Putting infection on the map: Using heatmaps to characterise within- and between-host distributions of trematode metacercariae.

The location of parasites within individual hosts is often treated as a static trait, yet many parasite species can occur in multiple locations or organs within their hosts. Here, we apply distributional heat maps to study the within- and between-host infection patterns for four trematodes (Alaria marcianae, Cephalogonimus americanus, Echinostoma spp. and Ribeiroia ondatrae) within the amphibian hosts Pseudacris regilla and two species of Taricha. We developed heatmaps from 71 individual hosts from six locations in California, which illustrate stark differences among parasites both in their primary locations within amphibian hosts as well as their degree of location specificity. While metacercariae (i.e., cysts) of two parasites (C. americanus and A. marcianae) were relative generalists in habitat selection and often occurred throughout the host, two others (R. ondatrae and Echinostoma spp.) were highly localised to a specific organ or organ system. Comparing parasite distributions among these parasite taxa highlighted locations of overlap showing potential areas of interactions, such as the mandibular inner dermis region, chest and throat inner dermis and the tail reabsorption outer epidermis. Additionally, the within-host distribution of R. ondatrae differed between host species, with metacercariae aggregating in the anterior dermis areas of newts, compared with the posterior dermis area in frogs. The ability to measure fine-scale changes or alterations in parasite distributions has the potential to provide further insight about ecological questions concerning habitat preference, resource selection, host pathology and disease control.

Systematics, distribution and morphology of the newt parasitic water mites of the subgenus Lurchibates Goldschmidt amp; Fu, 2011 (Acari, Hydrachnidia, Hygrobatidae, Hygrobates Koch, 1837), including the description of four new species and a key to all so far known species.

Four new water mite species of the genus Hygrobates, subgenus Lurchibates (Acari, Hydrachnidia, Hygrobatidae) are described. Hygrobates (Lurchibates) macrochela sp. nov. Goldschmidt, Nishikawa Shimano is described in male and female; Hygrobates (Lurchibates) malosimilis sp. nov. Goldschmidt, Nishikawa Shimano is described in male, both new water mite species were collected from newts of the species Pachytriton inexpectatus Nishikawa, Jiang, Matsui Mo, 2011; Hygrobates (Lurchibates) incognitus sp. nov. Goldschmidt, Nishikawa Shimano is described in female collected from newts of the species Paramesotriton guangxiensis (Huang, Tang and Tang, 1983); Hygrobates (Lurchibates) fragmentarius sp. nov. Goldschmidt, Nishikawa Shimano is described in a single severely fragmented specimen (sex could not be determined) collected from a newt of the species Paramesotriton yunwuensis Wu, Jiang, and Hanken, 2010. Principal differences between the subgenus Lurchibates and the nominal subgenus Hygrobates s. str. are illustrated and discussed as well as the sexual dimorphism of Lurchibates. Morphometric analyses confirmed the morphological differences of ten out of the eleven so far described species (H. (L.) fragmentarius could not be included in the analysis); two morphological groups became evident mainly based upon the shape of the anterior coxae. These analyses as well provide an idea of possible phylogenetic relationships among the species. A key to all currently known species of the subgenus Lurchibates is given. So far the subgenus is restricted to SE-Asia, a map showing the distribution of all species is presented.

Community disassembly and disease: realistic-but not randomized-biodiversity losses enhance parasite transmission.

Debates over the relationship between biodiversity and disease dynamics underscore the need for a more mechanistic understanding of how changes in host community composition influence parasite transmission. Focusing on interactions between larval amphibians and trematode parasites, we experimentally contrasted the effects of host richness and species composition to identify the individual and joint contributions of both parameters on the infection levels of three trematode species. By combining experimental approaches with field surveys from 147 ponds, we further evaluated how richness effects differed between randomized and realistic patterns of species loss (i.e. community disassembly). Our results indicated that community-level changes in infection levels were owing to host species composition, rather than richness. However, when composition patterns mirrored empirical observations along a natural assembly gradient, each added host species reduced infection success by 12-55%. No such effects occurred when assemblages were randomized. Mechanistically, these patterns were due to non-random host species assembly/disassembly: while highly competent species predominated in low diversity systems, less susceptible hosts became progressively more common as richness increased. These findings highlight the potential for combining information on host traits and assembly patterns to forecast diversity-mediated changes in multi-host disease systems.

Resistance and tolerance: A hierarchical framework to compare individual versus family-level host contributions in an experimental amphibian-trematode system.

