Sean A. Locke

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.

Morphological and Molecular Differentiation of Clinostomum complanatum and Clinostomum marginatum (Digenea: Clinostomidae) Metacercariae and Adults

abstract:  The separation of Clinostomum complanatum Rudolphi, 1814 and Clinostomum marginatum Rudolphi, 1819 has long been unclear. Recent data confirm the validity of the junior species, C. marginatum, by ∼1% differences in its 18S rDNA sequences. We collected adults and metacercariae of C. complanatum and C. marginatum and found reliable morphological differences in the genital complex at both developmental stages. In addition, we identified basic morphometrics (distance between suckers, body width) in metacercariae that may be useful for discriminating the species. The morphological differences were supported by the comparison of sequences of internal transcribed spacers of ribosomal DNA and of the mitochondrial gene cytochrome c oxidase I (COI) from 39 specimens. In 36 specimens, the average divergence between the species was 7.3% in ITS and 19.4% in COI sequences. Two specimens from North America and 1 from Europe had sequences that did not allow them to be clearly allied with either species.

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.

New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.

Status and prospects of DNA barcoding in medically important parasites and vectors

For over 10 years, DNA barcoding has been used to identify specimens and discern species. Its potential benefits in parasitology were recognized early, but its utility and uptake remain unclear. Here we review studies using DNA barcoding in parasites and vectors affecting humans and find that the technique is accurate (accords with author identifications based on morphology or other markers) in 94-95% of cases, although aspects of DNA barcoding (vouchering, marker implicated) have often been misunderstood. In a newly compiled checklist of parasites, vectors, and hazards, barcodes are available for 43% of all 1403 species and for more than half of 429 species of greater medical importance. This is encouraging coverage that would improve with an active campaign targeting parasites and vectors.

A large‐scale molecular survey of Clinostomum (Digenea, Clinostomidae)

Members of the genus Clinostomum Leidy, 1856 are parasites that mature in birds, with occasional reports in humans. Because morphological characters for reliable discrimination of species are lacking, the number of species considered valid has varied by an order of magnitude. In this study, sequences from the DNA barcode region of cytochrome c oxidase I (CO1) and/or internal transcribed spacer (ITS) from specimens from Mexico, Bolivia, Peru, Brazil, Kenya, China and Thailand were analysed together with published sequences from Europe, Africa, Indonesia and North America. Although ITS and CO1 distances among specimens were strongly correlated, distance‐based analysis of each marker yielded different groups. Putative species indicated by CO1 distances were consistent with available morphological identifications, while those indicated by ITS conflicted with morphological identifications in three cases. There was little overlap in sequence variation within and between species, particularly for CO1. Although ITS and CO1 distances tended to increase in specimens that were further apart geographically, this did not impair distance‐based species delineation. Phylogenetic analysis suggests a deep division between clades of Clinostomum inhabiting the New World and Old World, which parallels the distribution of their principal definitive hosts, the Ardeidae.

Vulnerability and diet breadth predict larval and adult parasite diversity in fish of the Bothnian Bay

Recent studies of aquatic food webs show that parasite diversity is concentrated in nodes that likely favour transmission. Various aspects of parasite diversity have been observed to be correlated with the trophic level, size, diet breadth, and vulnerability to predation of hosts. However, no study has attempted to distinguish among all four correlates, which may have differential importance for trophically transmitted parasites occurring as larvae or adults. We searched for factors that best predict the diversity of larval and adult endoparasites in 4105 fish in 25 species studied over a three-year period in the Bothnian Bay, Finland. Local predator–prey relationships were determined from stomach contents, parasites, and published data in 8,229 fish in 31 species and in seals and piscivorous birds. Fish that consumed more species of prey had more diverse trophically transmitted adult parasites. Larval parasite diversity increased with the diversity of both prey and predators, but increases in predator diversity had a greater effect. Prey diversity was more strongly associated with the diversity of adult parasites than with that of larvae. The proportion of parasite species present as larvae in a host species was correlated with the diversity of its predators. There was a notable lack of association with the diversity of any parasite guild and fish length, trophic level, or trophic category. Thus, diversity is associated with different nodal properties in larval and adult parasites, and association strengths also differ, strongly reflecting the life cycles of parasites and the food chains they follow to complete transmission.

Estimating diet in individual pumpkinseed sunfish Lepomis gibbosus using stomach contents, stable isotopes and parasites

The diets of 99 pumpkinseed sunfish Lepomis gibbosus from a pair of small, adjacent lakes in Ontario, Canada, were estimated from their stomach contents, trophically transmitted parasites and stable isotopes of carbon and nitrogen in fish tissue. The three methods provided virtually unrelated information. There was no significant correlation in the importance of any prey item across all three methods. Fish with similar diets according to one method of estimating diet showed no tendency to be similar according to other methods. Although there was limited variation in fish size and the spatial scale of the study was small, both fish size and spatial origin showed comparatively strong associations with diet data obtained with all three methods. These results suggest that a multidisciplinary approach that accounts for fish size and spatial origins is necessary to accurately characterize diets of individual fish.

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.

Altered trophic pathway and parasitism in a native predator (Lepomis gibbosus) feeding on introduced prey (Dreissena polymorpha)

Populations of invasive species tend to have fewer parasites in their introduced ranges than in their native ranges and are also thought to have fewer parasites than native prey. This ‘release’ from parasites has unstudied implications for native predators feeding on exotic prey. In particular, shifts from native to exotic prey should reduce levels of trophically transmitted parasites. We tested this hypothesis in native populations of pumpkinseed sunfish (Lepomis gibbosus) in Lake Opinicon, where fish stomach contents were studied intensively in the 1970s, prior to the appearance of exotic zebra mussels (Dreissena polymorpha) in the mid-1990s. Zebra mussels were common in stomachs of present-day pumpkinseeds, and stable isotopes of carbon and nitrogen confirmed their importance in long-term diets. Because historical parasite data were not available in Lake Opinicon, we also surveyed stomach contents and parasites in pumpkinseed in both Lake Opinicon and an ecologically similar, neighboring lake where zebra mussels were absent. Stomach contents of pumpkinseed in the companion lake did not differ from those of pre-invasion fish from Lake Opinicon. The companion lake, therefore, served as a surrogate “pre-invasion” reference to assess effects of zebra mussel consumption on parasites in pumpkinseed. Trophically transmitted parasites were less species-rich and abundant in Lake Opinicon, where fish fed on zebra mussels, although factors other than zebra mussel consumption may contribute to these differences. Predation on zebra mussels has clearly contributed to a novel trophic coupling between littoral and pelagic food webs in Lake Opinicon.