Lesser snow goose helminths show recurring and positive parasite infection-diversity relations.

The patterns and mechanisms by which biological diversity is associated with parasite infection risk are important to study because of their potential implications for wildlife population's conservation and management. Almost all research in this area has focused on host species diversity and has neglected parasite diversity, despite evidence that parasites are important drivers of community structure and ecosystem processes. Here, we assessed whether presence or abundance of each of nine helminth species parasitizing lesser snow geese (Chen caerulescens) was associated with indices of parasite diversity (i.e. species richness and Shannon's Diversity Index). We found repeated instances of focal parasite presence and abundance having significant positive co-variation with diversity measures of other parasites. These results occurred both within individual samples and for combinations of all samples. Whereas host condition and parasite facilitation could be drivers of the patterns we observed, other host- or parasite-level effects, such as age or sex class of host or taxon of parasite, were discounted as explanatory variables. Our findings of recurring and positive associations between focal parasite abundance and diversity underscore the importance of moving beyond pairwise species interactions and contexts, and of including the oft-neglected parasite species diversity in infection-diversity studies.

Cyclocoelid ( Morishitium sp.) Trematodes from an Air Sac of a Purple Sandpiper, Calidris maritima (Brünnich).

Like many shorebirds, purple sandpipers ( Calidris maritima [Brünnich] Charadriiformes: Scolopacidae) have experienced population declines in recent years, but causes of these declines have not been established. As part of a larger study to identify causes of these declines, we collected and examined 25 purple sandpipers in coastal Nova Scotia, Canada during March 2013. In the course of dissections to collect tissue samples, we detected a cluster of trematodes in the air sac of 1 bird that were subsequently identified as cyclocoelids belonging to the genus Morishitium Witenberg, 1928, which we believe is the first report of this genus of parasites in this host. Cyclocoelids have been reported from other scolopacids and other shorebird families, but we are unaware of reports of serious pathology arising from these trematodes. Given this and the low prevalence (4%) in our sample, our data cannot ascribe declines in purple sandpiper populations to these trematodes, but our sample is limited both spatially and temporally.

Diversity, specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of freshwater fish, as revealed by DNA barcodes.

Larvae (metacercariae) in some species of Diplostomidae (Platyhelminthes: Digenea) inhabit fish eyes and are difficult to identify to species based on morphology. DNA barcoding has clarified the diversity and life cycles of diplostomids in North America, Europe and Africa, but has seldom been used in parasites sampled in large numbers or at large spatial scales. Here, distance-based analysis of cytochrome c oxidase 1 barcodes and, in some specimens, internal transcribed spacer (ITS-1, 5.8S, ITS-2) sequences was performed for over 2000 diplostomids from Africa, the Middle East, Europe, Asia and the Americas. Fifty-two species of Diplostomum, Tylodelphys and Austrodiplostomum (Digenea: Diplostomidae) were distinguished. The 52 species comprise 12 identified species, six species in two species complexes and 34 putative species, and 33/52 had been delineated in previous studies. Most (23/40) of the unidentified, putative species distinguished by cytochrome c oxidase 1 distances were supported by at least one additional line of evidence. As the intensity of sampling of the 52 species increased, variation in cytochrome c oxidase 1 decreased between and increased within species, while the spatial scale at which species were sampled had no effect. Nonetheless, variation between species always exceeded variation within species. New life-cycle linkages, geographic and host records, and genetic data were recorded in several species, including Tylodelphys jenynsiae, Tylodelphys immer and Diplostomum ardeae. Species of Diplostomum inhabiting the lens are less host-specific and less numerous than those infecting other tissues, suggesting that reduced immune activity in the lens has influenced rates of speciation.

Community structure of Diplostomum spp. (Digenea: Diplostomidae) in eyes of fish: main determinants and potential interspecific interactions.

Parasite communities in freshwater fish vary annually and seasonally and can be influenced by the length, age, sex and phylogeny of hosts, and by associations among parasite species. We assessed the influence of these factors in species of Diplostomum in the eyes of 828 fish in 20 different species collected in a single lake in early summer or autumn over a 5year interval. Strong negative associations were found between five pairs of Diplostomum spp. and associations were strongest among abundant species. Most interspecific associations occurred among species inhabiting the lens, suggesting competitive interactions. However, the strongest association occurred between two Diplostomum spp. that inhabit different tissues (i.e., the vitreous humour and lens), indicating processes other than direct competition. Seasonal variation was small compared with inter-annual variation. Infection intensities were high in 2006 and decreased dramatically in 2010 and 2011. Infracommunity composition and structure showed no clear correspondence to the ecology or phylogeny of host species. Host length and age, but not sex, had significant effects on the structure of Diplostomum infracommunities in lenses. However, a significant amount of variance in lens infracommunities could not be explained, indicating the potential importance of other factors such as resistance or exposure in determining infracommunity structure.

