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 V?nern; 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.     

Demonstrating freedom from Gyrodactylus salaris (Monogenea: Gyrodactylidae) in farmed rainbow trout Oncorhynchus mykiss

This paper describes an approach to demonstrate freedom of individual rainbow trout farms from Gyrodactylus salaris Malmberg, 1957. The infection status of individual farms is relevant should G. salaris be introduced into a country or zone previously known to be free of the parasite. Trade from farms where G. salaris may have been introduced would be restricted until freedom had been demonstrated. Cage, fish and parasite sample sizes were calculated based on the minimum detectable prevalence (P*), test characteristics, population size, and Type I and II errors. Between 5 and 23 cages per farm would need to be sampled to demonstrate freedom at a cage level P* of 10%. The number of fish sampled per cage depended mainly on the test sensitivity (probability of correctly identifying an infected fish). Assuming a test sensitivity of 99% at the fish level, 59 fish per cage are needed (P* = 5%). Since G. salaris may exist in mixed infection with G. derjavini, testing a sample of gyrodactylid parasites may not result in the parasite being detected when present. Test sensitivity at the fish level depends on the number of gyrodactylids on the fish, the proportion of which are G. salaris and the number examined. Assuming a P* of 5% (i.e. G. salaris are at least 5% of the gyrodactylid population), between 20 and 73 parasites per fish would need to be sampled (depending on abundance) to maintain the Type I error at 0.01 (thus a fish level test sensitivity of 99%). This work identifies the critical information, and further research, needed to assess freedom from G. salaris with a known level of confidence; this is essential to provide a sound scientific basis for decision-making about disease control measures.     

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.     

The host specificity of Gyrodactylus salaris Malmberg (Platyhelminthes, Monogenea): susceptibility of Oncovhynchus mykiss (Walbaum) under experimental conditions

An experimental epidemiological approach was chosen to study the survival and infection dynamics of Gyrodactylus salaris on juvenile rainbow trout, Oncorhynchus mykiss , in the laboratory. A marked heterogeneity in the host stock was apparent. The rainbow trout could be divided into three groups on the basis of parasite survival and infection pattern on individually isolated fish: (1) hosts receptive to initial parasite attachment, but unreceptive to parasite establishment and reproduction; (2) hosts moderately susceptible to parasite establishment and reproduction, but which, after a period of restricted parasite population growth, responded, recovered and eliminated the parasites; and (3) hosts very susceptible to parasite infection and reproduction, but which, after a period of significant parasite population growth, responded, recovered and eliminated the parasites. These different patterns are considered to reflect genetic differences between host individuals. Parasite aggregation was also shown to be an important factor in the outcome of the host-parasite association. The parasites were finally eliminated on the individually isolated hosts, but not on hosts maintained in batches and so host population size and immigration of fresh. previously unexposed, hosts appeared to be important for growth and maintenance of the parasite population. The parasite was not found to cause host mortality. Rainbow trout was a suitable host for G. salaris , capable of transmitting the parasite to new localities as a consequence of stocking programmes or migratory behaviour.     

The susceptibility of Salvelinus fontinalis (Mitchill) to Gyrodactylus salaris Malmberg (Platyhelminthes; Monogenea) under experimental conditions

The host specificity of Gyrodaclylus Solaris is examined experimentally with respect to its ability to infect the brook trout, Salvelinus fontinalis . The parasite readily attached to and reproduced on parr of this host and infections grew for c. 20 days from first monitoring (c. 30 days from first infection) before declining. Parasites could persist on this host for up to 70 days before finally disappearing. The pattern of infection resembled that seen in many other gyrodactylid species on their normal hosts, and suggested the action of a host response, In this respect infections of G. salaris on parr of S. fontinalis , anadromous Salvelinus alpinus, Oncorhynchus mykiss, Thymallus thymallus and Baltic Salmo salar follow a normal pattern, while infections of Norwegian S. salar are unusual in a continued unchecked growth, until the host dies, under pooled laboratory conditions.     

Gyrodactylus gvozdevi n. sp. (Gyrodactylidae: Monogenea) from Noemacheilus dorsalis (Kessler) in Kazakhstan

Gyrodactylus gvozdevi n. sp. (Gyrodactylidae: Monogenea) is described from the skin of the freshwater fish Noemacheilus dorsalis (Kessler) (Cobitidae: Cypriniformes) from Kazakhstan. This species is most closely related to G. pseudonemachili Ergens & Bykhovsky, 1967 in the shape and size of the anchors and both the ventral and dorsal bars, but can be distinguished from it by the shape and size of the hookproper of the marginal hooks.     

Nominal species of the genus Gyrodactylus von Nordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species

The total diversity of the monogenean genus Gyrodactylus is evaluated. There are 409 potentially valid species names within the genus, recorded from c. 400 host species. Five species have been placed within Fundulotrema and an additional 51 Gyrodactylus species names represent synonyms, nomina nuda or have been reassigned to other non-viviparous monogenean genera. While the majority of Gyrodactylus species (59%) are recorded from single hosts, some have a much broader broad range.     

Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic Salmon Salmo salar L. in laboratory experiments

On parr of Atlantic salmon Salmo salar L. in laboratory experiments at different water temperatures, opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 differed in size. Their shape, on the other hand, varied only slightly. The opisthaptoral hard parts were largest at the lowest water temperatures and decreased in size with increasing water temperatures (1.5°C to 20.0°C). At both 18.0°C and 20.0°C the opisthaptoral hard parts of G. salaris were smaller than has been found under natural conditions. In the experiments, the mean size of the opisthaptor in the G. salaris micropopulation at each water temperature gradually increased or decreased compared to the mean size at the start of the experiments. At the end of the experiments the mean size of the opisthaptoral hard parts in the G. salaris micropopulations showed a significant regression to the different water temperatures.     

Gyrodactylus gemini n. sp. (Monogenea: Gyrodactylidae), a parasite of Semaprochilodus taeniurus (Steindachner) from the Venezuelan Amazon

Gyrodactylus gemini n. sp. (Monogenea, Gyrodactylidae) is described from the surface of the body and fins of the fish Semaprochilodus taeniurus (Steindachner) imported into Britain from the Venezuelan Amazon. The new species differs from other species of the genus, including those described from South and Central America, by having: (i) stout hamuli with straight shafts and diverging roots; (ii) marginal hooks with the sickle length larger than the width; (iii) a dorsal bar without a medial constriction; (iv) a rectangular ventral bar with short processes; (v) a triangular ventral bar membrane; and, most obviously, (vi) at least two generations which can develop two embryos simultaneously. This is the first known species of the genus Gyrodactylus from the Venezuelan Amazon and the first record of the subgenus Gyrodactylus (Gyrodactylus) from South America.     

Gyrodactylus longipes n. sp. (Monogenea: Gyrodactylidae) from farmed gilthead seabream (Sparus aurata L.) from the Mediterranean

Gyrodactylus longipes n. sp. (Monogenea, Gyrodactylidae) is described from the gills of farmed juvenile gilthead seabream (Sparus aurata L.) from two sites located in Italy and Bosnia-Herzegovina and represents the second species of Gyrodactylus to be described from S. aurata. Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 was the first gyrodactylid to be described from S. aurata, from populations cultured in Albania and Croatia. In the current study, G. longipes was found in a mixed infection with G. orecchiae on fish maintained in Latina Province, Italy, thus extending the reported distribution of the latter throughout the Mediterranean. The morphology of the opisthaptoral hard parts of G. longipes is compared to those of G. orecchiae, using light and scanning electron microscopy. Gyrodactylus longipes is characterised by having larger, elongated ventral bar processes and long, triangular-shaped toe region to their marginal hook sickles which, by comparison, are rhomboid in G. orecchiae. The marginal hook sickles of G. longipes are almost double the size of G. orecchiae which allows for their rapid discrimination from each other in mixed infections. A comparison of the DNA sequence of the ribosomal internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) of G. longipes with the corresponding sequence from G. orecchiae and with those available in GenBank, supports the separate species status of G. longipes. Part of this study necessitated an overview of the existing Gyrodactylus fauna from Italy and Bosnia-Herzegovina; a summary from each country is provided here to assist future investigations.     

Seasonal variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on parr of Atlantic salmon Salmo salar L. in the River Batnfjordselva,Norway

Seasonal variations in size and shape of the marginal hooks, the anchors and the ventral bar of the opisthaptor of Gyrodactylus salaris Malmberg, 1957 were studied. The G. salaris specimens were collected from parr of Atlantic salmon Salmo salar L. in the River Batnfjordselva, north-western Norway. Samples were taken at roughly monthly intervals during a two-year period. The marginal hooks, the anchors and the ventral bar showed considerable seasonal variation in size, but varied only slightly in shape. The variation in 12 of the 14 characters measured showed a significant regression to the variation in the water temperature. The total length of the marginal hooks of G. salaris can be considerably longer than the previously reported maximum of 40 m for the species.     

Seasonal variations in the prevalence and infestation intensity of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on Atlantic salmon parr, Salmo salar L., in the River Batnfjordselva, Norway

The prevalence of Gyrodactylus suluris Malmberg. 1957 on both yearlings and older parr of Atlantic salmon ( Sulmo sulur L.) in the River Batnfjordselva was 100% through most of the year. With one exception, uninfested fish were only found in the winter and spring after the water temperature had fallen to almost 0° C for 2–3 months. In general, the abundance (i.e. the mean number of parasites per investigated fish) of G. salaris increased during the warm period of the year (summer and early autumn). Abundanceas high as 1153 and 4418 in early autumn was found on yearlings and older parr, respectively. The abundance decreased during the cold period of the year (winter and early spring), in some cases to as low as two and four G. salaris on yearlings and older parr, respectively. About 86% of the G. salaris specimens were found on the fins of the salmon parr: mainly on the dorsal fin (34.4%) and the pectoral fins (27.0%). The remainder of the parasites were distributed on the body (7.8%), the head (3.5%), and the gills (2.6%).     

