Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.     

Differentiation between the urban mosquito Culex pipiens pipiens F. molestus and Culex torrentium (Diptera: Culicidae) by the molecular genetic methods

The molecular characters suitable for the differentiation between the autogenous form of Culex pipiens pipiens (the urban mosquito) and the species Culex torrentium, which was previously identified erroneously as an anautogenous form of C. pipiens pipiens (Shaikevich, Vinogradova, 2004), are given. These mosquitoes differ in the infection by the endosymbiotic bacteria Wolbachia pipientis, 6 transitions among 247 nucleotide sequences of 3' part of the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA), in the mtDNA fragments resulted from the restriction analysis, and in the lengths of second internal transcribed spacer (ITS2) sequences in the ribosomal DNA.     

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus (Diptera: Culicidae) in Petrozavodsk City

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus in Petrozavodsk city was studied for the first time. Immature mosquito stages were found in 21 of 68 basements flooded with polluted waters, the occurrence of the mosquitoes varied in different districts of the sity. The mean siphonal index of the mosquito larvae from 6 underground populations varied from 3.8 up to 4.2, i. e. within the range typical for C. p. p. f. molestus.     

Evolutionary history of a mosquito endosymbiont revealed through mitochondrial hitchhiking

Due to cytoplasmic inheritance, spread of maternally inherited Wolbachia symbionts can result in reduction of mitochondrial variation in populations. We examined sequence diversity of the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene in Wolbachia-infected (South Africa (SA), California and Thailand) and uninfected (SA) Culex pipiens complex populations. In total, we identified 12 haplotypes (A-L). In infected populations, 99% of individuals had haplotype K. In the uninfected SA population, 11 haplotypes were present, including K. Nuclear allozyme diversity was similar between infected and uninfected SA populations. Analysis of nuclear DNA sequences suggested that haplotype K presence in uninfected SA Cx. pipiens was probably due to a shared ancestral polymorphism rather than hybrid introgression. These data indicate that Wolbachia spread has resulted in drastic reduction of mitochondrial variability in widely separated Cx. pipiens complex populations. In contrast, the uninfected SA population is probably a cryptic species where Wolbachia introgression has been prevented by reproductive isolation, maintaining ancestral levels of mitochondrial diversity. Molecular clock analyses suggest that the Wolbachia sweep occurred within the last 47000 years. The effect of Wolbachia on mitochondrial dynamics can provide insight on the potential for Wolbachia to spread transgenes into mosquito populations to control vector-borne diseases.     

A comparative study on insemination dependency of circadian activity pattern in mosquitoes

Abstract. Two forms of mosquitoes in the Culex pipiens complex, C.p.pallens and C.p.molestus , were compared for the insemination-dependency of entrained female circadian activity in LD 16:8h. When virgin, Pallens is nocturnal and molestus is, in addition, diurnal. Insemination and injection of extracts of the male accessory gland affects virgin activity in that nocturnality is strengthened in both mosquitoes, and the diurnal activity of molestus is depressed. This was so whether the treatments were intra- or interspecific. These findings are compared with those already published for other mosquitoes. A coherent view is proposed that the mosquito, nocturnal/crepuscular, nocturnal and diurnal, or diurnal, has potentially a similar entrained waveform which is affected by insemination in a common way, and by lighting conditions in species specific ways.     

Spacing behaviour in resting Culex pipiens (Diptera, Culicidae): a computer modelling approach

Abstract. We compared two models based on Monte Carlo simulations with the spacing patterns of resting adults of the mosquito Cx pipiens L. (form molestus ) (Diptera, Culicidae). The comparison with the first model which assumed a distribution of complete spatial randomness showed that there was a tendency for mosquitoes to repel other mosquitoes which were too close, resulting in high local regularity. The second model took this repulsion into account. A final test of global patchiness using Model 2 indicated that the spacing behaviour of Cx pipiens was not sufficiently described by local regularity only, but also required a gregarious element. This supports the hypothesis that the molestus form of Cx pipiens shows spaced-out gregariousness in the sense of Kennedy & Crawley (1967).     

Molecular genetic methods for the identification of the urban mosquito Culex pipiens pipiens F. molestus (Diptera, Culicidae)

Molecular genetic methods for the identification of two ecotypes, or forms (pipiens and molestus) of the Culex pipiens pipiens mosquitoes, which are known as active bloodsuckers and vectors of various agents of diseases, are proposed. For the DNA analysis, two populations of the urban mosquitoes (the molestus ecotype) from St. Petersburg and Moscow and two populations from the overground reservoirs in the Leningrad Province and neighboring areas of Moscow (the pipiens ecotype) have been studied. These ecotypes differ by six transitions among 247 nucleotide sequences of 3' part of the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA), by the mtDNA fragments resulted from the restriction analysis and by the lengths of second internal transcribed spacer (ITS2) sequences in the ribosomal DNA.     

