Sulphonamide inhibition studies of the ß-carbonic anhydrase GsaCAß present in the salmon platyhelminth parasite Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Monogenean platyhelminth Gyrodactylus salaris, a fish parasite, GsaCAß, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAß inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9-139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAß inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with KIs in the range of 16.9-24.8 µM. Although no potent GsaCAß-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the inhibition profile of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.

Cloning, purification, kinetic and anion inhibition studies of a recombinant ß-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAß has a significant catalytic activity for the physiological reaction, CO2 + H2O ⇋ HCO3- + H+ with a kcat of 1.1 × 105 s-1 and a kcat/Km of 7.58 × 106 M-1 × s-1. This activity was inhibited by acetazolamide (KI of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAß at millimolar concentrations, but sulfamide (KI of 81 µM), N,N-diethyldithiocarbamate (KI of 67 µM) and sulphamic acid (KI of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAß is subsequently proposed as a new drug target for which effective inhibitors can be designed.

New Salinity Tolerant Species of Gyrodactylus (Platyhelminthes, Monogenea) on Intertidal and Supratidal Fish Species from the Chilean Coast.

PURPOSE: The intertidal and supratidal coastal zone challenges the osmoregulatory capacity of aquatic inhabitants. Four new species of Gyrodactylus ectoparasites on two intertidal fishes from Chile are described based on molecular and morphological analyses. METHODS: Monogeneans were found from two fish species, the clingfish Sicyases sanguineus Müller & Troschel, 1843 and the combtooth blenny Scartichthys viridis Valenciennes, 1836. The morphology was described by drawings, and minimal measurements. The parasites were barcoded via the sequencing of the ribosomal DNA over ITS1-5.8S-ITS2. RESULTS: The air-breathing clingfish S. sanguineus carried Gyrodactylus amphibius sp. nov., hiding in the ventral sucker formed by the modified pectoral fins of the fish. The intertidal combtooth blenny S. viridis carried three other new species: Gyrodactylus scartichthi sp. nov., Gyrodactylus viridae sp. nov., and Gyrodactylus zietarae sp. nov. CONCLUSION: The four new species were all phylogenetically related with the previously described G. chileani Zietara et al. 2012 on triplefin Helcogrammoides chilensis Cancino, 1960 in the same habitat. Thus, the five Chilean Pacific Gyrodactylus species formed a statistically well-supported (100%) monophyletic clade together with three geographically distant species recorded in Europe. The Chilean Pacific parasites are not related to G. salinae and G. magadiensis, parasites described in extreme osmotic stress environments earlier.

Population genomics reveals repeated signals of adaptive divergence in the Atlantic salmon of north-eastern Europe.

Our ability to examine genetic variation across entire genomes has enabled many studies searching for the genetic basis of local adaptation. These studies have identified numerous loci as candidates for differential local selection; however, relatively few have examined the overlap among candidate loci identified from independent studies of the same species in different geographic areas or evolutionary lineages. We used an allelotyping approach with a 220K SNP array to characterize the population genetic structure of Atlantic salmon in north-eastern Europe and ask whether the same genomic segments emerged as outliers among populations in different geographic regions. Genome-wide data recapitulated the phylogeographic structure previously inferred from mtDNA and microsatellite markers. Independent analyses of three genetically and geographically distinct groups of populations repeatedly inferred the same 17 haploblocks to contain loci under differential local selection. The most strongly supported of these replicated haploblocks had known strong associations with life-history variation or immune response in Atlantic salmon. Our results are consistent with these genomic segments harbouring large-effect loci which have a major role in Atlantic salmon diversification and are ideal targets for validation studies.

Horizontal transmission of the ectoparasite Gyrodactylus arcuatus (Monogenea: Gyrodactylidae) to the next generation of the three-spined stickleback Gasterosteus aculeatus.

