Genetic divergence of human pathogens Nanophyetus spp. (Trematoda: Troglotrematidae) on the opposite sides of the Pacific Rim.

Human and animal nanophyetiasis is caused by intestinal flukes belonging to the genus Nanophyetus distributed on both North American and Eurasian coasts of Northern Pacific. In spite of the wide geographical distribution and medical and veterinary importance of these flukes, the intra-generic taxonomy of Nanophyetus spp. remains unresolved. The two most widely distributed nominal species, Nanophyetus salmincola and Nanophyetus schikhobalowi, both parasitizing humans and carnivorous mammals, were described from North America and eastern Eurasia, respectively. However, due to their high morphological similarity their interrelationships remained unclear and taxonomic status unstable. In this study, we explored genetic diversity of Nanophyetus spp. from the Southern Russian Far East in comparison with that of samples from North America based on the sequence variation of the nuclear ribosomal gene family (18S, internal transcribed spacers, ITS1-5·8S-ITS2 and 28S). High levels of genetic divergence in each rDNA region (nucleotide substitutions, indels, alterations in the secondary structures of the ITS1 and ITS2 transcripts) as well as results of phylogenetic analysis provided strong support for the status of N. salmincola and N. schikhobalowi as independent species.

[Genomics and transcriptomics of the Chinese liver fluke Clonorchis sinensis (Opisthorchiidae, Trematoda)].

The review summarizes the results of first genomic and transcriptomic investigations of the liver fluke Clonorchis sinensis (Opisthorchiidae, Trematoda). The studies mark the dawn of the genomic era for opisthorchiids, which cause severe hepatobiliary diseases in humans and animals. Their results aided in understanding the molecular mechanisms of adaptation to parasitism, parasite survival in mammalian biliary tracts, and genome dynamics in the individual development and the development of parasite-host relationships. Special attention is paid to the achievements in studying the codon usage bias and the roles of mobile genetic elements (MGEs) and small interfering RNAs (siRNAs). Interspecific comparisons at the genomic and transcriptomic levels revealed molecular differences, which may contribute to understanding the specialized niches and physiological needs of the respective species. The studies in C. sinensis provide a basis for further basic and applied research in liver flukes and, in particular, the development of efficient means to prevent, diagnose, and treat clonorchiasis.

[(CLONORCHIASIS: GLOBAL AND REGIONAL EPIDEMIOLOGY)].

All over the world, much attention is given to the comprehensive studies of parasites and their induced diseases in order to devise effective methods for the diagnosis, treatment, and prevention of parasitic diseases. This review summarizes the information available in the literature on the epidemiology of clonorchiasis caused by the Chinese liver fluke Clonorchis sinensis, including global and regional risk factors for the infection and its transmission. The existing knowledge of this important, but far from the most studied zoonosis, the cause of severe human hepatobiliary diseases, is required for the success of future investigations of parasitic infections.

[Current treatments for clonorchiasis].

The paper briefly reviews the current techniques for treatment for human clonorchiasis, which are both widely used in medical practice and developed at experimental research laboratories. It describes specific examples of chemotherapy, including combined therapy, clonorchiasis vaccines and drug resistance. Particular emphasis is placed on the prospects of use of minor interfering RNA as a source of new-generation diagnostic and remedial agents.

[Current methods for the diagnosis of human and animal clonorchiasis].

The paper briefly reviews the current techniques for the diagnosis of human and animal clonorchiasis, which involve visual, immunological, and polymerase chain reaction-based methods, including those extensively used in medical practice and devised in experimental research laboratories.

[Genetic diversity and phylogenetic relationships of the Manchurian zokor Myospalax psilurus (Rodentia, Muridae) according to RAPD-PCR analysis].