几种蚊虫线粒体DNA-16SrRNA序列及其相互关系的研究

测定我国尖音库蚊复合组4亚种（尖音库蚊、淡色库蚊、致倦库蚊和骚扰库蚊）、三带喙库蚊、白纹伊蚊和中华按蚊的线粒体DNA 16S rRNA基因（mtDNA-16S rRNA)序列,发现我国尖音库蚊复合组4亚种mtDNA-16S rRNA序列基本一致,长度为555bp(554bp),GC含量为25．41％。该序列与其他3种蚊虫在种间存在差异,与三带喙库蚊的种间差异为0．54％;与白纹伊蚊的种间差异为5．77％;与中华按蚊的种间差异为9．62％。分子系统关系分析表明该序列与传统分类系统的同属或同亚科种类近似性相一致。     

Maternal lineages of Pinus densata,a diploid hybrid

Previous morphological, allozyme and chloroplast DNA data have suggested that Pinus densata originated through hybridization between P. tabuliformis and P. yunnanensis. In the present study, sequence and restriction site analyses of maternally inherited mitochondrial nad1 intron were used to detect variation patterns in 19 populations of P. tabuliformis, P. yunnanensis and P. densata. A total of three mitotypes (A, B, C) were detected. All but one of the populations of P. yunnanensis possessed mitotype B while all populations of P. tabuliformis had mitotype A. Pinus densata populations, on the other hand, harboured both mitotypes A and B, which are characteristic of P. tabuliformis and P. yunnanensis, respectively. This result gives strong additional evidence supporting the hybrid origin of this diploid pine. The distribution of mitotypes indicated very different mating compositions and evolutionary history among P. densata populations. It seems that local founder populations and backcrosses may have played important roles in the early establishment of P. densata populations. The uplift of the Tibetan Plateau had a significant impact on the distribution of maternal lineages of P. densata populations.     

Multiple Wolbachia determinants control the evolution of cytoplasmic incompatibilities in Culex pipiens mosquito populations

Wolbachia are maternally inherited endosymbionts that can invade arthropod populations through manipulation of their reproduction. In mosquitoes, Wolbachia induce embryonic death, known as cytoplasmic incompatibility (CI), whenever infected males mate with females either uninfected or infected with an incompatible strain. Although genetic determinants of CI are unknown, a functional model involving the so-called mod and resc factors has been proposed. Natural populations of Culex pipiens mosquito display a complex CI relationship pattern associated with the highest Wolbachia (wPip) genetic polymorphism reported so far. We show here that C. pipiens populations from La Réunion, a geographically isolated island in the southwest of the Indian Ocean, are infected with genetically closely related wPip strains. Crossing experiments reveal that these Wolbachia are all mutually compatible. However, crosses with genetically more distant wPip strains indicate that Wolbachia strains from La Réunion belong to at least five distinct incompatibility groups (or crossing types). These incompatibility properties which are strictly independent from the nuclear background, formally establish that in C. pipiens, CI is controlled by several Wolbachia mod/resc factors.     

MULTIPLE ORIGINS OF GENDER-ASSOCIATED MITOCHONDRIAL DNA LINEAGES IN BIVALVES (MOLLUSCA: BIVALVIA)

Previous studies have shown that marine mussels (genus Mytilus) and a freshwater mussel (Pyganodon grandis) contain two distinct gender-associated mitotypes, which is a characteristic feature of the phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA). Here we present evidence for the presence of distinct male (M) and female (F) mitotypes in three other bivalve species, the mytilid Geukensia demissa, and the unionid species P. fragilis and Fusconaia flava. Nucleotide sequences of a segment of the COI gene from the M and F mitotypes from each of the three mytilid species (M. edulis, M. trossulus, G. demissa) and three unionid species (P. grandis, P. fragilis, F. flava) were used for phylogenetic analysis. The analysis suggests three independent origins of M and F mitotypes for the six species examined; one for the three unionid species, one for the two Mytilus species, and one for Geukensia. The first of these F/M divergence events, while of uncertain age, predates the divergence of the two unionid genera and is likely older than either of the two F/M divergence events in the mytilid taxa. The most parsimonious explanation of multiple F/M divergence events is that they represent independent origins of DUI. Another possibility is that, in a given taxon, an F or M mitotype assumes the role of the opposite mitotype (by virtue of a mechanism that remains to be clarified) and subsequently was fixed within its new gender. The fixation of a mtDNA lineage derived from a mitotype of switched function would reset the divergence of the gender-associated lineages to zero, thereby mimicking a de novo split of F and M lineages from a preexisting mtDNA genome that was not gender specific. Further broad-scale taxonomic studies of the occurrence of distinct M and F mitotypes may allow for the evaluation of the latter hypothesis.     

Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens.

Reproductive incompatibilities called cytoplasmic incompatibilities are known to affect a large number of arthropod species and are mediated by Wolbachia, a maternally transmitted microorganism. The crossing relationships between strains of potential hosts define their incompatibility types and it is generally assumed that differences between strains of Wolbachia induce different crossing types. Among all the described host species, the mosquito, Culex pipiens, displays the greatest variability of cytoplasmic incompatibility crossing types. We analysed mitochondrial and bacterial DNA variability in Culex pipiens in order to investigate some possible causes of incompatibility crossing type variability. We sequenced fragments of the ftsZ gene, and the A + T-rich control region of the mtDNA. We also sequenced the second subunit of the mitochondrial cytochrome oxidase (COII) gene, in Culex pipiens and a closely related species, C. torrentium, in order to verify the usefulness of the A + T-rich region for the present purposes. No variability was found in the Wolbachia ftsZ gene fragment, and very limited variation of the mitochondrial marker whatever the compatibility type or the origin of the host. A low variability was found in the A + T-rich region and comparison of divergence of the A + T-rich region and COII gene between C. pipiens and C. torrentium did not reveal any special constraints affecting this region. In contrast to observations in other host species, variability of incompatibility crossing types is not due to multiple infections by distantly related Wolbachia strains.     

The postglacial history of Scots pine (Pinus sylvestris L.) in western Europe: evidence from mitochondrial DNA variation

The geographical structure of mitochondrial (mt)DNA variants (mitotypes) was investigated in 38 western European populations of Scots pine Pinus sylvestris using restriction fragment length polymorphism (RFLP) analysis of total DNA and a homologous cox1 probe. Three major mitotypes (designated a, b and d ) were detected. Within Spain all three major mitotypes were found, gene diversity was high, H_T = 0.586, and this diversity was distributed predominantly among rather than within populations (F_ST(M) = 0.813 for the seven Spanish populations). Mitotype d was present only in the most southerly population from the Sierra Nevada . Elsewhere in Europe, populations showed little or no mtDNA diversity within regions, but there were marked differences between regions. Italian populations were fixed for mitotype b ; populations from northern France, Germany, Poland, Russia and southern Sweden were fixed for mitotype a ; while populations in northern Fennoscandia were fixed for mitotype b . The isolated Scottish populations were predominantly of mitotype a , but mitotype b was present in three of the 20 populations scored. In Scotland, UK gene diversity (H_T = 0.120) and genetic differentiation among populations (F_ST(M) = 0.37) was much lower than in Spain. When interpreted in the light of complementary data from pollen analysis and nuclear genetic markers, the results suggest that present-day populations of P. sylvestris in western Europe have been derived from at least three different sources after glaciation.     

Impact of population age structure on Wolbachia transgene driver efficacy: ecologically complex factors and release of genetically modified mosquitoes

Wolbachia symbionts hold theoretical promise as a way to drive transgenes into insect vector populations for disease prevention. For simplicity, current models of Wolbachia dynamics and spread ignore ecologically complex factors such as the age structure of vector populations and overlapping vector generations. We developed a model including these factors to assess their impact on the process of Wolbachia spread into populations of three mosquito species (Anopheles gambiae, Aedes aegypti and Culex pipiens). Depending on the mosquito species, Wolbachia parameters, released mosquito life stage and initial age structure of the target population, the number of Wolbachia-infected mosquitoes that we predict would need to be released ranged from less than the threshold calculated by the simple model to a 10-30-fold increase. Transgenic releases into age-structured populations, which is an expectation for wild mosquitoes, will be difficult and depending on the circumstances may not be economically or logistically feasible due to the large number of infected mosquitoes that must be released. Our results support the perspective that understanding ecological factors is critical for designing transgenic vector-borne disease control strategies.     