In the parthenogenetic monogeneans of the genus Gyrodactylus Nordmann, 1832, the genetic diversity within or between hosts is determined by the relative roles of lateral transmission and clonal propagation. Clonality and limited transmission lead to high-amplitude metapopulation dynamics and strong genetic drift. In Baltic populations of the three-spined stickleback Gasterosteus aculeatus Linnaeus, the local mitochondrial diversity of Gyrodactylus arcuatus Bychowsky, 1933 is very high, and spatial differentiation weak. To understand the transmission dynamics in a single location, the transmission of the parasite from adults to next generation sticklebacks was investigated in a northern Baltic brackish water location. By sequencing 777 nt of cox1, as many as 38 separate mitochondrial haplotypes were identified. In August, the intensity of gyrodactylid infection on adult hosts was high, the haplotype diversity (h) was extreme and differentiation between fish was negligible (total h = 0.926, mean h = 0.938). In October, only 46% of the juvenile sticklebacks carried G. arcuatus. The number of parasites per young fish followed a Poisson distribution 0.92 ± 1.04 (mean ± SD) on October 2, and was clearly overdispersed 2.38 ± 5.00 on October 25. The total haplotype diversity of parasites on juveniles was nearly as high as in adults (h = 0.916), but the mean per fish was only h = 0.364 (FST = 0.60), due to low intensity of infection and rapid clonal propagation of early arrivals. The initial first come first served advantage of the first gyrodactylid colonisers will be lost during the host adulthood via continuous transmission. Nesting and polygamy are suggested as factors maintaining the high genetic diversity of the parasite population. The transmission dynamics and, consequently, the population structure of Baltic G. arcuatus is fundamentally different from that of G. salaris Malmberg, 1957, on the Baltic salmon Salmo salar Linnaeus.

Genomic signatures of parasite-driven natural selection in north European Atlantic salmon (Salmo salar).

Understanding the genomic basis of host-parasite adaptation is important for predicting the long-term viability of species and developing successful management practices. However, in wild populations, identifying specific signatures of parasite-driven selection often presents a challenge, as it is difficult to unravel the molecular signatures of selection driven by different, but correlated, environmental factors. Furthermore, separating parasite-mediated selection from similar signatures due to genetic drift and population history can also be difficult. Populations of Atlantic salmon (Salmo salar L.) from northern Europe have pronounced differences in their reactions to the parasitic flatworm Gyrodactylus salaris Malmberg 1957 and are therefore a good model to search for specific genomic regions underlying inter-population differences in pathogen response. We used a dense Atlantic salmon SNP array, along with extensive sampling of 43 salmon populations representing the two G. salaris response extremes (extreme susceptibility vs resistant), to screen the salmon genome for signatures of directional selection while attempting to separate the parasite effect from other factors. After combining the results from two independent genome scan analyses, 57 candidate genes potentially under positive selection were identified, out of which 50 were functionally annotated. This candidate gene set was shown to be functionally enriched for lymph node development, focal adhesion genes and anti-viral response, which suggests that the regulation of both innate and acquired immunity might be an important mechanism for salmon response to G. salaris. Overall, our results offer insights into the apparently complex genetic basis of pathogen susceptibility in salmon and highlight methodological challenges for separating the effects of various environmental factors.

Ancient and modern genome shuffling: Reticulate mito-nuclear phylogeny of four related allopatric species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae), ectoparasites on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae).