The population structuring and low genetic diversity of the Manchurian zokor Myospalax psilurus Milne-Edwards, 1874, an East Asian endemic included in the Red List of Russia, were demonstrated. Two separate geographical groups differing in the level of their genetic diversity were found on the territory of the Primorskii krai. The subpopulation located closest to the main area of this species was determined as ancestral. A subspecies differentiation of the Primorskii krai and Transbaikal M. psilurus populations was shown, as was the monophyletic origin of M. psilurus and its high divergence from M. aspalax. The animals from northern localities are recommended for reintroduction in nature under species recovery programs in Primorskii krai.

[Molecular genetic evidences of a deep phylogenetic discontinuity between Asian and European races of pygmy wood mouse based on the data of mtDNA cytochrome b gene variability].

The variability of mitochondrial DNA (mtDNA) cytochrome b gene sequences of pygmy wood mouse Sylvaemus uralensis (Pallas, 1811) from local populations of European regions of Russia, West Siberia, and neighboring countries (Moldova, Kazakhstan, Uzbekistan, and Turkmenistan) have been studied. Phylogenetic analysis both our results and data from GenBank revealed two clusters of haplotypes: "western" with reliable subdivision into two sequence groups and "eastern" without valid differentiation. Clusters correspond exactly to European and Asian races of pygmy wood mouse recognized earlier on the basis of biochemical and karyological variability. We suppose that Asian race can be considered as independent allopatric species. This is supported by following evidences: high divergence level (which proposes more than 1 mya of divergent evolution) between races, absence of common haplotypes and hiatus between main peaks of mismatch distribution, differences in frequencies of codon usage, fixed nucleotide substitutions in cyt b gene, and also changes in amino acid sequences of cytochrome b. Only specimens of the western phylogenetic linage can be considered as S. uralensis (Pallas, 1811) while according to the first description S. tokmak (Severtzov, 1873) may be considered as species united specimens of eastern phylogenetic linage.

The SSU rRNA secondary structures of the Plagiorchiida species (Digenea), its applications in systematics and evolutionary inferences.

The small subunit ribosomal RNA (SSU rRNA) is widely used phylogenetic marker in broad groups of organisms and its secondary structure increasingly attracts the attention of researchers as supplementary tool in sequence alignment and advanced phylogenetic studies. Its comparative analysis provides a great contribution to evolutionary biology, allowing find out how the SSU rRNA secondary structure originated, developed and evolved. Herein, we provide the first data on the putative SSU rRNA secondary structures of the Plagiorchiida species. The structures were found to be quite conserved across broad range of species studied, well compatible with those of others eukaryotic SSU rRNA and possessed some peculiarities: cross-shaped structure of the ES6b, additional shortened ES6c2 helix, and elongated ES6a helix and h39 + ES9 region. The secondary structures of variable regions ES3 and ES7 appeared to be tissue-specific while ES6 and ES9 were specific at a family level allowing considering them as promising markers for digenean systematics. Their uniqueness more depends on the length than on the nucleotide diversity of primary sequences which evolutionary rates well differ. The findings have important implications for understanding rRNA evolution, developing molecular taxonomy and systematics of Plagiorchiida as well as for constructing new anthelmintic drugs.

[Phylogenetic relationships of Amur sturgeon Acipenser schrenckii Brandt, 1869 based on 18S rDNA sequensing data].

The analysis of phylogenetic relationships based on 18S rDNA sequences of Amur sturgeon Acipenser schrenckii Brandt, 1869 with other acipenseriform species was performed in this study. Complete sequences (1746 b. p.) in seven individual clones of A. schrenckii 18S rRNA were determined. Mutation profile of Amur sturgeon 18S rDNA demonstrated high similarity with that of Lake Sturgeon A. fulvescens. Both presumably functional sequence and the specific mutation (insertion of adenine after position 658) of Amur sturgeon 18S rDNA were identified by structural-functional analyses. Phylogenetic reconstructions performed using different methods (NJ, MP, ML and Bayesian) support monophyly of the genus Acipenser and point to: 1) closer relationships Amur sturgeon with sterlet, than Baltic sturgeon, which is in agreement with Artyukhin's eco-morphological classification (Artyukhin, 1995, 2006); 2) sufficiently high differentiation between North-American (A. fulvescens) and Eurasian (A. schrenckii, A. ruthenus and A. sturio) sturgeons.