Hosts have two general strategies for mitigating the fitness costs of parasite exposure and infection: resistance and tolerance. The resistance-tolerance framework has been well developed in plant systems, but only recently has it been applied to animal-parasite interactions. However, difficulties associated with estimating fitness, controlling parasite exposure, and quantifying parasite burden have limited application of this framework to animal systems. Here, we used an experimental approach to quantify the relative influence of variation among host individuals and genetic families in determining resistance and tolerance within an amphibian-trematode system. Importantly, we used multiple, alternative metrics to assess each strategy, and employed a Bayesian analytical framework to compare among responses while incorporating uncertainty. Relative to unexposed hosts, exposure to the pathogenic trematode (Ribeiroia ondatrae) reduced the survival and growth of California newts (Taricha torosa) (survival: 93% vs. 74%; growth: 0.29 vs. -0.5 vs mm day -1). Similarly, parasite infection success (the inverse of resistance) ranged from 8% to 100%. Yet despite this broad variation in host resistance and tolerance among individual newts, we found no evidence for transmissable, among-family variation in any of the resistance or tolerance metrics. This suggests that opportunities for evolution of these traits is limited, likely requiring significant increases in mutation, gene flow, or environmental heterogeneity. Our study provides a quantitative framework for evaluating the importance of alternative metrics of resistance and tolerance across multiple time points in the study of host-parasite interactions in animal systems.

Helminth endoparasites of the smooth newt Lissotriton vulgaris: linking morphological identification and molecular data.

The helminth endoparasites of many European amphibian species are often known exclusively from morphological descriptions. A molecular library of DNA sequence data linked to morphological identifications is still in its infancy. In this paper, we aim to contribute to such a library on the smooth newt Lissotriton vulgaris, the intermediate and definitive host of 31 helminth parasites, according to evidence published so far. Newts (n = 69) were collected at two study sites in western Germany and examined for the presence of helminths. A total of five helminth species were detected in 56 (81%) of the newts, but only one or two species infected a single host. Four out of five helminth species were identified morphologically and based on DNA sequences as Parastrigea robusta (metacercariae), Oswaldocruzia filiformis, Megalobatrachonema terdentatum (adults and larvae) and Cosmocerca longicauda, and the corresponding sequences were provided subsequently. Oswaldocruzia molgeta was confirmed to be a junior synonym of O. filiformis. Molecular data on a fifth species (a cosmocercid nematode) that could not be identified at species level were added to GenBank. These findings increased the molecular library on morphologically identified smooth newt parasites significantly, from 12 to 15 entries.

Phylogenetic Relationships of Spiruromorph Nematodes (Spirurina: Spiruromorpha) In North American Freshwater Fishes.

Nematodes are common in the parasite communities of North American freshwater fishes, and the majority of them belong to 1 conventional order, Spirurida Chitwood, 1933. Within the Spirurida, the superfamilies Habronematoidea Chitwood and Wehr, 1932 and Thelazioidea Sobolev, 1949 have undergone considerable diversification. The dominant families of these 2 superfamilies, Cystidicolidae Skrjabin 1946 and Rhabdochonidae Railliet, 1916, respectively, are particularly common, widely distributed, and diverse, especially in North America, yet their phylogenetic relationships remain largely unexplored. In this study, we use near complete sequences of the 18S rRNA genes ( SSU rDNA) from species in 6 genera ( Capillospirura Skrjabin, 1924, Cystidicola Fischer, 1798, Salmonema Moravec, Santos and Brasil-Sato, 2008, Rhabdochona Railliet, 1916, Spinitectus Fourment, 1883, and a putative new cystidicolid in mooneyes, Hiodontidae), along with a species of Hedruris Nitzsch, 1812 from newts as a surrogate for the fish parasite Hedruris tiara VanCleave and Mueller, 1932, to explore their phylogenetic relationships. These sequences, together with available sequences from a range of other nematodes, including fish nematodes in other groups (Camallanoidea and 'Seuratoidea'), were analyzed using Bayesian inference and maximum likelihood. The results from both analyses indicate, for the first time, support for the close relationships of the sturgeon parasite Capillospirura with Ascarophis van Beneden, 1871 and Cystidicola; the relationship of the cystidicolid from Hiodontidae with Salmonema of salmonid fishes; the monophyly of the 2 dominant spiruridan genera of fishes, Rhabdochona and Spinitectus; and for previous relationships among Nearctic Spinitectus spp. The results also indicate a closer relationship of Rhabdochona and Spinitectus than is suggested by their conventional classification and reject the monophyly of Habronematoidea, Thelazioidea, and Cystidicolidae. Hedruridae appears to be an early branching lineage of spirurins. Finally, the pattern of association between the fish parasites in this study and their hosts indicates, with few exceptions, ecologically driven diversification events involving host shifting not related to the phylogenetic relationships of their hosts.

Noxious newts and their natural enemies: Experimental effects of tetrodotoxin exposure on trematode parasites and aquatic macroinvertebrates.