Phylogenetic analyses including four allopatric species of Gyrodactylus von Nordmann, 1832 on the Eurasian minnow Phoxinus phoxinus (L.) (Cyprinidae) revealed incongruence between the nuclear ITS1-5.8S-ITS2 and mitochondrial cox1 phylogenies due to ancient hybridisation. Gyrodactylus pannonicus Molnár, 1968 was sampled close to its type-locality, the upper reaches of River Tisza, tributary of Danube in the Black Sea Basin. Faunistic search detected three new related species with maximum composite likelihood distances in cox1 between 16.8-23.2% (tentatively 1.3 to 1.8 My of divergence). Gyrodactylus albolacustris n. sp. recorded in the White Sea Basin, eastern Baltic Basin and Mongolia was close to G. pannonicus in the nuclear ITS (divergence of 0.9%), but diverged in cox1 by 19.8%. The Mongolian isolate of G. albolacustris n. sp. diverged from the European isolates in cox1 by 8.9%, suggesting 0.7 My of isolation. The two other new species differed from G. pannonicus by >4% in ITS and some large indels in ITS1, and by >20% in cox1. Gyrodactylus danastriae n. sp. was found in River Strwiaz, a tributary of the River Dniester (Black Sea Basin) and was characterised by smaller size of anchors and by 29-41 bp dimorphic insertion in ITS1. Gyrodactylus botnicus n. sp. is considered endemic in the Baltic Basin, but was also found in the White Sea Basin as a postglacial immigrant, where it had hybridised with G. albolacustris n. sp. in spite of the high divergence in ITS (3.9%) and cox1 (22%). The discordant nuclear and mitochondrial phylogenies revealed an ancient mitochondrial introgression: G. albolacustris n. sp. was derived from a hybridisation combining proto-pannonicus ITS with proto-danastriae mitochondria, perhaps 1.3 My ago. The postglacial hybridisation of G. albolacustris n. sp. (as the donor of mtDNAalb and ITSalb) and G. botnicus n. sp. (donor of the ITSbot) offered a model of shuffling of the genomic components: the process of the homogenisation and stabilisation of nuclear ITS (concerted evolution) and the lineage sorting has hardly begun.

Genetic gradient of a host-parasite pair along a river persisted ten years against physical mobility: Baltic Salmo salar vs. Gyrodactylus salaris.

The Atlantic salmon, Salmo salar L., in the Tornio River in the Northern Baltic Sea basin accommodates a monogenean ectoparasite, Gyrodactylus salaris. The aim of the study was to understand the population structure of apparently co-adapted host-parasite system: no parasite-associated mortality has been reported. The parasite burden among salmon juveniles (parr) was monitored along 460km of the river in 2000-2009. Among the parr, 33.0% were infected (nfish=1913). The genetic structure of the parasite population was studied by sequencing an anonymous nuclear DNA marker (ADNAM1, three main genotypes) and mitochondrial CO1 (three clades, six haplotypes). During the ten years, the parasite population was strongly and stably genetically differentiated among up- and downstream nurseries (nADNAM1=411, FST=0.579; nCO1=443, FST=0.534). Infection prevalence among the smolts migrating to sea was higher than in the sedentary parr populations (82.2%, nfish=129). The spatial differentiation observed among the sedentary juveniles was reflected temporally in the smolt run: parasite genotypes dominating the upper part of the river arrived later than downstream dwellers (medians June 4 and June 2) to the trap 7km from the river mouth. The nuclear and mitochondrial markers were in stable disequilibrium which was not relaxed in the contact zone or among the smolts where the parasite clones often met on individual fish. Only five parasite specimens on smolts (nworms=217) were putative recent sexual recombinants. The contribution of extant salmon hatcheries into the infection was negligible. The host salmon population in Tornio River is known to show significant spatial differentiation (FST=0.022). The stable spatial genetic structure of the parasite against the high physical mobility suggested a possibility of local co-adaptation of the host-parasite subpopulations.

Displaced phylogeographic signals from Gyrodactylus arcuatus, a parasite of the three-spined stickleback Gasterosteus aculeatus, suggest freshwater glacial refugia in Europe.