[Analysis of genetic variation and differentiation in the pygmy wood mouse Sylvaemus uralensis (Rodentia, Muridae) aided by RAPD-PCR method].

The present work ascertains distinct differentiation of the pygmy wood mouse into two groups of populations, which correspond to the European and Asian races, but can not, however, be considered valid species due to the low values of genetic distances between them. Neither of the used statistical methods explicitly verifies genetic isolation of the East-European and South-European chromosomal forms, which are close to one another and together constitute the European race. However, polymorphism of these chromosomal forms/races of the pygmy wood mouse differs each of them from the others, that can be explained both by the history of their formation (the degree of proximity to the ancestral population complex, isolation, etc.) and by unequal current environmental conditions.

[Molecular identification and the features of genetic diversity in interspecific hybrids of Amur sturgeon (Acipenser schrenckii x A. baerii, A. baerii x A. schrenckii, A. schrenckii x A. ruthenus, and A. ruthenus x A. schrenckii) based on variability of multilocus RAPD markers].

The method of polymerase chain reaction with random primers (RAPD PCR) was used to identify the progeny of the crosses between three sturgeon species, Amur sturgeon (Acipenser schrenckii Brandt, 1869), Siberian sturgeon (A. baerii Brandt, 1869), and sterlet (A. ruthenus Linnaeus, 1758). Using ten primers, genetic variation in 70 yearlings, produced in seven individual crosses: Acipenser schrenckii x A. schrenckii, A. baerii x A. baerii, A. ruthenus x A. ruthenus, A. schrenckii x A. baerii, A. baerii x A. schrenckii, A. schrenckii x A. ruthenus, and A. ruthenus x A. schrenckii was described and evaluated. It was demonstrated that the samples composed of hybrids from individual crosses were more variable than the samples of parental species. On the other hand, pooled samples of hybrids from two cross directions were genetically less variable than the pooled samples of their parents. The three main features of the hybrid RAPD profiles identified included: (1) preservation of marker DNA fragments of both parents in one genome; (2) presence of specific DNA fragments, absent from both parents; and (3) dependence of the frequency of some DNA fragments from the cross direction. Multidimensional scaling clearly distinguishes in the space of three coordinates the individuals of original species and the hybrid progeny with differentiation in the groups of direct and backcross hybrids. Analysis of relationships (UPGMA and NJ) pointed to substantial differentiation between the species, as well as between the species and hybrid progeny. Close genetic relationships between direct and backcross hybrids were demonstrated. Multilocus RAPD markers in association with statistical methods are considered to be the useful tool for discrimination of interspecific hybrids of sturgeon. Possible reasons for the differences in the hybrid RAPD profiles are discussed.

[Discrimination of interspecific hybrids in natural populations of Amur sturgeon fishes using multilocus RAPD-PCR markers].

RAPD-PCR analysis of 46 individuals of sturgeons from Amur River has been carried out. Genetic status of Amur sturgeon Acipenser schrenckii Brandt, 1869 and kaluga Huso dauricus Georgi, 1775 native populations has been estimated. Genetic evidences of hybrid origin for two phenotypical hybrids were obtained; estimations of genetic distances between species and hybrids appeared to be at interspecific level. The exact test for differentiation of populations (Exact test) and multidimensional scaling (MDS) analysis were estimated to be the most effective for species and hybrid discrimination, respectively. According to data obtained populations of sturgeon fishes which inhabit Amur River maintained an essential level of genetic variability; the presence of hybrids is regarded as one of risk factors. Multilocus RAPD-PCR markers admit as the convenient and reliable tool for genetic monitoring of Amur River sturgeons to preserve their gene pool.

[Genetic variation and differentiation in striped field mouse Apodemus agrarius inferred from RAPD-PCR analysis].

