S. K. Semyenova

GENETIC DIFFERENTIATION IN EASTERN EUROPEAN AND WESTERN ASIAN POPULATIONS OF THE LIVER FLUKE, FASCIOLA HEPATICA, AS REVEALED BY MITOCHONDRIAL NAD1 AND COX1 GENES

Partial sequences of mitochondrial genes nad1 (316 bp) and cox1 (429 bp) were analyzed to estimate the variability of the liver fluke samples collected in 20 localities in Russia, Belarus, Ukraine, Bulgaria, Armenia, Azerbaijan, Georgia, Turkey, Turkmenistan, and China. The sequences had 4.1% (nad1) and 2.3% (cox1) of variable sites, and 13 and 10 haplotypes were identified among nad1 and cox1 genes, respectively. Spatial analysis of genetic and nucleotide diversity indicated little or no structuring of genetic variation between hosts or regions. The analysis of distribution of both separate and combined (nad1 + cox1) haplotypes revealed the existence of 2 well-defined lineages with 2 main haplotypes and a number of shared divergent haplotypes. Our study showed that the first lineage included the main N1–C1 haplotype, which was found in Australia, China, Georgia, Turkey, Armenia, Azerbaijan, and in all European populations (from Russia, Belarus, Ukraine, Bulgaria). The second lineage was found in all European populations and in populations from Armenia and Azerbaijan. It was suggested that one of the lineages (I) has an Asian origin. The possible source of mtDNA variability and associations between lineage divergence of parasite and its definitive hosts (cattle and sheep) are discussed.

Detection of European Trichobilharzia Schistosomes (T. franki, T. szidati, and T. regenti) Based on Novel Genome Sequences

Abstract The most frequent causative agents of cercarial dermatitis in Europe are the avian schistosomes of the genus Trichobilharzia. They preferably parasitize birds of the Anatidae. Trichobilharzia spp. schistosomes are also able to penetrate mammalian skin, posing a health risk to mammals, including humans. Currently several loci from nuclear and mitochondrial genomes are determined for European species of Trichobilharzia. Among them there is 1 genome sequence, ToSau3A, which is suitable for detection of Trichobilharzia spp. infection in aquatic systems. In the present paper, we used a PCR assay to obtain novel genome sequences from cercariae isolates of 3 European bird schistosome species (Trichobilharzia franki, Trichobilharzia szidati, and Trichobilharzia regenti) collected from freshwater ponds in Belorussia and Russia. We applied RAPD-fingerprinting using 1 random primer to differentiate 3 trichobilharzian species and subsequently cloned and sequenced putative species-specific RAPD fragments. One of them (410 bp in length), which was obtained for T. franki, revealed 64% homology with the repeat region of Schistosoma mansoni (GenBank FN357352) and turned out to be suitable for designing a specific primer pair (TR98F and TR98R) to detect 7 novel DNA sequences in the genome of 3 European Trichobilharzia species. The newly designed primer pair was found to be potentially suitable for PCR-based detection of trichobilharzian infection in snails. PCR primers TR98F and TR98R amplified only the DNA isolated from cercariae and sporocysts of 3 trichobilharzian species, but neither the DNA of 3 other digenean species (Bilharziella polonica, Apatemon sp., and Diplostomum sp.) nor the DNA of uninfected host snails (Lymnaea stagnalis, Radix auricularia, and Radix ovata).

Individual and population variation in cercariae of bird schistosomes of the Trichobilharzia ocellata species group as revealed with the polymerase chain reaction