We examined the global mitochondrial phylogeography of Gyrodactylus arcuatus, a flatworm ectoparasite of three-spined stickleback Gasterosteus aculeatus. In accordance with the suggested high divergence rate of 13%/million years, the genetic variation of the parasite was high: haplotype diversity h=0.985 and nucleotide diversity π=0.0161. The differentiation among the parasite populations was substantial (Φst=0.759), with two main allopatric clades (here termed Euro and North) accounting for 54% of the total genetic variation. The diversity center of the Euro clade was in the Baltic Sea, while the North clade was spread across the Barents and White Seas. A single haplotype within the North clade was found in the western and eastern Pacific Ocean. Divergence of main clades was estimated to be circa 200 thousand years ago. Each main clade was further divided into six distinct subclades, estimated to have diverged in isolation since 135 thousand years ago. This second division corresponds approximately to the Eemian interglacial predating the last glacial maximum. A demographic expansion of the subclades is associated with colonisation of northern Europe since the last glacial maximum, circa 15-40 thousand years ago. The parasite phylogeny is most likely explained by sequential isolated bottlenecks and expansions in numerous allopatric refugia. The postglacial intermingling and high variation in the marine parasite populations, separately in the Baltic and Barents Seas, suggest low competition of divergent parasite matrilines, coupled with a large population size and high rate of dispersal of hosts. The genetic contribution of the assumed refugial fish populations maintaining the parasite during the last glacial maximum was not detected among the marine sticklebacks, which perhaps were infected after range expansion.

Occurrence and morphogenetic characteristics of Gyrodactylus (Monogenea: Gyrodactylidae) from a rainbow trout farm (Lake Ladoga, Russia).

Gyrodactylus parasite infected juveniles on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from a fish farm in Lake Ladoga were investigated. The observed cases of infection in fish featured a high prevalence, when almost all of the fish were infected. However, if an outbreak of the monogenean infection is observed in spring, the intensity of the infection may be low, and when the infection occurs in the ice-covered period (late autumn - winter), the number of parasites on the fins of a single fish may exceed 3000 specimens. Molecular identification of the parasite demonstrated that the infecting clone was identical with rainbow trout specific strain of Gyrodactylus salaris RBT widely spread in Northern Europe, but a small proportion of the parasites were the hybrid clone Gyrodactylus pomeraniae x G. lavareti. Morphological variations of hooks and other opisthaptor parts in the monogenean Gyrodactylus depending on the intensity of infection in rainbow trout were demonstrated.

Footprints of directional selection in wild Atlantic salmon populations: evidence for parasite-driven evolution?

Mechanisms of host-parasite co-adaptation have long been of interest in evolutionary biology; however, determining the genetic basis of parasite resistance has been challenging. Current advances in genome technologies provide new opportunities for obtaining a genome-scale view of the action of parasite-driven natural selection in wild populations and thus facilitate the search for specific genomic regions underlying inter-population differences in pathogen response. European populations of Atlantic salmon (Salmo salar L.) exhibit natural variance in susceptibility levels to the ectoparasite Gyrodactylus salaris Malmberg 1957, ranging from resistance to extreme susceptibility, and are therefore a good model for studying the evolution of virulence and resistance. However, distinguishing the molecular signatures of genetic drift and environment-associated selection in small populations such as land-locked Atlantic salmon populations presents a challenge, specifically in the search for pathogen-driven selection. We used a novel genome-scan analysis approach that enabled us to i) identify signals of selection in salmon populations affected by varying levels of genetic drift and ii) separate potentially selected loci into the categories of pathogen (G. salaris)-driven selection and selection acting upon other environmental characteristics. A total of 4631 single nucleotide polymorphisms (SNPs) were screened in Atlantic salmon from 12 different northern European populations. We identified three genomic regions potentially affected by parasite-driven selection, as well as three regions presumably affected by salinity-driven directional selection. Functional annotation of candidate SNPs is consistent with the role of the detected genomic regions in immune defence and, implicitly, in osmoregulation. These results provide new insights into the genetic basis of pathogen susceptibility in Atlantic salmon and will enable future searches for the specific genes involved.

A new Gyrodactylus strain on brown trout (Salmo trutta) in Jänisjärvi, Russian Karelia, and a literature revision of salmonid parasites of the genus Gyrodactylus in North-Western Russia and adjacent areas.