Description of Gyrodactylus mediotorus n. sp. (Monogenea: Gyrodactylidae) infecting spottail shiner (Notropis hudsonius) from the St. Lawrence River, Canada.

Gyrodactylus mediotorus n. sp. is described from the body, fins, and buccal cavity of the spottail shiner, Notropis hudsonius (Cyprinidae) from the St. Lawrence River, Quebec, Canada. Gyrodactylus mediotorus n. sp. is the first species of Gyrodactylus Nordmann, 1832 described from N. hudsonius and is characterized by large hamuli, large medial process of the ventral bar, narrow linguiform ventral bar membrane, large anterolateral processes, and marginal hooks with long shafts and distinctly shaped sickle. The species that most resembles Gyrodactylus mediotorus n. sp. is Gyrodactylus protuberus Rogers and Wellborn, 1965 described from the stargazing shiner, Notropis uranoscopus Suttkus, 1959. The 2 species can be differentiated based on the larger hamuli (68.4 vs. 64) and ventral bar (38.4 vs. 24) of Gyrodactylus mediotorus n. sp. and the shape of the marginal hooks which for Gyrodactylus mediotorus n. sp. has a slightly larger toe and a point which is not as angled. The morphological description is supplemented with 436 sequenced base pairs of the 18S gene (including the V4 region) as well as 1,041 sequenced base pairs spanning the complete ITS-1, 5.8S, and ITS-2 regions. BLAST (Basic Local Alignment Search Tool) searches failed to provide any close matches for either regions of DNA, with Gyrodactylus colemanensis infecting Salvelinus fontinalis being the most genetically similar for both the 18S (∼91%, JF836090) and ITS (∼84%, JF836142) rDNA regions. Gyrodactylus mediotorus n. sp. has been found infecting spottail shiners in the St. Lawrence River in low prevalence and intensities periodically over the last 15 yr.

Mortality of American bullfrog tadpoles Lithobates catesbeianus infected by Gyrodactylus jennyae and experimentally exposed to Batrachochytrium dendrobatidis.

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis in postmetamorphic amphibians, has been linked to amphibian population declines. Different amphibian species, however, exhibit different susceptibility to Bd pathogenicity. At the same time, agricultural pesticides commonly found contaminating aquatic habitats have been reported to increase the susceptibility of amphibians to pathogens. To investigate whether certain pesticides are able to alter the pathogenicity of Bd to larval amphibians, we exposed larval American bullfrogs Lithobates catesbeianus to end-use formulations of the herbicides atrazine or glyphosate, and then exposed them to Bd. Following the experimental exposures, a preexisting infection of the tadpoles by the monogenean ectoparasite Gyrodactylus jennyae was detected in all experimental and control tadpoles. Gyrodactylus jennyae infection intensity varied, and individuals with heavy G. jennyae infections suffered more skin erosion due to grazing by the parasite. Tadpoles experimentally exposed to Bd, or to Bd and either herbicide, had significantly reduced survival rates compared with untreated tadpoles that were only infected by G. jennyae. Increased mortality was also correlated with degree of skin erosion; survival of tadpoles with severe skin erosion was significantly reduced compared with that of tadpoles with no, or mild, skin erosion. While infected with G. jennyae, the group of tadpoles with the lowest survival rate (exposed only to Bd) included significantly more individuals exhibiting severe skin erosion and significantly fewer individuals without skin erosion, compared with the control group. These results emphasize the potential pathogenicity of gyrodactylid infections in larval amphibian hosts and suggest that concomitant exposures to Bd may enhance infections and effects of G. jennyae in bullfrog tadpoles.

Molecular and morphological evidence for the Holarctic distribution of Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888 (Digenea: Leucochloridiidae).