Two new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) parasitizing Girardinichthys multiradiatus (Cyprinodontiformes: Goodeidae), an endemic freshwater fish from central Mexico

Gyrodactylus mexicanus n. sp. and Gyrodactylus lamothei n. sp. are described from the fins and skin of Girardinichthys multiradiatus, an endemic freshwater fish from central Mexico. Gyrodactylus mexicanus is compared to other Gyrodactylus species that parasitize Fundulus spp., the phylogenetically closest group to the Goodeidae from North America. Gyrodactylus mexicanus is distinguished by having large anchors with well-developed superficial roots, enlarged hooks with a proximally disrupted shank (ligament), and a ventral bar with 2 poorly developed anterolateral projections and a small medial process. Gyrodactylus lamothei is distinguished from G. mexicanus and from other species of Gyrodactylus on the North American continent by having anchors with a sclerite on the superficial root and robust hooks with a straight shaft and a recurved point.     

Four new species of Gyrodactylus von Nordmann, 1832 (Monogenea,Gyrodactylidae) on gobiid fishes: combined DNA and morphological analyses

Four Gyrodactylus species parasitising four closely related gobiid species in European coastal waters were studied and compared with G. arcuatus Bychowsky sensu Bychowsky & Poljansky (1953) from Gasterosteus aculatus . These were G. gondae n. sp. from Pomatoschistus minutus and P. lozanoi , G. flavescensis n. sp. from Gobiusculus flavescens , G. arcuatoides n. sp. from P. minutus and G. branchialis n. sp. from P. microps. Combined molecular and morphological analyses, as well as morphometric and statistical methods, were used. The ssrRNA V4 region and the complete ITS rDNA region were sequenced. Genetically the four new species are clearly distinct from G. arcuatus . From a morphological point of view, the haptoral hard parts of G. gondae n. sp., G. flavescensis n. sp. and G. arcuatoides n. sp. are related to those of G. arcuatus, while these parts of G. branchialis n. sp. are different, but related to those of G. quadratidigitus Longshaw, Pursglove & Shinn, 2003. For the latter two species, a new species group is formed. The V4 and ITS sequence analyses, however, indicate a close relationship between G. branchialis and the three G. arcuatus-like species.     

Gyrodactylus neili n. sp. (Monogenea: Gyrodactylidae), a parasite of chain pickerel Esox niger Lesueur (Esocidae) from freshwaters of New Brunswick, Canada

Gyrodactylus neili n. sp. (Monogenea: Gyrodactylidae) is described from the fins and body surface of Esox niger Lesueur (chain pickerel) (Esocidae) from the St. Croix River drainage, New Brunswick, Canada. G.␣neili n. sp. resembles most closely G. fryi Cone & Dechtiar, 1984, a parasite of E. masquinongy in North America, in having relatively large thin hamuli, well-developed marginal hook sickles with a relatively long, wide blade and short handle, a ventral bar with small antero-lateral processes and tongue-shaped membrane, and a cirrus with many small spines in two rows. The new species is easily separated from G. fryi by the length of the hamuli (70-76 m versus 92 m, respectively), by the distal width of the sickle (7–9 versus 14-16 m, respectively) and by subtle differences in the shape of the toe and heel of the marginal hook sickle. Sequence data (922 bp) of rDNA (internal transcribed spacers 1 and 2 and 5.8S) of G. neili n. sp. returned no identical matches in GenBank. The 5.8 sequence alone, however, was identical to morphologically similar gyrodactylids of the subgenus Gyrodactylus from cyprinid fishes in Eurasia. The discovery of G. neili n. sp. and features of its genetic makeup support the idea that this lineage parasitised ancestral cyprinids and that it radiated, possibly through predator/prey interactions, to an ancestor of contemporary Esox. It is concluded that DNA comparison of monogeneans on Holarctic freshwater hosts, such as E.␣lucius, may shed light on the nature of speciation of these parasites.     

Variations of opisthaptoral hard parts of Gyrodactylus salaris Malmberg, 1957 (Monogenea: Gyrodactylidae) on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) in a fish farm,with comments on the spreading of the parasite in south-eastern Norway

Studies were carried out on Gyrodactylus salaris Malmberg, 1957 on rainbow trout Oncorhynchus mykiss (Walbaum, 1792) from a fish farm in Lake Tyrifjorden, south-eastern Norway. The anchors were larger, and the shape of the anchors and the ventral bar differed slightly, as compared with the same parts of G. salaris on parr of Atlantic salmon Salmo salar L. in northern and north-western Norwegian rivers. At different water temperatures (0.8°C, 10.0°C, 18.0°C), the opisthaptoral hard parts of G. salaris on rainbow trout showed considerable variation in size, but varied only slightly in shape. It was found that the total length of the anchors of G. salaris on rainbow trout may considerably exceed the previously reported maximum of 80 m for the species. The spread of G. salaris to south-eastern Norway is described and discussed.