A Gyrodactylus parasite infected juveniles of brown trout (Salmo trutta L.) in a hatchery in Lake Janisjarvi, Russian Karelia. Molecular identification by ITS1-5.8S-ITS2 of ribosomal DNA indicated that the infection was caused by a non-segregating hybrid clone between unknown Gyrodactylus species and Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel & Vigneulle, 1999, described from brown trout collected in France. The mitochondrial CO1 was sequenced from the hybrid, but it is not available from pure G. teuchis from type locality. The mitochondrial DNA was an independent clade among the wageneri group parasites, differing from the nearest relative G. derjavinoides by 19%. Morphometric measurements of the Janisjarvi parasite were compared with separate (host specific) G. salaris strains from farmed rainbow trout and salmon in Lake Onega, and with available data on G. teuchis from Western Europe, Austria and Poland. All isolates were distinguishable by morphometry, and the measurements are a useful primary diagnostic tool for the salmonid parasites in Karelian great lakes and fish farms. Pure G. teuchis is not found on Russian territory, and the other parent of the hybrid has not been discovered yet in any other country. A mini review of Gyrodactylus on salmonids in the Russian Karelia is presented.

Temporal variation of genetic composition in Atlantic salmon populations from the Western White Sea Basin: influence of anthropogenic factors?

BACKGROUND: Studies of the temporal patterns of population genetic structure assist in evaluating the consequences of demographic and environmental changes on population stability and persistence. In this study, we evaluated the level of temporal genetic variation in 16 anadromous and 2 freshwater salmon populations from the Western White Sea Basin (Russia) using samples collected between 1995 and 2008. To assess whether the genetic stability was affected by human activity, we also evaluated the effect of fishing pressure on the temporal genetic variation in this region. RESULTS: We found that the genetic structure of salmon populations in this region was relatively stable over a period of 1.5 to 2.5 generations. However, the level of temporal variation varied among geographical regions: anadromous salmon of the Kola Peninsula exhibited a higher stability compared to that of the anadromous and freshwater salmon from the Karelian White Sea coast. This discrepancy was most likely attributed to the higher census, and therefore effective, population sizes of the populations inhabiting the rivers of the Kola Peninsula compared to salmon of the Karelian White Sea coast. Importantly, changes in the genetic diversity observed in a few anadromous populations were best explained by the increased level of fishing pressure in these populations rather than environmental variation or the negative effects of hatchery escapees. The observed population genetic patterns of isolation by distance remained consistent among earlier and more recent samples, which support the stability of the genetic structure over the period studied. CONCLUSIONS: Given the increasing level of fishing pressure in the Western White Sea Basin and the higher level of temporal variation in populations exhibiting small census and effective population sizes, further genetic monitoring in this region is recommended, particularly on populations from the Karelian rivers.

A monogenean fish parasite, Gyrodactylus chileani n. sp., belonging to a novel marine species lineage found in the South-Eastern Pacific and the Mediterranean and North Seas.

Gyrodactylus chileani n. sp. is the first Gyrodactylus species reported from Chile. It is an ectoparasite living on fins and skin of a small fish, the Chilean tidal pond dweller Helcogrammoides chilensis (Cancino) (Perciformes: Tripterygiidae). A phylogenetic analysis based on 5.8S+ITS2 of rDNA placed the new species close to marine Gyrodactylus species found in Europe: G. orecchiae Paladini, Cable, Fioravanti, Faria, Cave & Shinn, 2009 on gilthead seabream Sparus aurata L. from the Adriatic and Tyrrhenian Sea fish farms (Perciformes: Sparidae), and an undescribed species on the black goby Gobius niger L. from the North Sea (Perciformes: Gobiidae). A morphological description of the latter species is unavailable. These geographically distant parasite samples on different host families form a new well supported Gyrodactylus orecchiae lineage. Using molecular phylogenetics, it is shown that the marine species groups of Gyrodactylus may have a worldwide distribution.

Two new Antarctic Gyrodactylus species (Monogenoidea): description and phylogenetic characterization.

