Comparative analyses within Gyrodactylus (Platyhelminthes: Monogenea) mitochondrial genomes and conserved polymerase chain reaction primers for gyrodactylid mitochondrial DNA.

In this study, we describe the complete mitochondrial genomes of Gyrodactylus brachymystacis and Gyrodactylus parvae infecting rainbow trout (Oncorhynchus mykiss) and the invasive topmouth gudgeon (Pseudorasbora parva), respectively. The two circular genomes have a common genome organization found in other Gyrodactylus species. Comparative analyses of mitochondrial genomes from six Gyrodactylus species were carried out to determine base composition, codon usage, transfer RNA and ribosomal RNA genes, major non-coding regions, and nucleotide diversity within the genus. We also provide the first universal models of the secondary structures of rrnS and rrnL for this group thereby promoting utilization of these genetic markers. Universal primers provided herein can be used to obtain more mitochondrial information for pathogen identification and may reveal different levels of molecular phylogenetic inferences for this lineage.

Identification of transferrin in Atlantic cod Gadus morhua epidermal mucus.

The previously unreported presence of transferrin in Atlantic cod Gadus morhua epidermal mucus is described. A less destructive sampling method, which may result in decreased epidermal tissue damage, is discussed.

The placement of Gyrodactylus salmonis (Yin & Sproston) in the molecular phylogeny of studied members of the Gyrodactylus wageneri-group parasitizing salmonids.

The molecular phylogeny of Gyrodactylus salmonis from brook charr, Salvelinus fontinalis, rainbow trout, Oncorhynchus mykiss, cutthroat trout, O. clarkii, and Atlantic salmon, Salmo salar, in Canada is presented using sequences from ITS-rDNA and the mitochondrial COX 1 gene. Sequence variation among G. salmonis specimens from the different North American hosts was consistent with within-species variation reported for other Gyrodactylus. Sequence data are compared to those from other members of the wageneri group parasitizing salmoniform fishes in northern Europe (G. derjavini, G. derjavinoides, G. lavareti, G. salaris, G. salvelini, G. teuchis and G. truttae) and Asia (G. brachymystacis). Sequence divergence between G. salmonis and the recently described G. salvelini on Arctic charr, Salvelinus alpinus, in Finland was consistent with within-species levels of variation in Gyrodactylus; however, phylogenetic analyses and morphological comparisons provided evidence of their distinctiveness such that they appear to be sister species. They grouped with G. lavareti (a parasite of a coregonid) to form a clade separate from European and Asian species of the wageneri lineage known from salmonid fish. Further study of gyrodactylids from across salmonid, coregonid and thymallid fish in the northern hemisphere would shed more light on the phylogeography of these parasites and serves as an important backdrop to understanding the evolution of their emergent virulence.

Divergence between Asian, European and Canadian populations of the monogenean Pseudodactylogyrus bini indicated by ribosomal DNA patterns.

The monogenean Pseudodactylogyrus bini parasitizes the gills of eels belonging to the genus Anguilla. Circumstantial evidence suggests that the parasite has been spread accidentally from the Pacific area (East Asia) to Europe by the intercontinental eel trade. This is based on early descriptions of the parasites from Asian regions and the lack of records of the parasites in Europe before 1977. In addition, the susceptibility of European eels to infections with the parasite is significantly higher compared to that of Japanese eels, which could indicate that the European eel had not undergone co-evolution with this parasite. The present study was undertaken to elucidate the origin of the parasite by using molecular tools. Parasite samples were obtained from Europe (Germany), Asia (Taiwan) and Nova Scotia, the latter of which is the first record of P. bini in Canada. Sequencing of rDNA comprising part of the internal transcribed spacer 1 (ITS1) gene, 5.8S and part of ITS2 (1323 bp) showed that P. bini isolates from the first two regions showed high variability. One sequence was found both in a number of Asian parasites and with one to a few transitions in European parasites, which could indicate that they were split recently into the two regions. Other sequence variations suggested that one or a few genotypes of P. bini were imported on one occasion from Asia to Europe and that the two geographic isolates subsequently developed differently in the two regions. The Nova Scotian/Canadian isolates showed no variation and were found to be unique compared to the European and Taiwanese forms, indicating that this population is independent in origin. This could indicate that the Canadian parasites were introduced to North America on another occasion and independently of the European colonization.