Species of Urogonimus Monticelli, 1888 (Leucochloridiidae Poche, 1907) are difficult to distinguish using adult morphology, and their taxonomy has been repeatedly subjected to revision. Some Nearctic species have been regarded as synonymous with the Palearctic type species Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888. This implies that U. macrostomus is present in the Nearctic, but there is no additional evidence for this putative distribution. We collected trematodes morphologically indistinguishable from U. macrostomus from a house sparrow (Passer domesticus) in Edmonton, Alberta, Canada. Sequences 2958 bp in total length from the small and large subunits of ribosomal DNA from 2 specimens were 99.8-100% identical to those of U. macrostomus in the Ukraine and Japan. In light of the lack of morphological differences and small degree of genetic variation, we consider the specimens we collected to be conspecific with U. macrostomus in the Palearctic, and the Holarctic range of the species is thus supported. Sequences from a more rapidly evolving gene, cytochrome c oxidase 1, were obtained to aid future study of this and related species.

Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus (Digenea): species differentiation based on mtDNA (Barcode) and partial LSU-rDNA sequences.

Flukes belonging to Sphaeridiotrema are important parasites of waterfowl, and 2 morphologically similar species Sphaeridiotrema globulus and Sphaeridiotrema pseudoglobulus, have been implicated in waterfowl mortality in North America. Cytochrome oxidase I (barcode region) and partial LSU-rDNA sequences from specimens of S. globulus and S. pseudoglobulus, obtained from naturally and experimentally infected hosts from New Jersey and Quebec, respectively, confirmed that these species were distinct. Barcode sequences of the 2 species differed at 92 of 590 nucleotide positions (15.6%) and the translated sequences differed by 13 amino acid residues. Partial LSU-rDNA sequences differed at 29 of 1,208 nucleotide positions (2.4%). Additional barcode sequences from specimens collected from waterfowl in Wisconsin and Minnesota and morphometric data obtained from specimens acquired along the north shore of Lake Superior revealed the presence of S. pseudoglobulus in these areas. Although morphometric data suggested the presence of S. globulus in the Lake Superior sample, it was not found among the specimens sequenced from Wisconsin or Minnesota.

Linking larvae and adults of Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) using molecular data.

Because the taxonomy of trematodes is based on adults, the larval stages of most digeneans cannot be identified to species based on morphology alone. Molecular data provide a means of linking larval stages to known adults. We obtained sequences from the barcode region of cytochrome oxidase I (CO1) from adult and larval parasites of fish, frogs, birds, and mammals across North America. Sequences from adult Apharyngostrigea cornu, Hysteromorpha triloba, and Alaria mustelae (Diplostomoidea: Digenea) from definitive hosts matched those of meta- and mesocercariae from fish and frogs. These data provided new information on the distributions of all 3 parasite species. Metacercariae of A. cornu, which have not been previously reported in North American hosts, were found in Notemigonus crysoleucas, Pimephales notatus, and Catostomus commersonii in the St. Lawrence River. Metacercariae of H. triloba are reported in Canadian waters and in N. hudsonius for the first time. Alaria mustelae is reported for the first time in frogs from Quebec, Canada, and an additional species of Alaria was detected in California. Sequences of internal transcribed spacer rDNA from a subset of specimens support the same species boundaries indicated by CO1 divergence. There was little divergence in CO1 sequences from an unidentified diplostomid species sampled at a large spatial scale.

Experimental evidence of size/age-biased infection of Biomphalaria glabrata (Pulmonata: Planorbidae) by an incompatible parasite species: consequences for biological control.

Because the digenetic trematode Plagiorchis elegans can elicit a rapid, severe and permanent suppression of the reproductive output in the snail Biomphalaria glabrata, it is considered as a potential biological control agent of human schistosomiasis. This assumption however is derived from laboratory experiments that are poor approximations of what occurs in a natural ecosystem. In order to recreate conditions that resemble those found in nature, we exposed B. glabrata as individual populations composed of a young, juvenile and adult snails to various concentrations of P. elegans eggs to assess the probability of encountering the parasite eggs by the different snail sizes/age groups. We demonstrated that within populations composed of different size/age classes, larger/older snails displayed the negative effects typical of exposure to P. elegans, whereas smaller individuals appeared relatively unaffected, particularly at lower levels of exposure. These findings coupled with the difficulty of producing large quantities of parasite eggs suggest that P. elegans has limited efficiency as a biological control agent of human schistosomiasis.

Parasite fauna of Etheostoma nigrum (Percidae: Etheostomatinae) in localities of varying pollution stress in the St. Lawrence River, Quebec, Canada.