Genetic variation and differentiation of the trans-Palearctic species Apodemus agrarius (striped field mouse), whose range consists of two large isolates-European-Siberian and Far Eastern-Chinese, were examined using RAPD-PCR analysis. The material from the both parts of the range was examined (41 individual of A. agrarius from 18 localities of Russia, Ukraine, Moldova, and Kazakhstan); the Far-Eastern part was represented by samples from the Amur region, Khabarovsk krai, and Primorye (Russia). Differences in frequencies of polymorphic RAPD loci were found between the European-Siberian and the Far Eastern population groups of striped field mouse. No "fixed" differences between them in RAPD spectra were found, and none of the used statistical methods permitted to distinguish with absolute certainty animals from the two range parts. Thus, genetic isolation of the European-Siberian and the Far Eastern population groups of A. agrarius is not strict. These results support the hypothesis on recent dispersal of striped field mouse from East to West Palearctics (during the Holocene climatic optimum, 7000 to 4500 years ago) and subsequent disjunction of the species range (not earlier than 4000-4500 years ago). The Far Eastern population group is more polymorphic than the European-Siberian one, while genetic heterogeneity is more uniformly distributed within it. This is probably explained by both historical events that happened during the species dispersal in the past, and different environmental conditions for the species in different parts of its range. The Far Eastern population group inhabits the area close to the distribution center of A. agrarius. It is likely that this group preserved genetic variation of the formerly integral ancestral form, while some amount of genetic polymorphism could be lost during the species colonization of the Siberian and European areas. To date, the settlement density and population number in general are higher than within the European-Siberian isolate, which seems to account for closer interpopulation associations, intense genetic exchange, and "smoothing" of polymorphism within the Far Eastern population group of A. agrarius.

[Ancient DNA].

The review is devoted to molecular genetic studies of ancient DNA. The problems of DNA preservation and modification after cell death, as well as techniques of working with ancient DNA, including its retrieval, removal of inhibitors, PCR amplification, and phylogenetic analysis, are discussed in detail. The possibilities are considered of using ancient DNA in resolving issues of systematics and evolution of various animal taxa, population genetics of humans and rare species, taxonomic identification and paleontological reconstructions, geographic origin of populations, microbiological analysis of paleontological and archeological finds, as well as some humanitarian aspects of its use.

[Genetic evidence of hybridization between paletailed Spermophilus pallidicauda Satunin, 1903 and alashanic S. alaschanicus Buchner, 1888 ground squirrels in Mongolia].

Analysis of Spermophilus pallidicauda Satunin, 1903 from three localities in Mongolia using cytogenetic characteristics (chromosome number and morphology, as well as the number and localization of nucleolus organizer regions) revealed the presence of a first-generation (F1) hybrid animal (2n = 36), of the paletailed S. pallidicauda (2n = 34) and alashanic S. alaschanicus (2n = 38) ground squirrels in the contact zone of their ranges. Analysis of nuclear DNA from ten ground squirrels (from a set of karyologically examined animals) by RAPD-PCR with eight oligonucleotide primers (OPA10, OPA12, OPC02, OPC5, OPC08, OPC09, OPC12, and OPD05) revealed four hybrids in two sites. with one of the hybrid being F1. The position of the hybrids in phylogenetic reconstructions made for the subgenera Citellus and Colobotis species varied depending on the method used. In the UPGMA dendrogram of genetic similarity hybrids formed their own subcluster with high bootstrap index (949) within the cluster of Citellus species. In the NJ phylogenetic tree, hybrids also clustered with high boot-strap index (886). But in this case they were located between the Colobotis and Citellus species clusters. The mtDNA haplotypes of the three hybrids examined were highly similar to the Colobotis ground squirrels, albeit in phylogenetic reconstructions they were placed between Colobotis and Citellus. The sites of the hybrid animals identification were located more than 200 km apart. Hence, the contact zone between the S. pallidicauda and S. alaschanicus can encompass a large territory in Mongolia.