Taxonomy and host-specificity of Gyrodactylus aideni n. sp. and G. pleuronecti (Monogenea: Gyrodactylidae) from Pseudopleuronectes americanus (Walbaum) in Passamaquoddy Bay, New Brunswick, Canada.

Wild and cultured winter flounder Pseudopleuronectes americanus (Walbaum) from Passamaquoddy Bay were surveyed for species of Gyrodactylus Nordmann, 1832. Two species were found: G. pleuronecti Cone, 1981 and G. aideni n. sp, both members of Malmberg's 'groenlandicus group'. Although the hard parts in the haptor are very similar in the two species, hamuli of G. aideni are consistently shorter than those of G. pleuronecti. The two species differed by 35 base pairs in the ITS 1, 5.8 and ITS 2 region. A BLAST search identified a variety of species of Gyrodactylus from marine fishes in the Atlantic Ocean as closest matches, indicating the 'groenlandicus group' is part of a major marine lineage within Gyrodactylus (sensu lato) that has successfully radiated among coastal percid, pleuronectid, cottid and anarhichadid fishes. Exposure experiments suggested that winter flounder is the primary host of both species of parasites and that three other pleuronectid species in the bay may potentially serve only as occasional transport hosts.

First record of Anguillicoloides crassus (Nematoda) in American eels (Anguilla rostrata) in Canadian estuaries, Cape Breton, Nova Scotia.

In the summer of 2007, American eels, Anguilla rostrata, from 2 localities on Cape Breton Island, were found to be infected with the swim bladder nematode Anguillicoloides crassus. This is the first documented report of this highly invasive parasite in Canadian waters. More than half of the yellow eels in Mira River (6 of 10), and 1 eel (of 5) from Sydney Harbour were infected. Parasite intensity ranged from 1 to 11 worms per eel. The occurrence of A. crassus at these 2 localities suggests the need for a more extensive survey on the distribution of this exotic parasite in eel populations throughout Cape Breton Island.

Ectopic ossicles associated with metacercariae of Apophallus brevis (Trematoda) in yellow perch, Perca flavescens (Teleostei): development and identification of bone and chondroid bone.

This paper describes the development and tissues in mineralized ossicles in the musculature of Perca flavescens infected with metacercariae of the trematode Apophallus brevis. Analysis involved light microscopy, transmission and scanning electron microscopy, X-ray scanning electron microprobe analysis, and tetracycline labelling. Two to 14 days post-infection, fibroblast-like host cells stream towards the parasite cyst forming a fusiform cellular capsule. By 14 days post-infection the capsule differentiates into an inner hypertrophied layer, an extensive middle layer of fibroblast-like cells, and a thin outer layer of flattened fibroblast-like cells forming a fibrous sheath at the capsule/muscle interface. From 21-35 days post-infection, a bony tissue is deposited periosteally in an equatorial ring around the cyst. With time, additional tissue is secreted over the ring increasing its thickness and advancing the matrix front towards the poles of the ossicle. Plump osteoblast-like cells cover the developing ossicle and may become trapped within the matrix in lacunae encapsulated by collagen. By 63 days post-infection, medium-sized ossicles are morphologically similar to large cysts from perch captured in the wild; ovoid with two polarized canals, but lacking acellular or lamellar bone-like tissue. Mineralized ossicles contain calcium, phosphorus and oxygen. Large ossicles retrieved from perch given multiple doses of tetracycline revealed discrete fluorescent bands, indicative of incremental growth. Fully developed ossicles are composed of two skeletal tissues, an inner region of chondroid bone and an outer region of acellular, lamellar bone.

Annual cycle of Henneguya doori (Myxosporea) parasitizing yellow perch (Perca flavescens).

Henneguya doori Guilford, 1963 (Myxosporea) has an annual development cycle on the gills of adult Perca flavescens. The cycle involves a period of summer invasion, fall development of plasmodia and initiation of sporogenesis, winter completion of sporogenesis, and spring release of spores. An experiment revealed that rising water temperature in spring promotes cyst rupture and loss. The development of cysts is synchronized within individual fish and among members of the host population. As such, the parasite spends 8-9 mo in perch tissues to produce a single annual generation that culminates in a 4-6 wk period of spore dispersal. The parasite does not appear to be pathogenic. The observed cycle of H. doori repeated itself over 4 yr of sampling in Vinegar Lake, Nova Scotia.

Infrapopulation dispersal of Gyrodactylus colemanensis (Monogenea) on fry of Salmo gairdneri.

