Wolbachia, Spiroplasma, and Rickettsia symbiotic bacteria in aphids (Aphidoidea).

Aphids are a diverse family of crop pests. Aphids formed a complex relationship with intracellular bacteria. Depending on the region of study, the species composition of both aphids and their facultative endosymbionts varies. The aim of the work was to determine the occurrence and genetic diversity of Wolbachia, Spiroplasma and Rickettsia symbionts in aphids collected in 2018-2019 in Moscow. For these purposes, 578 aphids from 32 collection sites were tested by PCR using specific primers. At least 21 species of aphids from 14 genera and four families were identified by barcoding method, of which 11 species were infected with endosymbionts. Rickettsia was found in six species, Wolbachia in two species, Spiroplasma in one species. The presence of Rickettsia in Impatientinum asiaticum, Myzus cerasi, Hyalopterus pruni, Eucallipterus tiliae, Chaitophorus tremulae and Wolbachia in Aphis pomi and C. tremulae has been described for the first time. A double infection with Rickettsia and Spiroplasma was detected in a half of pea aphid (Acyrthosiphon pisum) individuals. For the first time was found that six species of aphids are infected with Rickettsia that are genetically different from previously known. It was first discovered that A. pomi is infected with two Wolbachia strains, one of which belongs to supergroup B and is genetically close to Wolbachia from C. tremulae. The second Wolbachia strain from A. pomi belongs to the supergroup M, recently described in aphid species. Spiroplasma, which we observed in A. pisum, is genetically close to male killing Spiroplasma from aphids, ladybirds and moths. Both maternal inheritance and horizontal transmission are the pathways for the distribution of facultative endosymbiotic bacteria in aphids.

Genetic characterization of Plasmodium vivax in the Kyrgyz Republic.

At the end of 2016, Kyrgyz Republic was certified by the World Health Organization as a malaria-free country, while only a decade ago this disease posed a serious health threat. The progress achieved by Kyrgyz Republic provides a unique example of tertian (Plasmodium vivax) malaria elimination. This success was based on an integrated approach, including measures for the treatment of infected people and disease prevention, vector control and the development of an effective national epidemiological surveillance system. Lower P. vivax msp-1, msp-3α, csp and dbpII genes polymorphism was revealed in Kyrgyz Republic in compare with that in Tajikistan. Molecular characterization of the causative agent found that P. vivax populations in Kyrgyz Republic was comprised by several lineages, highly divergent in the south-western and genetically homogeneous in the northern regions of Kyrgyz Republic, d. Such profile in the northern regions was compatible with several recent introductions rather than a long-term endemic circulation of the parasite. A low level of genetic variability suggested that the parasitic systems of tertian malaria, were not adapted, which, along with other factors, largely determined the possibility of malaria elimination in northern Kyrgyz Republic. Other determinants included environmental, social, and epidemiological factors that limited the spread of malaria. South-western Kyrgyz Republic, a region with a high level of interstate migration, requires considerable attention to prevent the spread of malaria.

[(THE FAUNA OF BLOODSUCKING MOSQUITOES IN THE NIZHNY NOVGOROD REGION, THEIR INFECTION WITH DIROFILARIA AND ENDOSYMBIOTIC BACTERIA)].

The fauna of bloodsucking mosquitoes in the Nizhny Novgorod Region is represented by 11 species from 5 genera of the family Culicidae. During 2014-2015, the predominant species were Ochlerotatus cantans and Aedes cinereus mosqui- toes in both a population aggregate and woodland. The infected mosquitoes accounted for 1.3% of their total number and were registered only in the village of Fokino. The investigators identified two human pathogenic nematode species: Diro- filaria immits and Dirofilaria repens (0.9% and 0.4% respectively). The effective carriers of Dirofilaria in the examined area can be Ae.cinereus and Och.cantans as only these species were found to have an invasive stage of the parasite. The symbiotic bacterium Wolbachia was detected in the mosquitoes that were not infected with dirofilariasis. This is the first study in Russia to investigate the effects of Wolbachia on the susceptibility of dirofilariasis vectors to infection.

[The spatial distribution and infection of Culex pipiens L. mosquitoes with Western Nile virus in the Volgograd region].