Transposable element polymorphism of Wolbachia in the mosquito Culex pipiens: evidence of genetic diversity, superinfection and recombination

Wolbachia is a group of maternally inherited endosymbiotic bacteria that infect and induce cytoplasmic incompatibility (CI) in a wide range of arthropods. In contrast to other species, the mosquito Culex pipiens displays an extremely high number of CI types suggesting differential infection by multiple Wolbachia strains. Attempts so far failed to detect Wolbachia polymorphism that might explain this high level of CI diversity found in C. pipiens populations. Here, we establish that Wolbachia infection is near to or at fixation in worldwide populations of the C. pipiens complex. Wolbachia polymorphism was addressed by sequence analysis of the Tr1 gene, a unique transposable element of the IS5 family, which allowed the identification of five C. pipiens Wolbachia strains, differing either by nucleotide substitution, presence or absence pattern, or insertion site. Sequence analysis also showed that recombination, transposition and superinfection occurred at very low frequencies. Analysis of the geographical distributions of each Wolbachia strain among C. pipiens populations indicated a strong worldwide differentiation independent from mosquito subspecies type, except in the UK. The availability of this polymorphic marker now opens the way to investigate evolution of Wolbachia populations and CI dynamics, in particular in regions where multiple crossing types coexist among C. pipiens populations.     

Coevolution of symbiotic bacteria Wolbachia and host mtDNA in Russian populations of the Culex pipiens mosquito complex

The mitochondrial DNA phylogenies of closely related forms of mosquitoes from the Culex pipiens complex and of strains of the endosymbiotic bacteria Wolbachia pipientis were compared. Based on the cytochrome oxidase subunit I gene polymorphism, six mitochondrial haplotypes and four W. pipientis groups were discovered in mosquitoes from geographically remote populations. A strict correlation between the COI type and the type of W. pipientis proves the stable coinheritance and distribution of both cytoplasmic components in the examined mosquito populations and suggests either the absence or rarity of horizontal transfer of the symbionts in the Culex pipiens complex.     

Population of the mosquitoes Culex pipiens molestus (Diptera: Culicidae) in dwellings of Lensk city

Culex pipiens molestus has been distributed in Siberia and the Russian Far East since 80s of XX century. In early 90s, C. p. molestus reached South Yakutia (Nerungri city), and nowadays it occurs in South-West Yakutia (Lensk city). The number of breeding places with larvae and pupae of mosquitoes was not numerous in Lensk; among 56 examined houses, the places with larvae and pupae mosquitoes were registered in seven houses, while the adult mosquitoes occurred in the majority of the houses. The occurrence of larvae varied from 12.5 to 33.3%, occurrence of adults-from 12.5 to 100%. The coefficient of variation of larvae in the samples was equal to 0.92, and adults had the following coefficients: 0.84 in the houses without basement; 0.92 in the houses with dry basements; 1.05 in the houses with pools in basements. This data are referred to the casual type, it means that the distribution of mosquitoes in space is dispersed. Imago spread actively in stone house of Lensk that provides mosquitoes with new places of breeding, and supports the stability of population in new areas.     

Cultivation of the mosquito Culex pipiens pipiens f. molestus (Diptera, Culicidae) without blood feeding

Optimal photoperiod and temperature conditions for rearing of the mosquito Culex pipiens pipiens f. molestus without blood feeding (through autogenous eggs) were experimentally substantiated. This method may be used in order to obtain relatively small quantities of mosquitoes or for preservation of a laboratory colony. Optimal conditions of rearing are the short-day photoperiod (12 h of light per day) at 20 degrees C, when 87 % of the females produce autogenous eggs rafts consisting of 52 eggs on the average. It should be emphasized that larval food must be adequate and abundant, because the quality and quantity of it has a pronounced effect on the autogeny rate.     

Generation of a novel Wolbachia infection in Aedes albopictus (Asian tiger mosquito) via embryonic microinjection

Genetic strategies that reduce or block pathogen transmission by mosquitoes are being investigated as a means to augment current control measures. Strategies of vector suppression and replacement are based upon intracellular Wolbachia bacteria, which occur naturally in many insect populations. Maternally inherited Wolbachia have evolved diverse mechanisms to manipulate host insect reproduction and promote infection invasion. One mechanism is cytoplasmic incompatibility (CI) through which Wolbachia promotes infection spread by effectively sterilizing uninfected females. In a prior field test, releases of Wolbachia-infected males were used to suppress a field population of Culex pipiens. An additional strategy would employ Wolbachia as a vehicle to drive desired transgenes into vector populations (population replacement). Wolbachia-based population suppression and population replacement strategies require an ability to generate artificial Wolbachia associations in mosquitoes. Here, we demonstrate a technique for transferring Wolbachia (transfection) in a medically important mosquito species: Aedes albopictus (Asian tiger mosquito). Microinjection was used to transfer embryo cytoplasm from a double-infected Ae. albopictus line into an aposymbiotic line. The resulting mosquito line is single-infected with the wAlbB Wolbachia type. The artificially generated infection type is not known to occur naturally and displays a new CI crossing type and the first known example of bidirectional CI in Aedes mosquitoes. We discuss the results in relation to applied mosquito control strategies and the evolution of Wolbachia infections in Ae. albopictus.