Manipulative positioning of Gyrodactylus colemanensis on individually isolated fry of Salmo gairdneri was used to examine the behavior of the parasite during colonization and the influence that site of invasion has on size and spatial distribution of ensuing infrapopulations. The parasite's initial response was to relocate posteriorly on the host's body; those that reached a fin usually end up on or adjacent to the fin's margin. Individuals monitored for up to 15 days postinfection moved both anteriorly and posteriorly on the body surface and relocated to new fins via the body surface. The parasite occurred most frequently on the caudal fin followed by the pectoral and pelvic fins, with length of the fin margin and fin activity appearing to be factors influencing the distribution. Infections originating from the head, flank, and caudal fin similarly rose and fell to extinction or near extinction on the host over 49 days at 10 C. The more posterior the site of invasion, the greater the proportion of parasites carried by the caudal fin. The study concludes that G. colemanensis is restricted in its distribution on the host and that the fin margins may serve as a reliable food source and favor transmission to new hosts.

Initial infection, distribution, and seasonal occurrence of Hoferellus gilsoni (Myxozoa) within a population of Anguilla rostrata (Anguillidae) in southern Nova Scotia.

Recently transformed elvers of Anguilla rostrata, entering the mouth of the East River in the Timber/Connaught Lake watershed (Nova Scotia) from the sea in mid-May, did not develop infections of the urinary bladder parasite Hoferellus gilsoni (Myxozoa) after nearly 3 mo in captivity. However, 40% of elvers collected from the mouth of the river in early June and 80% in late June exhibited patent infections by mid-August. It is speculated that given this initial level of infection, the summer run of elvers (estimated to be 208,000-963,000 individuals annually) represents a significant upstream dispersal of spores. Infections in yellow eel, which has a limited home range, probably contribute to spore dispersal within the home range and downstream through spore drift. Sporogenesis occurred in samples of yellow eel collected over an 8-mo period from April to November. Eel length was similar in 3 widely separated sampling locations in the watershed. Prevalences at these localities were 44, 36, and 57% in April and, by June, reached 76, 70, and 90%, respectively. Prevalences remained near the June levels until sampling ceased in November 1998. Studies on captive fish suggest that H. gilsoni can overwinter in the eel. Data from the study indicate that immediate infection of young elvers entering the system, effective spore dispersal, and a seasonally extended sporogenesis all contribute to the ubiquitous presence of the parasite in the eel population.

Myxozoan communities parasitizing Notropis hudsonius (Cyprinidae) at selected localities on the St. Lawrence River, Quebec: possible effects of urban effluents.

Late summer myxozoan infra- and component communities parasitizing 73 Notropis hudsonius at 5 sites on the St. Lawrence River upstream and downstream from the island of Montreal are described from study of histological sections of individual fish. Community membership included Myxobolus sp. A (intracellular in striated muscle fibers), Myxobolus sp. B (intracellular in striated muscle fibers), Myxobolus sp. C (brain), and Thelohanellus notatus (Mavor, 1916) Kudo, 1929 (loose connective tissue), all of which are histozoic myxobolids displaying strict tissue specificity for trophozoite development. Mean infracommunity richness in fish at the separate localities was estimated to be 0.4 +/- 0.5 to 1.3 +/- 0.7, with a maximum richness of 3 in any 1 fish. Component community richness in host samples was 2 to 4. It is argued that these are relatively high levels of diversity for freshwater fish parasites but that the values are probably conservative because of the study of only portions of individual fish. The percentage of fish infected with myxozoans of any species and infracommunity richness was significantly greater below the island of Montreal compared with above it. It is suspected that increased oligochaete populations at these sites, resulting from sewage-caused organic enrichment of sediments, may have accounted for the observed increased prevalence of infection.

Myxosporidan parasites for pumpkinseed (Lepomis gibbosus L.) from Ontario.