The infection of Culex pipiens f. pipiens and Cx. pipiens f. molestus mosquitoes with Western Nile virus was studied. Their taxonomic status was determined from the type of mitochondrial DNA. Among 424 females individually examined, there were 4 infected specimens: 3 pipens females gathered in the urban and suburban open biotopes and one molestus female caught in the open urban biotope. Molestus mitotype females were first detected in the suburban open biotopes. The possible reasons for the wide colonization of molestus mosquitoes and its epidemic consequences are discussed.

[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.

[Comparative characteristics of intergenic spacers of ribosomal RNA gene cluster in mosquitoes of the genus Culex (Diptera: Culicidae)].

Nucleotide sequences of intergenic spacer of ribosomal RNA gene cluster (rIGS) were identified in mosquitoes Culex modestus, Culex torrentium and Culex pipiens pallens. The level of interpopulation variability of the rIGS in the subspecies C. pipiens pipiens (form pipiens--mosquitoes that inhabit the open waters, and form molestus--mosquitoes that inhabit basements) living in Russia was estimated. No extensive repetitive sequences characteristic of the rIGS of all previously described species of mosquitoes were found within the rIGS of Culex mosquitoes. At the same time, evolutionarily conserved motifs and relatively short degenerate sequences of different classes of transposable elements, as well as multiple blocks of variable microsatellite repeats were identified. Our data demonstrated that the rIGS of Culex mosquitoes can be considered as a promising molecular marker for the analysis of population and phylogenetic relationships within this group of insects.

[Role of the mosquitoes Culex pipienis f. pipiens and Cx. pipiens f. molestus (Diptera, Culicidae) in the spread of West Nile virus in the south of Russia].

Cx. pipiens is one of the major vectors of West Nile virus in the south of Russia and it is represented by the autogenic form, molestus, and the non-autogenic form, pipiens. The spatial distribution of its larvae and food preferences of females was investigated to assess the potential role of each form in the spread of the virus. The taxonomic status of the mosquitoes was determined from their capacity for autogenicity (543 specimens) and the type of mitochondrial DNA (348 specimens). The mosquitoes of the pipiens form were shown to develop in the open urban and rural reservoirs and the females were non-autogenic. Cx. pipiens form molestus was found only in the urban biotopes, mainly in the flooded basements and, in some years, also in the open reservoirs where it formed a mixed population with Cx. pipiens form pipiens. Both autogenic (85-95.2%) and non-autogenic specimens were identified among the females with the mitotype in form molestus. Genetic analysis of Cx. pipiens females collected under the Berezantsev bell and in the trap with a bird showed that both forms were attracted to man and the bird. The findings suggest that Cx. pipiens form pipiens can transmit West Nile virus to humans both in the town and in its suburb and Cx. pipiens form molestus can only in urban areas.

[Polymorphism of mitochondrial DNA and distribution of cytoplasmic symbionts in the populations of two-spot ladybird beetle Adalia bipunctata].

In geographically distant populations of ladybird beetle Adalia bipunctata from Eurasia mitotypes and infection with symbiotic bacteria Spiroplasma and Rickettsia were determined. All populations examined demonstrated mtDNA polymorphism and striking differences in prevalence of bacteria (from about 50% of individuals infected with Spiroplasma in St.-Petersburg population and 50% of the Rickettsia prevalence in Kem' population to complete absence of bacteria in the population from Archangelsk). In the populations studied a total of 14 mitotypes were discovered, including two mitotypes that were remarkably different from the others in nucleotide composition. Mitotype 10, which was the most different from all the others, was found in all populations from Germany to Transbaikalia, excluding the population from Tashkent. Linkage disequilibrium between mitotype 10 and the Rickettsia infection was confirmed. Infection with the Spiroplasma bacteria was typical of the individuals with haplotype 1 and relative to it. The results obtained supported the conclusion on the association between infection with Spiroplasma and Rickettsia and certain mitotype of A. bipunctata, which was the consequence of either absence or rare horizontal transfer of symbionts and ancientness of the first contact between the bacteria and A. bipunctata ladybird beetles.

[Study of the pathogen populations of tertian malaria and possibilities of its establishment in Kyrgyzstan].

A genetic analysis has indicated that the population of the tertian malaria causative agents Plasmodium vivax in Kyrgyzstan is heterogeneous and consists of various isolates resulting from multiple carriages of the pathogens from the areas of the neighboring countries. Low genetic variability and a weak gene flow between the isolates do not contribute to the establishment of malaria in this area. Experience in controlling the epidemic outbreaks shows that active and residual foci are easily sanitized when antimalaria measures are timely and qualitatively implemented. This creates the necessary for eliminating tertian malaria in the Republic of Kyrgyzstan in the near future.

