Polymorphism of mitochondrial COI and nuclear ribosomal ITS 2 in the Culex pipiens complex and in Culex torrentium ( Diptera : Culicidae )

Polymorphism of the mtDNA gene COI encoding cytochrome C oxidase subunit I was studied in the mosquitoes Culex pipiens Linnaeus, 1758 and C. torrentium 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 ecophysiological differences between its morphologically similar members. The full-size DNA sequence 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 sequences was similar in infected C. p. pipiens and C. p. pipiens f. molestus and uninfected 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.


INTRODUCTION
Mosquitoes of the genus Culex Linnaeus, 1758 are active bloodsuckers and serve as vectors of many human and animal infections, such as West Nile fever, fi lariasis, encephalitis, etc.In this respect, the Culex pipiens species complex is of special interest, since the species show notable behavioral, morphological and physiological polymorphism in different parts of the inhabited area and have different epidemiological signifi cances.The complex includes widely distributed species: C. p. pipiens Linnaeus, 1758, C. p. quinquefasciatus Say, 1823, C. p. pallens Coquillett, 1898and C. p. australicus Dobrotworsky et Drummond 1953; a classifi cation also exists in which the closely related species C. torrentium Martini, 1925 andC. vagans Wiedemann, 1828 are included in this complex (Vinogradova, 2000).No  C. torrentium and C. p. pipiens seem to be closely related species.Under laboratory condition, these two taxa can cross, but the hybrids are not viable (E.B.Vinogradova, personal communication).C. torrentium and C. p. pipiens have similar biological features: both are non-autogenous, diapausing and eurygamous.C. torrentium is widely distributed in the Palearctic, and both species often inhabit the same water bodies.Only minor morphological differences exist in their male genitalia.Similarities are often results in misidentifi cation of these two species.However, genetic differences allow precise distinction of these species using the appropriate DNA markers (Fedorova, Shaikevich, 2007).
Mitochondrial DNA is often used in phylogenetic studies of insects, including mosquitoes (e.g., Morlais, Severson 2002;Rasgon et al., 2006;Cywinska et al., 2006;Kumar et al., 2007;Paudan, Ribolla, 2008).It is believed that nucleotide substitutions in mtDNA occur with about constant rate in the evolutionary time, and this allows one to estimate the time of a divergence (DeSalle et al., 1987).The gene encoding cytochrome oxydase I (COI) is the largest and the most conserved of the three mitochondrial genes coding for cytochrome oxydase subunits (Beard et al., 1993).The insect cells usually have one mitochondrial haplotype per individual, though heteroplasmia has been reported (White et al., 2008;Paudan, Ribolla 2008;Savamura et al., 2008).
It is known that C. pipiens are infected with intracellular symbiotic bacteria, Wolbachia Hertig, 1936.These bacteria, fi rst discovered in mosquitoes of the genus Culex (Hertig, Wolbach, 1924), are maternally transmitted and may cause cytoplasmic incompatibility (e.g., Guillemaud et al., 1997;Sinkins et al., 2005;Walker et al., 2009).If the infected males are crossed with uninfected females, all the offspring die at the embryo stage, while a reciprocal cross produces normal progeny.Consequently, the infected females in mosquito populations possess reproductive advantage compared with uninfected females.Similar to mitochondria, the intracellular symbiotic bacteria are transmitted transovarily, while horizontal transmission of the symbionts is rare.Correlation of Wolbachia occurrence with a certain variant of mitochondrial haplotype was detected as "linkage disequilibrium" in some insect species (Montchamp-Moreau et al., Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics 1991; Schulenburg et al., 2002;Jiggins 2003;Rasgon et al., 2006).It was suggested that the lines of an insect host species formed after infection gain some competitive advantage(s).
As a result of expansion of the special mtD-NA variant, which is spread with the bacteria, polymorphism in the infected insect population will be lower than in the uninfected populations.This work aimed to analyze mtDNA variability basing on polymorphism study in the mtDNA gene COI in Culex pipiens com-plex and C. torrentium and to discuss evolutionary signifi cance of Wolbachia infection in these mosquitoes.

MATERIAL AND METHODS
The mosquitoes of the C. pipiens complex were collected in geographically distant areas (Table 1).The mosquitoes were identifi ed by E.B. Vinogradova and M.V. Fedorova.DNA was isolated from ethanol-preserved mosquitoes using the DIAtom™ DNA  (Tamura et. al. 2007).The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of Culex complex.The evolutionary distances were computed using the Neighbor-Joining method with Maximum Composite Likelihood substitution model.