The polymerase chain reaction with arbitrary (RAPD-PCR) or specific primers was used to study the population variation and to identify the species in cercariae of schistosomes of the Trichobilharzia ocellata species group (Trematoda, Schistosomatidae). In total, 28 cercariae were obtained from two spontaneously invaded mollusks Lymnaea stagnalis (LS) and L. ovata (LO), which were collected in different ponds of Moscow. RAPD-PCR was carried out with two arbitrary primers, OPA9 and OPB11, which each detected different levels of individual and among-group variation and revealed considerable genetic differentiation of cercariae from different host mollusks. To check whether the cercariae of the two samples belong to one species, sequencing was performed with a region corresponding to intergenic transcribed spacer 2 (ITS2), which was earlier proposed for cercaria identification in three European species of bird schistosomes of the genus Trichobilharzia (T. franki, T. regenti, and T. szidati). The ITS2 sequences of two LO cercariae were identical, each consisted of 319 bp, and showed 100% homology to the T. franki ITS2 sequence. The ITS2 sequences of two LS cercariae were identical, each consisted of 323 bp, and showed 99.4% homology to the T. szidati counterpart. The causes of genetic variation in cercariae and prospects of using RAPD markers to study different stages of the life cycle in trematodes are discussed.

Polymorphism of the ND1 and CO1 Mitochondrial Genes in Populations of Liver Fluke Fasciola hepatica

Polymorphism of fragments of the ND1 and CO1 mitochondrial genes was for the first time found in four liver fluke Fasciola hepatica samples from Ukraine, Belarus, Moscow region, and Mordovia. The ND1 and CO1fragments were respectively 292 and 433 bp in size, with polymorphic sites amounting to 2.7 and 0.9% of the total sequence. Seven haplotypes were found in the four samples; two haplotypes (A and B) were most common (29.1 and 45.8%, respectively) in the pooled sample. The haplotype frequency distribution differed among the four populations. Haplotype B prevailed in the Mordovian and Moscow region samples. In addition, these samples had a higher number of unique haplotypes (A2, A3, B2). The results testify to genetic differences of the four geographically distant populations of F. hepatica.

Genetic diversity and differentiation of Russian common carp (Cyprinus carpio L.) breeds inferred from RAPD markers

Polymorphic components of the common carp Cyprinus carpio L. genome were examined by means of polymerase chain reaction with random primers (RAPD-PCR). Using four primers, genetic diversity estimates were obtained for 12 populations and seven strains of Russian common carp breeds, as well as for European Hungarian common carp and Amur wild common carp (N = 87). The highest number of polymorphic loci was revealed in Angelinskii common carp, as well as in the samples of Altai common carp and Amur wild common carp (P = 23.8−18.7%), while the lowest number (12.8%) of polymorphic loci was in the BB strain of Ropsha common carp. The index of genetic diversity, H, was high (11%) in Amur wild common carp, as well as in Altai and Angelinskii common carps. In the remaining breeds, the value of this index varied from 4 to 8%. Based on summarized RAPD profile (132 bands), a dendrogram of genetic differences was constructed. In this dendrogram, all breeds examined grouped into two clusters. One of the clusters was formed by Hungarian and Angelinskii common carps, and the three samples of Altai common carp. The second cluster was formed by the group consisting of the representatives of Cherepetskskii, Stavropol, and Ropsha common carps, along with the differing from them Amur wild common carp. The observed differentiation was confirmed by the analysis of the polymorphic markers variance by the method of principle components. Evolutionary history and the reasons for genetic differentiation of Russian common carp breeds are discussed.

RAPD Variation in Two Trematode Species (Fasciola hepatica and Dicrocoelium dendriticum) from a Single Cattle Population