Two new species of Gyrodactylus Nordmann, 1832 (Gyrodactylidae, Monogenoidea) are described, i.e., Gyrodactylus coriicepsi n. sp. from gills of black rockcod, Notothenia coriiceps Richardson, 1844, and Gyrodactylus nudifronsi n. sp. from gills of gaudy notothen, Lepidonotothen nudifrons Lönnberg, 1905, collected in waters surrounding the South Shetland Islands, Antarctica. Gyrodactylus coriicepsi n. sp. is characterized by a long, narrowing membrane of the ventral bar and a marginal hook sickle with beveled heel and toe. Gyrodactylus nudifronsi n. sp. is characterized by a conspicuous ventral bar process and barely curved marginal hook sickle with curved heel and toe. Definitive species identification and phylogenetic position were based on sequences of the rDNA region (ITS1-5.8S-ITS2). Both new species belong to a monophyletic group (bootstrap support 99%) that includes the European marine species Gyrodactylus perlucidus Bykhovskiy and Polyanskiy, 1953 (on Zoarces viviparus), Gyrodactylus flesi Malmberg, 1957 and G. robustus Malmberg, 1957 (both on Platichthys flesus), and the freshwater species Gyrodactylus hrabei Ergens, 1957 and Gyrodactylus mariannae (both on Cottus poecilopus). The observations suggest an evolutionary continuum of the marine Gyrodactylus spp. fauna in the northern and southern hemispheres, which indicates the marine origin of some freshwater Gyrodactylus species.

Molecular faunistics of accidental infections of Gyrodactylus Nordmann, 1832 (Monogenea) parasitic on salmon Salmo salar L. and brown trout Salmo trutta L. in NW Russia.

Salmon Salmo salar L. and brown trout S. trutta L. juveniles were examined for the presence of accidental monogenean ectoparasitic species of Gyrodactylus Nordmann, 1832 in the Baltic and White Sea basins of Russian Karelia in order to estimate the frequency of host-switching attempts on an ecological timescale. To collect phylogeographical information and for exact species identification, the parasites were characterised by nuclear internal transcribed spacer sequences of rDNA (ITS) and, for some species, also by their mitochondrial DNA (CO1 gene) sequences. Four accidental Gyrodactylus species were observed on salmon and brown trout. A few specimens of G. aphyae Malmberg, 1957, the normal host of which is the Eurasian minnow Phoxinus phoxinus (L.), were observed on lake salmon from the Rivers Kurzhma (Lake Kuito, White Sea basin) and Vidlitsa (Lake Ladoga, Baltic basin). G. lucii Kulakovskaya, 1952, a parasite of the northern pike Esox lucius L., was observed on salmon in the Kurzhma. In the River Vidlitsa, two specimens of G. papernai Ergens & Bychowsky, 1967, normally on stone loach Barbatula barbatula (L.), were found on salmon. On anadromous White Sea salmon in the River Pulonga in Chupa Bay, a few salmon parr carried small colonies of G. arcuatus Bychowsky, 1933, which were shown to have originated from the local three-spined stickleback Gasterosteus aculeatus L. consumed as prey. No specimens of Gyrodactylus salaris Malmberg, 1957 were observed, although the Pulonga is the nearest salmon spawning river to the River Keret', which is heavily infected with introduced G. salaris. In the River Satulinoja, Lake Ladoga, three specimens of G. lotae Gusev, 1953, from burbot Lota lota (L.), were collected from a single brown trout S. trutta. All nonspecific gyrodactylid infections on salmonids were judged to be temporary, because only a few specimens were observed on each of the small number of infected fishes. The prevalence of endemic G. salaris was also low, only 1% (Nfish = 296) in Lake Onega and 0.7% (Nfish = 255) in Lake Ladoga, while brown trout specific Gyrodactylus species were not observed on any of the 429 trout examined from the Ladoga basin. The host-specific and unspecific burden of Gyrodactylus spp. on these 'glacial relict' populations of salmon and brown trout was very low, suggesting a generalised resistance against the co-evolved freshwater parasite community, or some kind of 'vaccination' effect. These hypotheses deserve further testing.

Escape from an evolutionary dead end: a triploid clone of Gyrodactylus salaris is able to revert to sex and switch host (Platyhelminthes, Monogenea, Gyrodactylidae).