Polymorphism of COI and Wolbachia infection in mosquitoes of the C. pipiens complex and C. torrentium
Not less than 20 mosquitoes from a population were analyzed to evaluate infection with Wolbachia using the PCR primers complementary to the gene wsp which encodes a Wolbachia cell wall protein.The mosquitoes from all studied populations of C. p. pipiens and C. p. pipiens f. molestus, C. p. quinquefasciatus and C. p. pallens were infected with Wolbachia (Table 1).The proportion of infected mosquitoes varied between the populations, being however always high (90-Prep Kit (Isogen, Moscow, Russia) and the polymerase chain reaction was run in the thermocycler GeneAmp R PCR System 2700 (Applied Biosystems, Foster City, CA, USA) with the amplifi cation kits GenePak™ PCR Core (Isogene, Moscow, Russia).
PCR amplifi cation reactions were carried out in a fi nal volume of 25 μl with PCR buffer (Isogen, Russia), 200μM of each dNTP, 2.5 mM MgCl2, one unit of Taq DNA polymerase, 5 pmol of each primer, and 0.1 μg of the isolated DNA.PCR conditions were the following: primary denaturing, 5 min at 94°С; 35 cycles: denaturing at 94°С, 30 s, annealing at 55°С, 40 s, elongation at 72°С, 40 s; fi nal elongation at 72°С, 10 min.The amplicons were revealed by electrophoresis in 1% agarose gel (Sigma, St. Louis, Mo, USA).The amplifi ed DNA fragments were isolated from the gel using JETQUICK Gel Extraction Spin Kit (Genomed, Löhne, Germany) for subsequent sequencing.Ten DNA samples were sequenced for each native population of C. p. pipiens and C. p. pipiens f. molestus, C. torrentium, and two DNA samples for laboratory cultures: C. p. quinquefasciatus and C. p. pallens.The sequencing was run We obtained the full-size DNA sequence of the gene COI encoding cytochrome oxydase subunit I in mosquitoes of the C. pipiens complex and C. torrentium.The gene COI is fl anked by tRNA-Tyrosine at the 5' end and by tRNA-Leucine at the 3'end.Since amplifi cation and subsequent DNA sequencing were run using the primer TY-J-1460 complementary to tRNA-Tyr located upstream of the COI gene, it was possible to identify the hexanucleotide ATTTAA, followed by the codon TCG which is the start codon for the gene COI.The primer UEA10 complementary to tRNA-Leu located downstream of the gene COI allowed us to determine the termination codon TAA.The   1).In total, we found 64 variable nucleotide sites (4 %) in the 36 studied DNA sequences; among these 54 (3.5 %) were considered informative under the conditions of parsimony (Fig. 1).
The subspecies of C. pipiens have 7 variable sites in the gene COI (Fig. 1).All the detected nucleotide substitutions were transitions A↔G located at the third codon position and not changing the amino acid sequence.The mosquitoes of C. p. pipiens have three variable sites characterizing three haplotypes: A, B, and C.These haplotypes were not population-specifi c (Fig. 2).Haplotype A was found in mosquitoes from two Moscow Region populations, PIP1 (PIP1-6, PIP1-7) and PIP2 (PIP2-7), and from a Volgograd population PIP4 (PIP4-17); this haplotype was also detected in two studied individuals of C. p. pallens.Other mosquitoes from the population PIP2 (PIP2-16, PIP2-19) had haplotype B which differs from haplotype A by two transitions.Haplotype C was found in mosquitoes from PIP1, Moscow Region (PIP1-3, PIP1-4, PIP1-8) and from the PIP4 population, Volgograd (PIP4-13).All the studied mosquitoes C. p. pipiens f. molestus were identical and formed cluster D. The urban mosquitoes studied in this work were collected from geographically remote locations in Petozavodsk, St. Petersburg, Moscow, Nizhniy Novgorod, and Volgograd.All the C. p. pipiens f. molestus mosquitoes differ from C. p. pipiens by the two fi xed nucleotide substitutions in positions 270 (G→A) and 1047 (A→G) (Fig. 1).The mosquitoes C. p. quinquefasciatus from the laboratory lines Hyderabad and Pondicherry (India) are identical and differ from the other subspecies by two transitions (A→G) in positions 357 and 999 (Fig. 1).They form haplotype cluster E.
The detected nucleotide substitutions for C. p. pipiens f. molestus and C. p. quinquefasciatus are strictly subspeciesspecifi c.Consequently, the haplotype has no correlation with geographical coordinates of the area, being correlated with subspecies within the species complex.
The mosquitoes of the Wolbachiauninfected species C. torrentium showed considerable polymorphism of the gene COI.Each of the ten studied individual mosquitoes from three populations: Leningrad Region (TOR1-1, TOR1-10, TOR1-29), Moscow Region (TOR2-13, TOR2-14, TOR2-27, TOR2-28), and Saratov Region (TOR3-4, TOR3-5, TOR3-7) have a different haplotype (F-O, Fig. 2).The DNA sequence of the gene COI for an individual C. torrentium differs by 1-13 nucleotide substitutions from COI sequence of other individual mosquitoes, both within the same population and between populations (Fig. 1).Among 21 variable sites, 19 nucleotide substitutions are at the third codon positions, one at the fi rst and one at the second.Only one transition G→ A at position 744 is common for all three studied mosquitoes from population TOR1, Leningrad Region, and not detected in mosquitoes from the Moscow and Saratov Regions.Translation to amino acid sequence showed that C. torrentium, compared with subspecies C. pipiens, has amino acid substitutions at four sites.Two valine-isoleucine substitutions (Val↔Ile, Ile↔Val) are specifi c for all mosquitoes of this species.An additional amino acid substitution, valine for alanine (Val↔Ala)    TOR1.One more substitution of methionine with leucine (Met↔Leu) was found in mosquito TOR1-1 (Fig. 1).