Parasite communities were examined in johnny darters (Etheostoma nigrum) collected from five localities in the St. Lawrence River in southwestern Quebec: two reference localities, one polluted locality upstream of the Island of Montreal and downstream of industrial and agricultural activity, and two polluted localities downstream of the Island of Montreal in the plume from the wastewater treatment facility. Twenty-four helminth species were found. Fish from the upstream polluted locality had the highest parasite species richness and total parasite numbers, and fish from the downstream polluted localities the lowest. Nonmetric multivariate analyses were conducted using square-root-transformed Bray-Curtis dissimilarity index. An analysis of similarity, dendrogram of centroids, and a permutational multivariate analysis of variance with contrasts all showed that fish from the reference localities had different parasite community composition than those from the polluted localities, and fish from the upstream polluted locality had different parasite communities than fish from the downstream polluted localities. Differences between reference and polluted localities were mainly due to higher abundances of the brain-encysting trematode, Ornithodiplostomum sp., at the reference localities. Differences between upstream and downstream polluted localities were mainly due to a higher diversity and abundance of trematodes in fish at the upstream locality.

Diversity and specificity in Diplostomum spp. metacercariae in freshwater fishes revealed by cytochrome c oxidase I and internal transcribed spacer sequences.

In this study, sequences from the barcode region of cytochrome c oxidase I (COI) were used to distinguish Diplostomum spp. in a sample of 497 metacercariae collected from diverse fishes of the St. Lawrence River, Canada and findings were corroborated with internal transcribed spacer (ITS) regions of rDNA. Twelve species were detected based on sequences and metacercarial specificity for hosts and tissues. Although this is an unusually high diversity, additional species are likely to exist in the study area. Two species were indistinguishable with ITS data and there is evidence that they may be undergoing hybridization and/or have recently diverged. The ITS sequences of another species are similar to those of Diplostomum pseudospathaceum from Europe, but ITS data are insufficient to show that they are conspecific. Diplostomum spp. that infect tissues other than the lens are more host-specific than species inhabiting the lenses of fishes, which is attributed to the enhanced immunological privilege of the lens site compared with other tissues. Overall, COI sequences were superior to more commonly used ITS markers for delineating species of this important and taxonomically difficult pathogen.

Plagiorchis elegans (Trematoda) and incompatible snail hosts: implications for snail life history traits and biocontrol of human schistosomiasis.

We examined the effect of Plagiorchis elegans on egg production and survival on Bulinus truncatus and Helisoma trivolvis trivolvis. Both species are incompatible hosts for P. elegans. Helisoma t. trivolvis occurs sympatrically with P. elegans; B. truncatus does not. Overall, P. elegans had no effect on survivorship or egg production in H. t. trivolvis or on the survivorship of B. truncatus. Its effect on egg production in B. truncatus was transitory; egg production was reduced by 50% for 5 wk following exposure but returned to normal thereafter. Neither egg production nor survivorship was affected in adult H. t. trivolvis. Egg production ceased at 14 wk post-exposure (PE), but resumed when the snails were paired. Young H. t. trivolvis also produced eggs after exposure, but later than the adults and only after they had been paired with another snail. This suggests that a need for periodic cross-fertilization in H. t. trivolvis rather than the effect of the parasite is responsible for the cessation of egg production in this species. Survivorship in young H. t. trivolvis was significantly higher in exposed snails between wk 7 to 10 PE than in controls.

Morphology and molecular taxonomy of Gyrodactylus jennyae n. sp. (Monogenea) from tadpoles of captive Rana catesbeiana Shaw (Anura), with a review of the species of Gyrodactylus Nordmann, 1832 parasitising amphibians.

Gyrodactylus jennyae n. sp. is described from the body surface and mouthparts of tadpoles of the bullfrog Rana catesbeiana Shaw imported presumably from Missouri, USA, into a federal government facility in Moncton, New Brunswick, Canada. Its morphology resembles most closely that of G. chologastris Mizelle, Whittaker & McDougal, 1969 described from two amblyopsids (blind cave fishes) in Kentucky and North Carolina. Both species have long slender hamuli, a ventral bar with a relatively long membrane and small anterolateral processes, a cirrus with two rows of small spines and marginal hooks with a well-developed sickle heel and short handle. The two species differ morphologically; G. jennyae has a marginal hook sickle with a more pronounced heel than that found in G. chologastris. A BLAST search using a 945 base pair sequence that included the nuclear ribosomal DNA internal transcribed spacers 1 and 2 and the 5.8S rRNA gene from G. jennyae n. sp. showed that the overall similarity with other Gyrodactylus sequences on GenBank was relatively low. The ITS1 region was similar to that of G. misgurni Ling, 1962; however, no ITS2 and 5.8S rRNA sequences are available for that species. A separate search using 5.8S sequences revealed that G. markakulensis Gvosdev, 1950 and G. laevis Malmberg, 1957 were the closest to G. jennyae (1 and 2 bp differences, respectively). These species are parasites of cyprinids (or their predators) and are similar to G. jennyae and G. chologastris in having a double row of small hooks on the cirrus and overall similar morphologies of the haptoral hard parts. There are now five species of Gyrodactylus described exclusively from amphibians and this appears to have involved at least three separate host-switches from fishes.

