J. Daniel McLaughlin

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.

DNA barcodes show cryptic diversity and a potential physiological basis for host specificity among Diplostomoidea (Platyhelminthes: Digenea) parasitizing freshwater fishes in the St. Lawrence River, Canada.

Diplostomoid metacercariae parasitize freshwater fishes worldwide and cannot be identified to species based on morphology. In this study, sequences of the barcode region of cytochrome c oxidase subunit 1 (CO1) were used to discriminate species in 1088 diplostomoids, most of which were metacercariae from fish collected in the St. Lawrence River, Canada. Forty‐seven diplostomoid species were detected, representing a large increase in known diversity. Most species suggested by CO1 sequences were supported by sequences of internal transcribed spacer (ITS) of rDNA and host and tissue specificity. Three lines of evidence indicate that physiological incompatibility between host and parasite is a more important determinant of host specificity than ecological separation of hosts and parasites in this important group of freshwater fish pathogens. First, nearly all diplostomoid species residing outside the lens of the eyes of fish are highly host specific, while all species that occur inside the lens are generalists. This can be plausibly explained by a physiological mechanism, namely the lack of an effective immune response in the lens. Second, the distribution of diplostomoid species among fish taxa reflected the phylogenetic relationships of host species rather than their ecological similarities. Third, the same patterns of host specificity were observed in separate, ecologically distinctive fish communities.

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.

LINKING LARVAE AND ADULTS OF APHARYNGOSTRIGEA CORNU, HYSTEROMORPHA TRILOBA, AND ALARIA MUSTELAE (DIPLOSTOMOIDEA: DIGENEA) USING MOLECULAR DATA

abstract:  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.

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.

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.

Effects of herbicides and the chytrid fungus Batrachochytrium dendrobatidis on the health of post-metamorphic northern leopard frogs (Lithobates pipiens)

Effects of exposure to contaminants such as pesticides along with exposure to pathogens have been listed as two major contributors to the global crisis of declining amphibian populations. These two factors have also been linked in explanations of the causes of these population declines. We conducted a combined exposure experiment to test the hypothesis that exposure to two agricultural herbicides would increase the susceptibility of post-metamorphic northern leopard frogs (Lithobates pipiens) to the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed the independent and interactive effects of these exposures on the health and survival of the frogs. Wild-caught frogs underwent a 21-day exposure to a nominal concentration of either 2.1 μg/L atrazine (Aatrex(®) Liquid 480) or 100 μg a.e./L glyphosate (Roundup(®) Original), followed by Bd, and then were observed until 94 days post-initial exposure to the herbicides. Actual levels of atrazine were between 4.28 ± 0.04 μg/L and 1.70 ± 0.26 μg/L while glyphosate degraded from 100 μg a.e./L to approximately 7 μg a.e./L within 6 days of initial exposure to the herbicides. Compared to controls, the glyphosate formulation reduced the snout-vent length of frogs during the pesticide exposure (at Day 21), and the atrazine formulation reduced gain in mass up to Day 94. No treatment affected survival, splenosomatic or hepatosomatic indices, the densities and sizes of hepatic and splenic melanomacrophage aggregates, the density and size of hepatic granulomas, proportions of circulating leucocytes, the ratio of neutrophils to lymphocytes, or the ratio of leucocytes to erythrocytes. Histological assessment of samples collected at Day 94 revealed no evidence of Bd infection in any Bd-exposed frogs, while real-time PCR detected only one case of light infection in a single atrazine- and Bd-exposed frog. Frogs exposed to Bd shed their skin significantly more frequently than Bd-unexposed frogs, which may have helped them resist or clear infection, and could explain why no interaction between the herbicides and Bd was detected. The results suggest that these frogs were resistant to Bd infection and that pre-exposure to the herbicides did not alter this resistance. The effects seen on the growth following herbicide exposure is a concern, as reduced growth can lower the reproductive success and survival of the amphibians.

Associations between body composition and helminths of lesser snow geese during winter and spring migration.

Costs of parasitism are predicted to be higher with greater parasite intensities and higher inter-parasite competition (diversity). We tested whether greater helminth intensities and diversity were associated with poorer body composition (whole-body fat, protein, mineral and true body mass) in lesser snow geese, Chen caerulescens caerulescens. As part of a larger study on nutritional ecology, 828 wintering or migrating geese were shot between January and May 1983 in 27 different date-locations (samples) during their northward migration through mid-continental North America. A large proportion of overall variation in body composition and parasite communities was among samples, so we analyzed data within each of the 27 samples, controlling for structural body size (the first principal component of 10 body size measurements), sex and the age of geese. There was no compelling evidence that cestodes, trematodes or helminth diversity were associated with variation in body composition but nematodes had several negative associations with fat reserves. However, negative associations between fat reserves and nematodes occurred most often in geese collected between March and May when nematode prevalences and intensities were relatively low. This suggests several possibilities: that the most common nematodes (Heterakis dispar and Trichostrongylus tenuis) were more virulent at this time, that infected individuals had been chronically infected and suffered cumulative nutrient deficits that lasted until late in the spring migration, or that geese became more vulnerable to the effects of parasites at this time of year, possibly because they redirected resources away from immunity toward fat storage in preparation for reproduction.

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.

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.