Heteroplasmia in C. torrentium
Among mosquitoes collected in the Leningrad Region, two individuals with heteroplasmia were found.Both were C. torrentium collected at Station Skachki (TOR4).Using the PCR-RFLP method (Shaikevich 2007), two individual mosquitoes (TOR4-14 and TOR4-15) from C. torrentium populations were found to have more than one mtDNA sequence (Table 3).Treatment of the amplifi ed DNA products with HaeIII restriction endonuclease produced three fragments (725, 455, and 270 bp).Cloning of the PCR products showed that the HaeIII restriction site was present in some clones and absent in other clones.After amplifi cation and subsequent restriction, some clones were digested into two fragments, 455 and 270 bp, while other clones remained uncut (725 bp).Nucleotide sequences of the 5-end of the gene COI were obtained for clones from two individual mosquitoes, TOR4-14 and TOR4-15.We found 10 variable sites and 4 types of mtDNA gene COI sequence: clones TOR4-14-1 and TOR4-14-2, TOR4-15-1 and TOR4-15-2.Each individual mosquito has two types of sequences.Nine transitions were found: four G↔A and three Т↔С at the third codon position, one G↔A at the fi rst position, and one Т↔С at the second position.One transversion A↔T is located at the fi rst codon position.All substitutions at the third codon position do not affect the amino acid sequence.Transition G↔A at the fi rst position results in Val substitution with Ala in TOR4-14-1.Transition Т↔С at codon position 2 results in Thr substitution with Ala, and transversion A↔T substitutes Phe with Ile in TOR4-15-2 (Table 3).
Comparing the DNA sequences of the 5' ends of the COI gene of the two heteroplasmic mosquitoes from the population TOR4 and all the other studied C. torrentium specimens,  3).A deviation from the main type (an amino acid substitution) was found only in one haplotype (TOR1).In two individuals from population TOR4, heteroplasmia results in two more variants of mtDNA, TOR4-15-2 with two amino acid substitutions and TOR4-14-1 with one.

Ribosomal DNA variability
The DNA variations between infected and uninfected mosquitoes were evaluated by comparison of 478bp nucleotide sequence of  1; sample numbers are also shown.