Diploid parthenogenesis, with rare sex, is considered as the basic mode of reproduction among the hermaphroditic and viviparous Gyrodactylus. A particular strain of the monogenean parasite Gyrodactylus salaris (RBT clone) was recognized by an invariable, unique mitochondrial DNA haplotype in rainbow trout (Oncorhynchus mykiss) farms. The RBT clone was shown to be triploid and asexual by analyzing a 493 bp sequence of a nuclear DNA marker. Three alleles were present as heterozygous in all 237 individuals sampled in years 2001-2005 from five isolated Finnish farms. The triploid clone probably originated from a diploid oocyte fertilized by a non-self hermaphrodite, most probably in a fish farm. Identical mitochondrial COI gene (1606 bp) was also found in G. salaris parasites on landlocked salmon (Salmo salar) in two rivers draining to the lake Kuitozero, Russian Karelia. In the river Pisto, the clone was triploid, but the diagnostic "short" nuclear allele of the RBT clone was replaced by an allele typical for salmon specific parasites in the Lake Onega. The clone in the river Kurzhma was diploid, having lost the "short" allele, but still heterozygous for the other two alleles of the RBT clone. Evidently, the triploid parthenogenetic RBT clone had produced diploid oocytes, when (as a female) stimulated by a non-self mate in the new environment. The genetic reorganization coincided with a switch to the salmon host. Participation of triploids into the gene pool of the species is rarely reported in animals, and the triploidy is generally considered as an irreversible dead-end of the evolution. Liberalism in ploidy level may significantly add to the evolutionary options available for a parasite in ever-changing environments.

Initial steps of speciation by geographic isolation and host switch in salmonid pathogen Gyrodactylus salaris (Monogenea: Gyrodactylidae).

To test the hypothesis that host-switching can be an important step in the speciation of gyrodactylid monogenean flatworms, we inferred the phylogeny within a cluster of parasites morphologically close to Gyrodactylus salaris Malmberg 1957, collected from Atlantic, Baltic and White Sea salmon (Salmo salar), farmed rainbow trout (Oncorhynchus mykiss), and grayling (Thymallus thymallus) from Northern Europe. The internal transcribed spacer region of the nuclear ribosomal gene was sequenced for taxonomic identification. Parasites on grayling from the White Sea Basin differed from the others by one nucleotide (0.08%), the remainder were identical to the sequence published earlier from Norway (G. salaris on salmon), England (Gyrodactylus thymalli on grayling), and the Czech Republic (unidentified salaris/thymalli on trout). For increased resolution, 813 nucleotides of the mitochondrial COI gene of 88 parasites were sequenced and compared with 76 published sequences using phylogenetic analysis. For all tree building algorithms (NJ, MP), the parasites formed a star-like phylogeny of six definite sister clades, indicating nearly simultaneous radiation. Average K2P distances between clades were 1.8-2.6%, and internal mean distances 0.2-1.1%. The genetic distance to the nearest known relative, Gyrodactylus lavareti Malmberg, was 24%. A variable salmon-specific mitochondrial Clade I was observed both in the Baltic Basin and in pathogenic populations introduced to the Atlantic and White Sea coasts. An invariable Clade II was common in rainbow trout farms in Sweden, Denmark and Finland; the same haplotype was also infecting salmon in a landlocked population in Russian Karelia, and in Oslo fjord and Sognefjord in Norway. Four geographically vicariant sister clades were observed on graylings: Clade III in the Baltic Sea Basin; Clade IV in Karelian rivers draining to the White Sea; Clade V in Norwegian river draining to Swedish lake Vänern; and Clade VI in rivers draining to Oslo fjord. The pattern fitted perfectly with the postglacial history of grayling distribution. Widely sampled clades from salmon and Baltic grayling had basal haplotypes in populations, which were isolated early during the postglacial recolonisation. The divergence between the six clades was clear and linked with their hosts, but not wide enough to support a species status for them. Parasites from the Slovakian type population of G. thymalli were not available, so this result does not mean that G. salaris and G. thymalli are synonyms. It is suggested that the plesiomorphic host of the parasite cluster was grayling, and the switch to salmon occurred at least once when the continental ice isolated Baltic salmon in an eastern freshwater refugium, 130,000 years ago. At the same time, parasites on grayling were split geographically and isolated into several allopatric refugia. The divergence among the parasite clades allowed a tentative calibration of the evolutionary rate, leading to an estimate of the divergence of 13.7-20.3% per million years for COI coding mtDNA. The results supported the hypothesis that parallel to the allopatric mode, host switch and instant isolation by host specificity can be operated as a speciation mechanism.