[Molecular genetic analysis of malaria mosquitoes of the Anopheles maculipennis (Diptera, Culicidae) complex in Azerbaijan].

Molecular genetic analysis of malaria vectors in the Republic of Azerbaijan has identified three species of malaria mosquitoes of the Anopheles maculipennis complex: An. maculipennis, An. sacharovi, and An. persiensis. An. melanoon has not been found. An. sacharovi has been ascertained to predominate in the low-lying areas of the country. An. maculipennis prevails in the north, on the foothills of the Great Caucasus and it is also observed in the south, on the Talysh foothills and mountains. An. persiensis has been first recorded for the malaria mosquito fauna in the CNS. This species has been detected only in the south of the republic (Lenkoran and Astar districts).

[Identification of Culex mosquitoes (Diptera, Culicidae) by the restriction assay of amplification products].

Mosquitoes of the Culex genus are active bloodsuckers and vectors of many dangerous human and animal diseases. Special interest has been shown in the Culex pipiens complex in recent years. The complex comprises closely related mosquito species. The specific feature of the complex is that there are significant ecophysiological differences between morphologically similar species and subspecies. Taxonomy of the complex by classical morphological methods is hardly feasible and therefore molecular-genetic markers are being actively searched for to identify the members of the Culex pipiens complex. The proposed polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay based on the analysis of the nucleotide composition of the 5'-end of the mitochondrial DNA cytochrome oxidase I gene permits differences in the nucleotide sequences of this region to be found and serves as a rapid way of differentiating Cx. pipiens (f. pipiens and f. molestus), Cx. quinquefasciatus, Cx. torrentium, as well as Cx. modestus.

[Comparison of Chironomus usenicus and Chironomus curabilis with species of the group plumosus (Diptera) inferred from the mitochondrial DNA gene COI and by the polytene chromosomes banding pattern].

The sequence of a 595-bp fragment of the mitochondrial COI gene was determined for the species Chironomus usenicus and Chironomus curabilis of the genus Chironomus. Phylogenetic reconstructions based on the analysis of the COI gene sequence coincide on the whole with cytogenetic data, permitting Ch. usenicus and Ch. curabilis to be regarded as members of the group plumosus. Chironomus usenicus and Ch. plumosus have identical COI gene sequences. Two hypotheses explaining this identity are considered: inheritance of mtDNA from one of the parental species during hybridogenesis and horizontal transfer of mitochondrial genes.

[Anopheles mosquitoes (Diptera, Culicidae) of the Tien Shan: morphological, cytogenetic, and molecular genetic analysis].

Morphological, cytogenetic, and molecular genetic studies of the Anopheles fauna in the valley and foothills of the Tien Shan identified 5 species of malaria mosquitoes: An. artemievi Gordeev et al., An. messeae Fall, An. claviger Meigen, An. hyrcanus Pallas, An. pulcherrimus Theobald, and superpictus Grassi. An. claviger, An. hyrcanus, and An. messeae were prevalent in the Northern Tien-Shan. An. artemievi, An. claviger, An. hyrcanus, An. messeae, and An. superpictus were detected in the Western Tien Shan. An. artemievi was first recorded in Kazakhstan. An. artemievi, An. claviger, and An. superpictus were noted in the Inferior Tien Shan. An. messeae was first observed in the Issyk Kul hollow. An. artemievi, An. claviger, and An. superpictus were habitants of the foothills of the South-Western Tien Shan. An. artemievi, An. hyrcanus, An. superpictus, and An. pulcherrimus were in the plain. An. pulcherrimus and An. superpicts mosquitoes are regarded as important vectors in the new malaria foci of the Fergana regions. The role of An. artemievi in the transmission of malaria is to be specified.

[Removing endosymbiotic Wolbachia specifically decreases lifespan of females and competitiveness in a laboratory strain of Drosophila melanogaster].