Comp. Cytogenet., 2010 4(2)
Comparative Cytogenetics the region of the second internal transcribed spacer (ITS2) in ribosomal RNA of C. p. pipiens (both forms) and C. torrentium.This region is variable and convenient for phylogenetic modeling, since a large set of data is available for the ITS2 nucleotide sequences for the mosquitoes of the genus Culex from all over the world.The mosquitoes C. p. pipiens from Petrozavodsk, Saint-Petersburg, Moscow, Nizhniy Novgorod and Volgograd urban underground populations on one hand, and from open suburban habitats from Moscow and Volgograd regions on the other hand differ in ACG, CGT and CA repeats, located in the downstream part of the studied fragment.Single nucleotide indels are also common for this fragment.The highest ITS2 genetic distance between specimens of C. p. pipiens and C. p. pipiens f. molestus from various studied populations was found to be 2.3 % (Fig. 3).Such a high level of ITS2 genetic variability is common for mosquitoes from both ecotypes and, contrary to mtDNA, ITS2 cannot serve as a marker for detection of species and subspecies of Wolbachia-infected representatives of the complex.Similar high level within-species ITS2 genetic distances (about 3%) was earlier found in C. p. pipiens f. molestus from four Russian towns (Vinogradova, Shaikevich, 2007).This fi ts well with the results obtained for American mosquitoes of the C. pipiens complex (Miller et. al. 1996).
The ITS2 region from C. torrentium is 52-54 bp shorter than in C. p. pipiens; this difference is explained by multiple deletions in C. torrentium.The 3' end of the ITS2 region in C. torrentium contains dinucleotide repeats in various combinations (Fedorova, Shaikevich, 2007).Variability of the ITS2 region in the studied specimens of C. torrentium is 2 %, and the difference between C. p. pipiens and C. torrentium reaches 12.5 % (Fig. 3).

DISCUSSION
For the fi rst time we obtained the sequence of the complete mitochondrial gene COI from mosquitoes of the C. pipiens complex and C. torrentium.In earlier studies, the initiation codon TCG with the upstream ATTTAA sequence was detected in the gene COI from many species of Diptera, e.g.Anopheles gambiae Giles, 1902, A. quadrimaculatus Say, 1824and Aedes aegypti (Linnaeus, 1762); however, the termination codon for these species was represented by single nucleotide T (Lunt еt al., 1996;Bernasconi et al., 2000;Morlais, Severson, 2002).We have shown that according to the invertebrate mtDNA genetic code the TAA triplet is a terminating codon for the gene COI in all studied representatives of the C. pipiens complex and C. torrentium.The full-size gene COI spans 1548 bp and encodes 516 amino acids.Mutations are evenly distributed along the whole sequence, and more variable regions cannot be defi ned.
In 16 populations of the studied mosquito species 15 haplotypes were found (A-O), ten of these were detected in the Wolbachia -uninfected population of C. torrentium.Monomorphic haplotypes were found in C. p. quinquefasciatus (E) and C. p. pipiens f. molestus (D).Three haplotypes were detected for the C. p. pipiens (A, B, C); these haplotypes were not population-specifi c (Fig. 2 (Rasgon et al., 2006).In the studied gene COI, C. p. pipiens and C. p. quinquefasciatus differ by two fi xed nucleotide substitutions.
Among C. pipiens subspecies, all detected substitutions were found to be A↔G transitions located at the third codon position and not affecting the amino acid sequence.It is known that the guanine bases are under maximal mutation pressure in human mtDNA, and the mutation spectra are shifted toward G→A transitions (Malyarchuk, 2005).This is probably also true for insects, and this explains why in our study the transitions A↔G are much more frequent than other mutations.
The Neighbor-Joining dendrogram demonstrating the relation of haplotypes within the studied Culex shows two main clusters: the fi rst is formed by Wolbachia-infected subspecies of C. pipiens, and the second includes Wolbachia -uninfected C. torrentium.It should be noted that polymorphism of the mtDNA gene COI in C. torrentium includes not only nucleotide differences, but also some amino acid substitutions.Two cases of heterolasmia are found in this species.The presence of two distinct mtDNA gene sequences in one organism can be explained by amplifi cation of nuclear pseudogenes, mutations in mtDNA in the cell or the presence of paternal mtDNA.The essential feature of a pseudogene is a stop-codon affecting translation into amino acid sequence.No stop codons were detected in the studied variants of the gene COI from C. torrentium.Multiple mtDNA differences in two individual C. torrentium mosquitoes with heteroplasmia (three and eight nucleotides) give evidence for two-parent mtDNA transfer in these cases.The presence of paternal mtDNA was recorded in the cells of Drosophila simulans Sturtevant, 1919(Satta, Chigusa, 1991) and Aedes aegypti (Paudan, Ribolla, 2008).
Our data show considerable decrease of mtDNA polymorphism in Wolbachia-infected population of C. pipiens compared with uninfected C. torrentium.The number of haplotypes in C. pipiens was low, and same haplotypes were found in distant populations.At the same time C. torrentium has numerous haplotypes and each individual has a unique haplotype.These data correspond to the observations of Rasgon et al. (2006), though these authors compared more closely related forms of the genus Culex.Polymorphism in nuclear sequence (ITS2) was similar in infected C. pipiens and uninfected C. torrentium.It can be suggested that the reduction of mitochondrial polymorphism is more likely to be due to the selective sweep of the Wolbachia-infected haplotype than to a passage of C. pipiens through a bottleneck before the spread around the world.The relation between Wolbachia infection and decreased mtDNA polymorphism has been reported earlier (Montchamp-Moreau et al., 1991;Schulenburg et al., 2002;Jiggins 2003;Rasgon et al., 2006).It is considered that Wolbachia provides its host with some selective Comp. Cytogenet., 2010 4(2) Comparative Cytogenetics advantage, and the infected individuals are favoured by natural selection.Consequently, the bacterium provides dissemination of a certain mtDNA haplotype.This phenomenon is called genetic hitchhiking (Barton, 2000).
The existing strict subspecies-specifi c pattern of haplotypes among Wolbachiainfected populations of mosquitoes C. pipiens, suggests that the infection of mosquitoes with the endosymbiotic bacteria occurred earlier than the divergence of subspecies.Probably, bacterial infection played a key role in subspecies formation, since it is known that Wolbachia induces cytoplasmic incompatibility in C. pipiens.
The hypothesis that the divergence of C. pipiens subspecies in physiological characters and biotopic preference occurred in post-glacial time after introduction of Wolbachia infection is also favored by the data on similarity of studied genes of Wolbachia pipientis in mosquitoes C. pipiens.Earlier we obtained the nucleotide sequence of the bacterial gene wsp from underground mosquitoes in Moscow, Petersburg, and Volgograd (Vinogradova et al., 2003).The GenBank contains 21 sequences of the gene wsp from endosymbionts of C. p. pipiens (both forms), C. p. quinquefasciatus, and C. p. pallens from geographically distant populations.The wsp gene was found to be identical for all studied bacteria, and these data correspond well with the results showing the absence of polymorphism in the other studied genes of Wolbachia, e.g.ftsZ (Guillemaud et al., 1997) and 16S rRNA gene (Pidiyar et al., 2003).Only differences in the prophage-associated ankyrin repeat domain gene between Wolbachia from C. p. pipiens f. molestus and C. p. quinquefasciatus laboratory strains have been found (Walker et al., 2009).The absence of DNA polymorphism in bacteria from geographically remote mosquito populations indicates that Wolbachia infection occurred rather recently and spread rapidly after introduction by one or a few infected females.
In conclusion, our results suggest that Wolbachia probably could play an essential role in divergence of the two species: C. pipiens and C. torrentium, because of reproductive isolation of infected and uninfected species.Additional evidence is provided by the fact that viable hybrids of these closely related species can be obtained in the laboratory only if infected females of C. pipiens are crossed with uninfected males of C. torrentium.The reciprocal crosses produced no viable progeny (E.B.Vinogradova, personal communication).The hybrids were not detected in nature, though both species often inhabit the same areas.