Five species of myxosporideans were found in pumpkinseed (Lepomis gibbosus L.) from Ryan Lake, Algonquin Park, Ontario. Myxobolus osburni Herrick 1936 was found in pancreatic tissue. Myxobilatus ohioensis (Herrick 1941) Davis 1944 was found in the ureters and urinary bladder. Three new species are described, namely, Myxobolus uvuliferis sp. n. (in the fibrous capsule surrounding the metacercariae of Uvulifer ambloplitis (Hughes 1927), Myxobolus magnaspherus sp. n. (in the parietal peritoneum) and Myxobolus dechtiari sp. n. (in the gills). Myxobolus uvuliferis sp. n. is most similar to M. osburni, but its spores are subspherical and not spherical. Myxobolus magnaspherus sp. n. is most similar to Myxobolus gigas Auerbach 1906, but its spores are wider than long, and they have exceedingly long polar filaments. Myxobolus dechtiari sp. n. is similar to Myxobolus karelicus Petruschewsky 1940 but it lacks an intercapsular appendix.

Settling rates and density of spores of Henneguya doori (Myxosporea) in water.

Using Henneguya doori from Perca flavescens (yellow perch), the current study provides a method for examining settling rates, attitudes, and densities of myxosporean spores. Fresh and fixed spores settled similarly at 8.34 x 10(-6) m/sec and 8.53 x 10(-6) m/sec, respectively. The spores settled in the anterior end first, did not rotate, and had a specific gravity of 1.062. Comparison of settling rates proved more effective for determining spore density than direct chemical estimation. The slow rate of settling permits effective dispersal in open water.

Myxobolus mississippiensis n. sp. (Myxosporea) from gills of Lepomis macrochirus in Mississippi.

Myxobolus mississippiensis n. sp. is described from gill lamellae of the bluegill (Lepomis macrochirus) inhabiting the Pascagoula River System, Mississippi. Fresh spores measure 16.4-18.7 microns long, 3.9-6.2 microns wide, and 4.7-6.2 microns thick. Spore width to length ratio is 1:3.2. Polar capsules are 5.5-7.8 microns long and 1.5-2.3 microns wide, with 9-10 filament coils that when extruded measure 42.1 +/- 4.2 microns. This parasite is unique among known species of Myxobolus in having spores that are lenticular in frontal view.

On the distribution and abundance of eel parasites in Nova Scotia: influence of pH.

The geographic distribution of metazoan parasites of American eels (Anguilla rostrata) was determined from 28 sites in the Southern Upland and adjacent regions of Nova Scotia. Twelve parasite species were encountered. Component community diversity as measured by species richness, Shannon-Wiener index (H') and Hill's number (N1) decreased when pH < 5.4. Seven species (Azygia longa, Crepidostomum brevivitellum, Bothriocephalus claviceps, Proteocephalus macrocephalus, Paraquimperia tenerrima, Echinorhynchus salmonis, Ergasilus celestis) were found in both the Southern Upland and adjacent watersheds to the north. Three species (Pseudodactylogyrus anguillae, Diplostomum sp., Pomphorhynchus bulbocolli) were found only at sites beyond the Southern Upland, which flowed into the Bay of Fundy and had high pH values ( > 5.4). Two species (Daniconema anguillae, Echinorhynchus lateralis) were limited to sites within the Southern Upland. Digeneans were absent from rivers in the southwest with pH values < 4.7. Variation in occurrence of parasite species was observed among rivers within a single watershed and among years within individual sites. Nevertheless, the impact of acidity at the local level was observed regionally across the Southern Upland of Nova Scotia. These data support the hypothesis that parasite assemblages may be good indicators of environmental stress.

Species of Myxobolus (Myxozoa) from the bulbus arteriosus of centrarchid fishes in North America, with a description of two new species.

Three species of Myxobolus (Myxozoa, Myxosporea) occur in heart tissue of centrarchids. Myxobolus paralintoni Li and Desser, 1985 from Lepomis gibbosus in Algonquin Park and in Lake Erie, Ontario, has subcircular spores (in plane of spore length) in frontal view (11-13 microm long, 9-10 microm wide, and 5 microm thick) with a width-to-length ratio of 1:1.2. Myxobolus jollimorei n. sp. from Lepomis macrochirus in Lake Erie and in the Pascagoula River System, Mississippi, has subcircular spores (in plane of spore width) in frontal view (10.0-11.5 microm long, 12.0-14.5 microm wide, and 6.5-8.0 microm thick) with a width-to-length ratio of 1:0.8. Myxobolus manueli n. sp. from Pomoxis nigromaculatus in Lake Erie has spores (10-11 microm long, 8-10 microm wide, and 6.5-7.0 microm thick) that are nearly circular in frontal view but that have 2 distinct sublateral knobs along the sutural ridge and a width-to-length ratio of 1:1.2. All 3 species occur in the bulbus arteriosus of their hosts where they form small, saucer-shaped pseudocysts. Free spores were found free in the lumen of the heart and bulbus arteriosus, in bile, and in kidney tissue presses.