The method of random DNA amplification by PCR with arbitrary primers (RAPD–PCR) was used for the description and estimation of genetic variation in two trematode species, Fasciola hepatica (n = 21) and Dicrocoelium dendriticum (n= 8). The studied trematodes were liver parasites of five cattle individuals belonging to the same herd. To study the F. hepatica population, five primers were selected, which revealed 230 RAPD markers in five samples of parasites isolated from five different host individuals. Using 87 RAPD markers, a comparison of variation was conducted betweenF. hepatica and D. dendriticum samples from the same host individual. Based on the estimates of RAPD variation for the individual samples of parasites collected from each of five host individuals and for the total F. hepatica population, standard indices of genetic similarity (S), diversity (H), polymorphism (P), and population subdivision (FST) were calculated. From the indices of similarity in pairs (S), dendrograms were constructed, which reflect genetic relationship between the representatives of two species and between F. hepatica individuals isolated from the same or different host individuals. It was revealed that polymorphism level (P) varied within a range of 35.5 to 83.2% in the studiedF. hepaticapopulation and reached 95.1% in the studied D. dendriticum population. Two different trematode species that simultaneously parasitize the same host animal were characterized by similar estimates of polymorphism and genetic diversity and by similar topology of genetic similarity dendrograms. The degree of genetic similarity between F. hepatica andD. dendriticum was significantly lower (20%) than between five F. hepatica samples (41.4%) that formed two unequal clusters. Each of these clusters represents a heterogeneous group consisting of parasites collected from three or four host individuals. In the individual samples of parasites related to each of the studied host individuals, the indices of genetic similarity (S) and diversity (H) varied within a range of 43.3 to 64.8% and 25.1 to 56.6%, respectively. In the total F. hepatica sample, the estimates of intraspecific variation, the topology of dendrograms, and the FST index (7.4%) indicate the absence of clear genetic differentiation between the samples of parasites isolated from different host individuals. Possible reasons for the high level of genetic variation in the studied trematode populations and the genetic consequences of host–parasite interaction are discussed.

Genetic differentiation of cercariae infrapopulations of the avian schistosome Trichobilharzia szidati based on RAPD markers and mitochondrial cox1 gene

Avian schistosome Trichobilharzia szidati is a member of the largest genus within the family Schistosomatidae (Trematoda). Population genetic structure of Trichobilharzia spp. schistosomes, causative agents of cercarial dermatitis in humans, has not been studied yet. The knowledge of the genetic structure of trichobilharzian populations is essential for understanding the host–parasite coevolutionary dynamics and epidemiology strategies. Here we examined genetic diversity in three geographically isolated local populations of T. szidati cercariae inhabiting Russia based on nuclear (randomly amplified polymorphic DNA, RAPD) and mt (cox1) markers. We analyzed T. szidati cercariae shed from seven naturally infected snails of Lymnaea stagnalis. Using three random primers, we demonstrated genetic variation among populations, thus posing genetic structure across geographic sites. Moreover, T. szidati cercariae have been genetically structured among hosts (infrapopulations). Molecular variance analysis was performed to test the significance of genetic differentiation within and between local populations. Of total parasitic diversity, 18.8% was partitioned between populations, whereas the higher contribution (48.9%) corresponds to the differences among individual cercariae within infrapopulations. In contrast to RAPD markers, a 1,125-bp fragment of cox1 mt gene failed to provide any significant within-species structure. The lack of geographic structuring was detected using unique haplotypes which were determined in the current work for Moscow and Western Siberian local populations as well as obtained previously for European isolates (Czech Republic and Germany). All T. szidati/Trichobilharzia ocellata haplotypes were found to be mixed across their geographical origin.

Multilocus variation in cercariae, parthenogenetic progeny of different species of the class Trematoda.

Parthenogenesis, a form of sexual reproduction, is not a rare event in many invertebrate, including lower Crustacea, Rotifera, insects, corals, worms, and molluscs. Parthenogenesis is advantageous because it increases the species reproduction rate. It has been previously assumed that parthenogenetic progeny originating from a fertilized ovicell is a population (clone) of genetically homogenous individuals. However, in some ascidium, Daphnia, and bee species, rapid genetic changes within parthenogenetic clones have been found using different genetic markers (RAPDs, AFLPs, miniand microsatellites, and some locus-specific sequences). Parthenogenetic species show significant genotypic and phenotypic variations, including those within clones [1]. The molecular mechanisms underlying this variation remain unknown.

Polymorphism of microsatellite markers in Russian common carp (Cyprinus carpio L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.

Clonal Diversity and Clone Formation in the Parthenogenetic Caucasian Rock Lizard Darevskia dahli

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.