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Polymorphism of Mitochondrial COI and Nuclear Ribosomal ITS2 in the Culex Pipiens Complex and in Culex Torrentium (Diptera: Culicidae)

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Polymorphism of Mitochondrial COI and Nuclear Ribosomal ITS2 in the Culex Pipiens Complex and in Culex Torrentium (Diptera: Culicidae) © Comparative Cytogenetics, 2010 . Vol. 4, No. 2, P. 161-174. ISSN 1993-0771 (Print), ISSN 1993-078X (Online) Polymorphism of mitochondrial COI and nuclear ribosomal ITS2 in the Culex pipiens complex and in Culex torrentium (Diptera: Culicidae) E.V. Shaikevich, I.A. Zakharov N.I. Vavilov Institute of General Genetics, 119991 Moscow, Russia E-mails: [email protected], [email protected] Abstract. Polymorphism of the mtDNA gene COI encoding cytochrome C oxidase subunit I was studied in the mosquitoes Culex pipiens Linnaeus, 1758 and C. torren- tium Martini, 1925 from sixteen locations in Russia and in three laboratory strains of subtropical subspecies of the C. pipiens complex. Representatives of this complex are characterized by a high ecological plasticity and there are signifi cant ecophysiologi- cal differences between its morphologically similar members. The full-size DNA se- quence of the gene COI spans 1548 bp and has a total A+T content of 70.2 %. The TAA is a terminating codon in all studied representatives of the C. pipiens complex and C. torrentium. 64 variable nucleotide sites (4 %) were found, fi fteen haplotypes were detected, and two heteroplasmic specimens of C. torrentium were recorded. COI haplotype diversity was low in Wolbachia–infected populations of the C. pipiens complex. Monomorphic haplotypes were found in C. p. quinquefasciatus and C. p. pipiens f. molestus. Three haplotypes were detected for the C. p. pipiens, but these haplotypes were not population-specifi c. On the other hand, each of the ten studied Wolbachia-uninfected C. torrentium individuals from three different populations had unique mitochondrial haplotypes. Polymorphism of the 478-bp ITS2 nucleotide se- quences was similar in infected C. p. pipiens and C. p. pipiens f. molestus and unin- fected C. torrentium specimens. The ITS2 genetic distance between C. p. pipiens and C. torrentium reached 12.5 %. Possible effects of Wolbachia invasion on C. pipiens populations are discussed. Key words: Culex pipens, C. torrentium, Wolbachia, cytochrome C oxidase I, mtDNA, ITS2, polymorphism. INTRODUCTION includes widely distributed species: C. p. Mosquitoes of the genus Culex Linnaeus, pipiens Linnaeus, 1758, C. p. quinquefasciatus 1758 are active bloodsuckers and serve as Say, 1823, C. p. pallens Coquillett, 1898 and vectors of many human and animal infections, C. p. australicus Dobrotworsky et Drummond such as West Nile fever, fi lariasis, encephalitis, 1953; a classifi cation also exists in which the etc. In this respect, the Culex pipiens species closely related species C. torrentium Martini, complex is of special interest, since the species 1925 and C. vagans Wiedemann, 1828 are show notable behavioral, morphological and included in this complex (Vinogradova, physiological polymorphism in different 2000). No samples of C. p. australicus and C. parts of the inhabited area and have different vagans were available for this work. Though epidemiological signifi cances. The complex morphological variations between the species http://pensoftonline.net/compcytogen Comparative Cytogenetics doi: 10.3897/compcytogen.v4i2.45 162 E.V. Shaikevich, I.A. Zakharov and between the forms are negligible, notable species. However, genetic differences allow ecophysiological difference does exist. C. p. precise distinction of these species using pipiens includes two biotypes, these are C. p. the appropriate DNA markers (Fedorova, pipiens and C. p. pipiens f. molestus Forskal, Shaikevich, 2007). 1775. The mosquitoes of these two forms have Mitochondrial DNA is often used in different biological features. C. p. pipiens are phylogenetic studies of insects, including non-autogenous (unable to lay the fi rst portion mosquitoes (e.g., Morlais, Severson 2002; of the eggs without taking a blood meal), Rasgon et al., 2006; Cywinska et al., 2006; diapausing (have a reproductive diapauses) Kumar et al., 2007; Paudan, Ribolla, 2008). and eurygamous mosquitoes (need large It is believed that nucleotide substitutions space for mating with swarming), while C. p. in mtDNA occur with about constant rate in pipiens f. molestus are autogenous (lay the fi rst the evolutionary time, and this allows one to portion of the eggs without preceding blood estimate the time of a divergence (DeSalle meal), non-diapausing (active all-year-round), et al., 1987). The gene encoding cytochrome and stenogamous (able to copulate within a oxydase I (COI) is the largest and the most limited space, without swarming). Within the conserved of the three mitochondrial genes temperate zone, these forms are biotopically coding for cytochrome oxydase subunits isolated: C. p. pipiens mosquitoes inhabit (Beard et al., 1993). The insect cells usually the open water bodies, while C. p. pipiens f. have one mitochondrial haplotype per molestus mosquitoes prefer underground water