Myxobolus petenensis n. sp (Myxosporea) from the circumorbital integument of Dorosoma petenense (Clupeidae) in Kentucky Lake.

Myxobolus petenensis n. sp. is described from the circumorbital integument of threadfin shad (Dorosoma petenense) in Kentucky Lake, Kentucky. Preserved spores are subspherical in frontal view and measure 10.1-13.1 microm long and 12.0-15.8 microm wide. The polar capsules are pyriform and often unequal in size, with the larger capsule measuring 6.6-8.0 microm long and 4.8-5.7 microm wide and the smaller capsule 5.7-7.4 microm long and 4.2-5.5 microm wide. Examination of several clupeids, skipjack herring (Alosa chrysochloris), gizzard shad (D. cepedianum), and threadfin shad revealed M. petenensis to infect only threadfin shad. This report of M. petenensis is the first known species of Myxobolus infecting a species of freshwater fish of the family Clupeidae in North America.

On the distribution and abundance of eel parasites in Nova Scotia: local versus regional patterns.

Site-to-site variation in macroparasite species distribution and richness was observed in eels (Anguilla rostrata) among 7 sites in the Timber Lake drainage system, Nova Scotia. There was a positive relationship between maximum local species distribution, as measured by maximum prevalence at a site, and regional distribution, as measured by the number of sites in which a parasite was found within the watershed and among 28 sites across Nova Scotia. Species richness increased with spatial scale. However, the species richness of host-specific parasites approached a maximum at the spatial scale of the watershed, whereas that of generalist parasites continued to increase with increasing scale. The near-peak in species richness of eel specialists at the watershed level suggests that in a functional sense for these parasites it is the spatial scale of the watershed that serves as a species pool from which local species richness is derived.

Myxobolus cognati n. sp. (Myxosporea) from the opercular integument of Cottus cognatus (Cottidae) in Lake Michigan.

A myxosporean producing aggregations of white pseudocysts in skin covering the posterior margin of the operculum and perioral tissue of Cottus cognatus in Lake Michigan is described as Myxobolus cognati n. sp. (Myxosporea). Histological sections revealed that the parasite develops in vascularized regions of the dermis and with development protrudes beyond the surrounding skin surface. Fixed spores are oval in front view, measure 12.0-14.0 microns long and 9.5-10.5 microns wide, and contain 8-11 filament coils wound tightly and perpendicular to the longitudinal axis of the capsule. Myxobolus cognati resembles Myxobolus rhinichthidis Fantham, Porter and Richardson, 1939 from subepithelial tissue of the operculum of the cyprinid Rhinichthys cataractae. Spores in the 2 species are the same size and shape. However, in contrast to those of M. cognati, spores of M. rhinichthidis have filament coils wound loosely at 45 degrees to the axis of the polar capsule and have no sutural ridge folds.

The demise of a phylum of protists: phylogeny of Myxozoa and other parasitic cnidaria.

The notion that members of the phylum Myxozoa Grassé, 1970 do not properly belong in classifications of protists has frequently been suggested because the infective spores of these parasites are not unicellular. Systematists have failed to be decisive about myxozoan phylogenetic affinities, either finding the suggestion of a cnidarian connection to be preposterous or considering the recent suggestion of a relationship with nematodes to be an obvious failure of molecular phylogenetics. Thus, the group has remained in classifications as a protistan phylum in its own right. The ultrastructure of the development of myxozoans was critically re-examined in order to more fully explore the possibility of morphological synapomorphies with metazoan taxa. These morphological characters, in combination with small ribosomal subunit gene sequences, were used in a phylogenetic analysis in order to assess myxozoan origins. The results unequivocally support the inclusion of myxozoans as a clade of highly derived parasitic cnidarians, and as sister taxon to the narcomedusan Polypodium hydriforme. Reassessment of myxozoans as metazoans reveals terminal differentiation, typical metazoan cellular junctions, and collagen production. Their "polar capsules" are redescribed as typical nematocysts bearing atrichous isorhiza. Insofar as taxa cannot be contained within other taxa of equal rank, the phylum Myxozoa is abandoned and it is recommended that the group as a whole be removed from all protistan classifications and placed in a more comprehensive cnidarian system.