[Genetic evidence of extensive introgression of short-tailed ground squirrel genes in a hybridization zone of Spermophilus major and S. erythrogenys, inferred from sequencing of the mtDNA cytochrome b gene].

We have completely sequenced the mtDNA cytochrome b gene of ground squirrels from the zone of overlapping ranges of Spermophilus major and S. erythrogenys in the Tobol-Ishim interfluve, which is a putative hybridization zone of these species. The results of the sequencing showed extensive introgression of mtDNA genes of the short-tailed ground squirrel S. e. brevicauda, whose haplotype had fully replaced the S. major haplotype. All of the ground squirrels from the Tobol-Ishim interfluve had a variant of the S. e. brevicauda mtDNA haplotype that was specific for this zone. On average, 119 substitutions (10.44%) were found between S. major from Ul'yanovsk oblast and S. e. brevicauda from the northern Kazakhstan, the mean genetic distance (D) between them being 0.115, which conforms to the corresponding parameters for the S. e. brevicauda-S. pygmaeus pair (122 substitutions, D = 118). Insignificant differences (seven substitutions, D = 0.043) were found between the S. major and S. pygmaeus haplotypes, which suggest that these species have similar mitochondrial haplotypes. Five to ten nucleotide substitutions (0.44--0.88%) were detected between the animals from the Tobol--Ishim interfluve and S. e. brevicauda. The mtDNA haplotype divergence D within the genus Spermophilus (ten species) for all codon positions ranged from 0.035 to 0.158. Phylogenetic reconstructions (MP, ML, and NJ trees) showed two well-differentiated clusters with high bootstrap support. However, there was different branching topology within the cluster and their species composition varied. The maximum likelihood tree, ML, differentiating the species into two subgenera, Citellus and Colobotis, most reliably reflected taxonomic relationships of the species from the genus Spermophilus, inferred from morphological and genetic biochemical data. The morphologically pure S. major (subgenus Colobotis) animals, used in the analysis, proved to carry the haplotype of another species, S. pygmaeus (subgenus Citellus). This poses a question on the existence of the specific haplotype of S. major, the reason of its replacement by haplotype of other species, and possible consequences of this phenomenon for survival of the species.

[RAPD-PCR analysis of ground squirrels from the Tobol-Ishim interfluve: evidence for interspecific hybridization between ground squirrel species Spermophilus major and S. erythrogenys].

Populations of two ground squirrel species, Spermophilus major and S. erythrogenys, from the interfluvial area of the Tobol and Ishim rivers, where their ranges overlap, have been examined using RAPD-PCR. We have identified 253 loci, which included taxon-specific markers for S. major and S. erythrogenys as well as markers for geographic populations. Estimation of genetic diversity and construction of phylogenetic relationships were performed using software programs POPGENE, TEPGA, and TREECON. In all, based on morphological traits, animals from the Tobol-Ishim interfluve were assigned to the two parental morphotypes and showed similar levels of genetic variability (H, n(a), n(e)). However, the total polymorphism level proved to be higher in ground squirrels with the major morphotype (P = 40.32%, P95 = 27.27%) than in animals with the erythrogenys morphotype (P = 32%, P95 = 22.13%). Nevertheless, the number of rare alleles was high in both cases, constituting about 70% of the total number. By contrast, interpopulation differentiation was considerably higher in S. major (6 = 0.50) than in S. erythrogenys (delta = 0.41). The genetic differentiation between local samples from the Tobol-Ishim interfluvial area was lower than that between the parental species. A significant part of the genetic diversity of the species examined and animals from the zone of overlapping ranges was accounted for by intrapopulation variability. Animals from the northern and southern parts of the Tobol-Ishim interfluve were characterized by the core traits of S. major and S. erythrogenys, respectively, falling into two distinct clusters in the UPGMA and NJ reconstructions. In addition to three hybrid individuals, identified by the bioacoustic method, three hybrid animals were distinguished using RAPD analysis. These animals earlier were thought to be "pure" species and formed their own clusters in phylogenetic reconstructions. Thus, the RAPD-PCR results directly showed the existence of stable hybridization (20% genetic hybrids) between S. major and S. erythrogenys in the Tobol-Ishim interfluvial area, which is more extensive than inferred previously from morphological and bioacoustic data.