Combined effects of agricultural activity and parasites on biomarkers in the bullfrog, Rana catasbeiana.

Agricultural contaminants can have devastating impacts on amphibian survival and development, particularly considering their sensitivity to environmental perturbation. However, it is commonly overlooked that amphibians are infected with various parasites that can influence the overall health of the animal when exposed to a stressful environment. We investigated the interaction of agriculture and parasitism on the health of bullfrogs (Rana catesbeiana) in the field. Nine physiological and immunological biomarkers were related to naturally acquired parasite infections, along a gradient of agricultural activity. Most health biomarkers were affected by agriculture, parasitism, or both. Although bullfrogs residing in agricultural areas were infected with fewer parasite species, reflecting environmentally compromised ecosystems, certain persistent parasites interacted with agricultural disturbance to alter the physiology and immune competence of bullfrogs. The consequences of the combination for animal health highlight the importance of parasitism in ecotoxicological studies. Consideration of parasitism is warranted when evaluating the influence of anthropogenic disturbance on amphibian declines and environmental health.

Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths.

The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9-25%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.

Short-term seasonal changes in parasite community structure in northern leopard froglets (Rana pipiens) inhabiting agricultural wetlands.

Parasite community structure can change seasonally with shifts in host habitat and in diet. However, anthropogenic activity may influence the natural changes in transmission dynamics of different parasite species. Effects of seasonal and agricultural activity on the parasite communities of newly metamorphosed northern leopard frogs (Rana pipiens) were investigated in July and September 2001 in 5 wetlands, 3 of which were exposed to pesticide runoff from surrounding agriculture. Nineteen parasite taxa were found. Component community richness was consistently high at the pristine reference wetland, whereas the communities at a managed reference wetland remained depauperate. Infracommunity richness increased throughout the season, but more so in frogs resident in agricultural wetlands. Digeneans using frogs as intermediate hosts dominated the communities, although many species were much lower in abundance in September, suggesting mortality of heavily infected frogs. Mean abundance of Haematoloechus spp. was positively related to that of odonate naiads in the frog diet, which appeared to reflect differential second intermediate host availability between reference and agricultural wetlands. Although virtually absent from wetlands in July just after frog metamorphosis, monoxenous nematodes were more prevalent and abundant at agricultural wetlands as the season progressed. Our results suggest that agricultural activity may further facilitate the transmission of monoxenous nematodes as frogs become more terrestrial.

Morphological, developmental and ecological evidence for a progenetic life cycle in Neochasmus (Digenea).

Seven species of fishes, Catostomus commersonii (Lacépède), Etheostoma nigrum Rafinesque, Micropterus dolomieu Lacépède, Notemigonus crysoleucas (Mitchill), Notropis hudsonius (Clinton), Perca flavescens (Mitchill), and Percina caprodes (Rafinesque) from the St. Lawrence River, Quebec, Canada, were found infected with progenetic specimens of Neochasmus spp. in the orbits and/or the body musculature. Worms displayed varying degrees of maturation. Eggs occupied the entirety of the worm in late stages of development and persisted as distinct clusters in situ after worm death. Populations of parasites were studied monthly in E. nigrum from one site between May and October in order to follow parasite recruitment, development and maturation. Recruitment of parasites was observed in young-of-the-year fish primarily in July and continued through October. Worms matured rapidly, displaying egg production within a month. Later developmental stages, in which eggs occupied most of the worm, and clusters of eggs became abundant by September. Infections in overwintered fish collected in May consisted mainly of worms in early stages of egg production and of clusters of eggs. When hatched artificially, eggs from the clusters released viable miracidia, indicating that they survive beyond the lifespan of the adult worm. It is suggested that progenesis is a fixed characteristic of the life cycle of these species, that egg dispersal requires the death of the host and that it is facilitated by predation. All prior records of Neochasmus spp. are examined, leading us to conclude that the role of the putative definitive host (primarily basses) has been reduced to that of a dispersal agent. Current hypotheses concerning the evolution and maintenance of progenesis are considered, but it is concluded that they do not apply to this host-parasite system.