The crossroads of molecular, typological and biological species concepts: two new species of Gyrodactylus Nordmann, 1832 (Monogenea: Gyrodactylidae).

Nucleotide sequences of nuclear ribosomal DNA internal transcribed spacers (ITS) were used to confirm morphological identification of Gyrodactylus species in Fennoscandia. Three pairs of morphologically similar or cryptic species are compared in this study. G. branchicus Malmberg, 1964 and G. rarus Wegener, 1910, hosted by the sticklebacks Gasterosteus aculeatus L. and Pungitius pungitius (L.), respectively, displayed a genetic divergence of 0.9-1.3% along 774 nucleotides of ITS (Jukes & Cantor distance). G. branchicus isolates from the Baltic, White Sea and Scottish North Sea were invariable, but a Belgian North Sea population with a 0.4% divergence in ITS sequence has been collected. The species status of G. branchicus and G. rarus is supported by host-specificity in sympatric habitats and consistent morphological difference in the marginal hook sickles. Among Gyrodactylus on burbot Lota lota (L.) specimens were collected with their 792 bp long ITS sequences differing by 8.5%. This has led to the splitting of G. lotae Gusev, 1953 into two species, G. lotae and G. alexgusevi n. sp. The species are morphologically separable, and a similar range of variation was found in both the anchors and the marginal hooks in other collections and in the original description. Among G. macronychus-like parasites of minnow Phoxinus phoxinus (L.), divergent ITS sequences indicate two cryptic species. By comparison with the type-specimens, one was identified as G. macronychus Malmberg, 1957; the other is here designated as G. jussii n. sp., differing by 21.8% in 950-973 bp long ITS. The consequences of these findings for the species concept and species identification of Gyrodactylus are discussed.

Speciation by host switch and adaptive radiation in a fish parasite genus Gyrodactylus (Monogenea, Gyrodactylidae).

Four hundred Gyrodactylus species have been formally described, but the estimated number of species in this fish ectoparasite genus of Monogenean Platyhelminthes is more than 20,000. The unusually high species richness has lead to the hypotheses of speciation and adaptive radiation via host switching. These hypotheses were tested by reconstructing a molecular phylogeny for the subgenus G. (Limnonephrotus) which is a group of freshwater parasites, including five species infecting wild and farmed salmonids. The highly variable ITS1 and ITS2 segments and the conservative 5.8S ribosomal gene were sequenced in 22 species plus two species representing the subgenus G. (Paranephrotus) as an outgroup. The phylogeny was compared with host systematics: the species were collected from six fish families (Cyprinidae, Salmonidae, Percidae, Esocidae, Gasterosteidae, and Gobitidae). The phylogenetic analysis demonstrated that G. (Limnonephrotus) is a monophyletic group that was originally hosted by cyprinids. The speciation has occurred in two episodes, the older one manifested in genetic distances 25-33% (4-6 Myr BP). The latter speciation burst occurred in one clade only, perhaps one million years ago. This clade has been morphologically identified as a wageneri species group. It is a monophyletic group of 18 species [studied here] and contains all five salmonid parasites, but also parasites, on cyprinids, percids, esocids, and gasterosteids. In G. (Limnonephrotus), eight host switches crossing the host family barrier were observed, and at least three of them were followed by repetitive speciation. Seven host-switch events were statistically confirmed by bootstrapping. The suggested model of speciation by host switch was accepted, and interestingly the adaptive radiation seems to be a consequence of host switch to a new family (key innovation model). The molecular and ecological evolution rate of Gyrodactylus parasites is manyfold in comparison to host species, and the phylogenies are largely independent and disconnected.