region, although it has been regarded as the only representative of the genus Anopheles at the Russian Far East. The new data on the species composition of this genus in the region in question suggest that it is purposeful to conduct additional morphological, cytogenetic, and molecular genetic studies of the Anopheles hyrcanus complex. This study was supported by a research grant from Russian Fund of Basic Research, "The genetic mechanisms of adaptation of sibling-species of malaria mosquitoes of Anopheles hyrcanus complex in Russia" (11-04-00716-а), 2011 – 2013.