To understand specific symbiotic relationships ensuring stable existing of the bacterium Wolbachia in laboratory strains of Drosophila melanogaster, the imago lifespan and senescence rate, as well as competitiveness, have been evaluated as components of fitness in females from the following laboratory strains: (1) inbred strain 95 infected with Wolbachia; (2) two uninfected strains obtained by tetracycline treatment that were genetically similar to strain 95; and (3) two control, uninfected, wild-type laboratory strains that were used to assess the possible effects of the antibiotic on the studied characters in the absence of Wolbachia. The results have shown that infected females have longer lifespan and competitiveness than females with the same genotype uninfected with Wolbachia. The increase in the senescence and mortality rates with age was also slower in infected females. It is noteworthy that tetracycline does not affect the lifespan of females from the two control, uninfected, wild-type strains. Therefore, the antibiotic is not the cause of the positive changes in fitness that were observed in infected females. The obtained results are the first direct evidence that the relationship s in the Wolbachia-D melanogaster symbiotic system are mutualistic rather than parasitic, at least in micropopulations adapted to laboratory conditions.

[The malaria mosquitoes (Diptera, Culicidae, Anopheles) of the Amudarya river valley].

Five malaria mosquitoes were identified in the Amudarya river valley. An. pulcherrimus, An. martinius, An. superpictus, and An. hyrcanus inhabit in the plain of the valley. An. superpictus, An. pulcherrimus, An. hyrcanus, and An. claviger occur in the head of the Amudarya river. The nucleotide composition of the second inner transcribed spacer (ITS2) of ribosomal DNA and the locus of cytochromoxidase I (COI) of mitochondrial DNA was determined in the closely related species An. martinius and An. artemievi. It is shown that the loci in question may be used to diagnose twin species of the maculipennis complex.

[Description of the new species Anopheles artemievi sp.n. (Diptera, Culicidae)].

The paper describes the new species Anopheles artemievi belonging to a complex of the species maculipennis that may play an important role in the transmission of malaria in both the Fergana area and the Fergana valley. In terms of the morphological signs of imagoes, larvae, and the composition of the egg exochorion, the new species is the twin of An. sacharovi Favre and An. mihrtinius Shingarev. According to the pattern of polytene chromosomal disks, the described type is identical to An. maculipennis Meigen. The structure of the second internal transcribed spacer (ITS2) of a cluster of ribosomal genes in An. artemievi is species-specific and, in its nucleotide composition, differs from that in related species. The sequence of ITS2 in the new species has been registered at the GeneBank under No. AJ849886 and it is 419 p.n. The length of ITS2 in An. maculipennis and A. martinius is equal to 422 p.n. (AY238435) and 447 p.n. (AJ849885), respectively. The sequences of ITS in An. artemievi and An. maculipennis are 91% homologous; those in An. artemievi and An. martinius are 87% homologous. The nucleotide composition of ITS2 in An. artemievi differs from that in An. maculipennis in three deletions, 1 insertion, and 18 point substitutions and from that in An. martinius in 12 insertions, 2 deletions, and 40 point substitutions. The standard-type series is as follows: Kyrghyzstan, Batkensk Region, Leileksky district, Alga settlement, a cowshed, 1 male-holotype, l female-allotype. The paratypes are 2 males, 6 females.

[Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium Wolbachia pipientis in mosquitoes of the Culex pipiens 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 mosquito ecotype 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.

[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.

[Molecular genetic methods for the identification of the urban mosquito Culex pipiens pipiens F. molestus (Diptera, Culicidae)]. / Molekuliarno-geneticheskie metody identifikatsii gorodskogo komara 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.

[Supplement to the list of Anopheles (Diptera, Culicidae) mosquitoes of Tadjikistan and the predominant types of vectors in the current foci of malaria in the republic]. / Dopolnenie k spisku komarov roda Anopheles (Diptera, Culicidae) Tadzhikistana i dominiruiushchie vidy perenoschikov v sovremennykh ochagakh malarii v respublike.

The species composition of and the indices of predominance of Anopheles mosquitoes were determined in the current foci of malaria in the Republic of Tadjikistan. The predominant types of malaria mosquitoes were An. superpictus, An. pulcherrimus, and An. hypcanus were found to be in the Khatlon Region of Tadjikistan and in the contiguous areas of Uzbekistan. An. superpictus prevailed in the study foci of malaria and, as An. pulcherrimus, were the most dangerous vectors of malaria in the south of the republic. The main vectors of malaria were shown to be An. maculipennis sensu stricto and An. superpictus in the environs of the town of Khudjand, Sogdi Region (Tadjikistan). An. maculipennis was first recorded for Tadjikistan and should be included into the list of malaria mosquitoes of the republic. It is suggested that An. pulcherrimus plays an important role in the spread of malaria in the north of Tadjikistan.