Divergence of two families of repeated DNA in wood and field mice of the genus Apodemus (Muridae, Rodentia). / Divergentsiia dvukh semeistv povtoriaiushcheisia DNK lesnykh i polevykh myshei roda APodemus (Muridae, Rodentia).

The nDNA of six species of a wood and field mice of the genus Apodemus (2n = 48): peninsulae, sylvaticus, flavicollis, microps, speciosus, agrarius, grey rat Rattus norvegicus as well, and house mouse Mus musculus were studied using EcoRI and HindIII restriction endonucleases with an agarose and polyacrylamide gel electrophoresis and a microdensitometry. It is shown for dispersed sequences that a quarter of 1.85 kb EcoRI genus specific family of repeated DNA is constant but a quarter of 1.35 kb EcoRI repeats, which are specific for more large taxon unit, is variable. The HindIII satDNA of the various species differs in the length of monomer, size of more representative multimer and digestion of the monomers by the same endonuclease of restriction; HindIII satDNA quantity correlates with 1.35 kb EcoRI repeat content. Some nonidentified components are found in Apodemus genomes. The possible ways evaluation of the repetitive DNA are briefly discussed. It was supposed that the evolutionary age of the wood mice is directly proportional to the length of HindIII multimer and inversely proportional to the amount of HIndIII satDNA with a 1.35 kb EcoRI repeats.

[Differentiation of GC-rich restriction sites in the highly repeated DNA in Apodemus (Muridae, Rodentia]. / Differentsiatsiia GTs-bogatykh saitov restriktsii v chastopovtoriaiushcheisia DNK Apodemus (Muridae, Rodentia).

nDNAs of six species of wood and field mice of the genus Apodemus (peninsulae, flavicolis, microps, speciosus, and agrarius), grey rat Rattus norvegicus as well, and house mouse Mus musculus were digested with restriction endonucleases (MspI (CCGG), BspRI (GGCC) and MvaI (CCA/TGG). Two highly representative satellites are found in A.peninsulae (8%) and M.musculus (20%) genomes using BspRI and MvaI. The length of restriction fragments of these components are equal to n x 30 and n x 230 nb, respectively. Based on the results of PDRF of nDNA in rodent interspecies, genetic distances are estimated and phylogenetic tree reconstructed. Among wood mice examined A.sylvaticus and A.flavicollos are most closely related, A.peninsulae is more diverged. Field mouse has approx. equal differences from the european and asian species of wood mice. A middle value of genetic distances is about 7.12% in Apodemus genus, 12.69 and 13.2% between Apodemus with M.musculus and R.norvegicus, respectively.

[DNA restriction polymorphism in European and Asiatic species of wood mice of the genus Apodemus]. / Osobennosti restriktsionnogo polimorfizma DNK evropeiskikh i aziatskikh vidov lesnykh myshei roda Apodemus.

Comparative restriction analysis of genomic DNA from six wood mice species of the genus Apodemus as well as from the house mouse Mus musculus and a Norway rat Rattus norvegicus was conducted by means of AluI, TagI, Csp6I, and BglII endonucleases. The data from RFLP and UPG analyses allowed division of all the forms studied into two groups, evolutionarily young and old, including European (sylvaticus, flavicollis, and microps) and Asiatic (peninsulae, speciosis, and agrarius, together with the striped field mouse) wood mice species, respectively. The first group is characterized by low interspecies and intragenomic RFLPs of the repeated DNA sequences, while these indices were higher in the second group. The interspecies genetic distances averaged around 1.2% within the first group, around 5.88% within the second group, and around 9.49% between the groups. The divergence of A. agrarius from the Asiatic species of wood mice was 5.33%, twofold lower than divergence from the European species (9.17%).