Fig. 2 .
Fig. 2. The similarity dendrogram obtained for nucleotide sequences of the gene COI from mosquitoes of the C. pipiens complex.The Neighbor-Joining method was used.Numbers are bootstrap coeffi cients calculated for 1000 repeats.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches.The haplotypes are marked by letters on the right.The nucleotide sequence of homologous fragment of the gene COI from C. tarsalis (AF425847) is used as an external reference.
samples of C. p. australicus and C. vagans were available for this work.Though morphological variations between the species isolated: C. p. pipiens mosquitoes inhabit the open water bodies, while C. p. pipiens f. molestus mosquitoes prefer underground water bodies (usually in basements of multistoried town buildings); on the other hand, both forms are found together in the subtropical zone.Both forms are active bloodsuckers and act as disease transmission vectors.C. p. quinquefasciatus and C. p. pallens are stenogamous, like C. p. pipiens f. molestus, and non-autogenous like C. p. pipiens.Diapause is absent in C. p. quinquefasciatus, but C. p. pallens may diapause.

Table 1 .
Sampling locations and Wolbachia infection in the studied mosquito populations.

Table 2 .
Base composition in the C. pipiens COI gene at the three codon positions (%).
Comparative Cytogenetics 100%).The lowest percentage of infected individuals was in one of the studied laboratory cultures of C. p. quinquefasciatus.The DNA of all the uninfected individuals was positive for PCR amplifi cation using primers for COI, indicating satisfactory DNA template quality.No infection was detected in C. torrentium.

Table 3 .
Variable sites at the 5' region of the gene COI from C. torrentium.
. pipiens from South Africa.The fragment of the gene ND4 showed no difference for the infected C. p. pipiens and C. p. quinquefasciatus p