individual, though heteroplasmia has been bodies (usually in basements of multistoried reported (White et al., 2008; Paudan, Ribolla town buildings); on the other hand, both 2008; Savamura et al., 2008). forms are found together in the subtropical It is known that C. pipiens are infected zone. Both forms are active bloodsuckers with intracellular symbiotic bacteria, Wolba- and act as disease transmission vectors. C. chia Hertig, 1936. These bacteria, fi rst discov- p. quinquefasciatus and C. p. pallens are ered in mosquitoes of the genus Culex (Hertig, stenogamous, like C. p. pipiens f. molestus, and Wolbach, 1924), are maternally transmitted non-autogenous like C. p. pipiens. Diapause and may cause cytoplasmic incompatibil- is absent in C. p. quinquefasciatus, but C. p. ity (e.g., Guillemaud et al., 1997; Sinkins et pallens may diapause. al., 2005; Walker et al., 2009). If the infected C. torrentium and C. p. pipiens seem to males are crossed with uninfected females, all be closely related species. Under laboratory the offspring die at the embryo stage, while condition, these two taxa can cross, but the a reciprocal cross produces normal progeny. hybrids are not viable (E.B. Vinogradova, Consequently, the infected females in mosqui- personal communication). C. torrentium to populations possess reproductive advantage and C. p. pipiens have similar biological compared with uninfected females. Similar to features: both are non-autogenous, diapausing mitochondria, the intracellular symbiotic bac- and eurygamous. C. torrentium is widely teria are transmitted transovarily, while hori- distributed in the Palearctic, and both species zontal transmission of the symbionts is rare. often inhabit the same water bodies. Only Correlation of Wolbachia occurrence with a minor morphological differences exist in certain variant of mitochondrial haplotype was their male genitalia. Similarities are often detected as “linkage disequilibrium” in some results in misidentifi cation of these two insect species (Montchamp-Moreau et al., Comparative Cytogenetics Comp. Cytogenet., 2010 4(2) Polymorphism of mitochondrial DNA in Culex 163 Table 1. Sampling locations and Wolbachia infection in the studied mosquito populations. Form Population origin Abbrevi- Biotope Latitude/ Provided by Wolbachia ation longitude Infection 1 C. p. pipiens Russian, Iksha, PIP1 Open, 55°10' N/ 37°31' E M. Fedorova . 100% Moscow Region suburban 2 C. p. pipiens Russian, Luzki, PIP2 Open, 55°51' N/ 36°57' E M. Fedorova 90% Moscow Region. suburban 3 C. p. pipiens Russian, Sarepta, PIP3 Open, 48°31' N/ 44°30' E M. Fedorova 100% Volgograd Region. suburban 4 C. p. pipiens Russian, Liteishik, PIP4 Open, 48°45' N/ 44°42' E M. Fedorova 100% Volgograd Region suburban 5 C. p. pipiens f. Russian, Moscow MOL1 Basement, 55°55' N/ 37°20' E E. Vinogradova 96% molestus urban 6 C. p. pipiens f. Russian, Nizhniy MOL2 Basement, 56°20' N/ 44°00' E E. Vinogradova 100% molestus Novgorod urban 7 C. p. pipiens f. Russian, Saint- MOL3 Basement, 59°56' N/ 30°15' E E. Vinogradova 94% molestus Petersburg urban 8 C. p. pipiens f. Russian, MOL4 Basement, 61°47' N/ 34°20' E E. Vinogradova. 90% molestus Petrozavodsk urban 9 C. p. pipiens f. Russian, Volgograd, MOL5 Basement, 48°44' N/ 44°31' E M. Fedorova 100% molestus ul Rokossovskogo urban 10 C. p. pipiens f. Russian, Volgograd, MOL6 Open, 48°31' N/ 44°30' E M. Fedorova 100% molestus ul. Aldanskaya urban 11 C. p. quinque- India, Hyderabad QUIN1 Lab 17°37' N/ 78°48' E E. Vinogradova 80% fasciatus culture 12 C. p. quinque- India, Pondicherry QUIN2 Lab 12°25' N/ 80°41' E E. Vinogradova 42% fasciatus culture 13 C. p. pallens Japan, Ohno-cho, PAL1 Lab 34°24' N/132°27'E E. Vinogradova 90% Hiroshima culture 14 C. torrentium Russian, St.69 km, TOR1 Open, 60°49' N/ 30°10' E E. Vinogradova 0% Leningrad Region suburban 15 C. torrentium Russian, TOR2 Open, 56°01' N/ 37°10' E M. Fedorova 0% Chashnikovo, suburban Moscow Region 16 C. torrentium Russian, Saratov TOR3 Open, 51°34' N/ 46°02' E N. Polukonova 0% Region suburban 17 C. torrentium Russian, St. TOR4 Open, 59°45' N/ 30°07' E E. Vinogradova 0% Skachki, Leningrad suburban Region 1991; Schulenburg et al., 2002; Jiggins 2003; plex and C. torrentium and to discuss evolu- Rasgon et al., 2006). It was suggested that the tionary signifi cance of Wolbachia infection in lines of an insect host species formed after in- these mosquitoes. fection gain some competitive advantage(s). As a result of expansion of the special mtD- MATERIAL AND METHODS NA variant, which is spread with the bacteria, The mosquitoes of the C. pipiens complex polymorphism in the infected insect popula- were collected in geographically distant areas tion will be lower than in the uninfected popu- (Table 1). The mosquitoes were identifi ed lations. This work aimed to analyze mtDNA by E.B. Vinogradova and M.V. Fedorova. variability basing on polymorphism study in DNA was isolated from ethanol-preserved the mtDNA gene COI in Culex pipiens com- mosquitoes using the DIAtom™ DNA Comparative Comp. Cytogenet., 2010 4(2) Cytogenetics 164 E.V. Shaikevich, I.A. Zakharov Prep Kit (Isogen, Moscow, Russia) and the Table 2. Base composition in the C.
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