The dermal glands of many amphibian species secrete toxins or other noxious substances as a defense strategy against natural enemies. Newts in particular possess the potent neurotoxin tetrodotoxin (TTX), for which the highest concentrations are found in species within the genus Taricha. Adult Taricha are hypothesized to use TTX as a chemical defense against vertebrate predators such as garter snakes (Thamnophis spp.). However, less is known about how TTX functions to defend aquatic-developing newt larvae against natural enemies, including trematode parasites and aquatic macroinvertebrates. Here we experimentally investigated the effects of exogenous TTX exposure on survivorship of the infectious stages (cercariae) of five species of trematode parasites that infect larval amphibians. Specifically, we used dose-response curves to test the sensitivity of trematode cercariae to progressively increasing concentrations of TTX (0.0 [control], 0.63, 3.13, 6.26, 31.32, and 62.64 nmol L-1) and how this differed among parasite species. We further compared these results to the effects of TTX exposure (0 and 1000 nmolL-1) over 24 h on seven macroinvertebrate taxa commonly found in aquatic habitats with newt larvae. TTX significantly reduced the survivorship of trematode cercariae for all species, but the magnitude of such effects varied among species. Ribeiroia ondatrae - which causes mortality and limb malformations in amphibians - was the least sensitive to TTX, whereas the kidney-encysting Echinostoma trivolvis was the most sensitive. Among the macroinvertebrate taxa, only mayflies (Ephemeroptera) showed a significant increase in mortality following exogenous TTX exposure, despite the use of a concentration 16x higher than the maximum used for trematodes. Our results suggest that maternal investment of TTX into larval newts may provide protection against certain trematode infections and highlight the importance of future work assessing the effects of newt toxicity on both parasite infection success and the palatability of larval newts to invertebrate predators.

Pathological and phylogenetic characterization of Amphibiothecum sp. infection in an isolated amphibian (Lissotriton helveticus) population on the island of Rum (Scotland).

Outbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infected L. helveticus in France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction from Amphibiocystidium spp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that the L. helveticus-infecting pathogen represents a single, novel species; Amphibiothecum meredithae.

Why are the prevalence and diversity of helminths in the endemic Pyrenean brook newt Calotriton asper (Amphibia, Salamandridae) so low?

A cornerstone in parasitology is why some species or populations are more parasitized than others. Here we examine the influence of host characteristics and habitat on parasite prevalence. We studied the helminths parasitizing the Pyrenean brook newt Calotriton asper (n= 167), paying special attention to the relationship between parasites and ecological factors such as habitat, sex, ontogeny, body size and age of the host. We detected two species of parasites, Megalobatrachonema terdentatum (Nematoda: Kathlaniidae) and Brachycoelium salamandrae (Trematoda: Brachycoeliidae), with a prevalence of 5.99% and 1.2%, respectively. Marginally significant differences were found in the prevalence between sexes, with females being more parasitized than males. The present results show significant differences in the body length of paedomorphic and metamorphic individuals, the former being smaller. Nevertheless, no significant correlations between parasite prevalence and either newt body length, ontogenetic stage or age were found. In comparison with other Salamandridae living in ponds, prevalence and diversity values were low. This may be due to a long hibernation period, the species' lotic habitat and its reophilous lifestyle, which probably do not allow for a high parasite load.

Metacercariae of Clinostomum complanatum (Trematoda: Digenea) in European newts Triturus carnifex and Lissotriton vulgaris (Caudata: Salamandridae).

Adults of Clinostomum spp. are digenetic trematodes found in fish-eating birds, reptiles and occasionally mammals, including humans. Freshwater snails serve as first intermediate hosts and many fish species and amphibians as second intermediate hosts. To date, amphibian hosts of Clinostomum metacercariae include members of urodele and anuran families in North America, but no data are available on infections of European amphibians, including newts. In this study, we characterize infections of Clinostomum complanatum metacercariae in four smooth (Lissotriton vulgaris) and 18 Italian crested newts (Triturus carnifex) from an artificial pond located in a protected area in Tuscany, Italy. Parasites were surgically removed from the infected newts and identified both morphologically and using sequences of a mitochondrial gene, cytochrome c oxidase I, and the ribosomal markers, internal transcribed spacers. This is the first record of C. complanatum in European newts and, more generally, in amphibians in Europe.

Parasite transmission in complex communities: predators and alternative hosts alter pathogenic infections in amphibians.

While often studied in isolation, host-parasite interactions are typically embedded within complex communities. Other community members, including predators and alternative hosts, can therefore alter parasite transmission (e.g., the dilution effect), yet few studies have experimentally evaluated more than one such mechanism. Here, we used data from natural wetlands to design experiments investigating how alternative hosts and predators of parasites mediate trematode (Ribeiroia ondatrae) infection in a focal amphibian host (Pseudacris regilla). In short-term predation bioassays involving mollusks, zooplankton, fish, larval insects, or newts, four of seven tested species removed 62-93% of infectious stages. In transmission experiments, damselfly nymphs (predators) and newt larvae (alternative hosts) reduced infection in P. regilla tadpoles by -50%, whereas mosquitofish (potential predators and alternative hosts) did not significantly influence transmission. Additional bioassays indicated that predators consumed parasites even in the presence of alternative prey. In natural wetlands, newts had similar infection intensities as P. regilla, suggesting that they commonly function as alternative hosts despite their unpalatability to downstream hosts, whereas mosquitofish had substantially lower infection intensities and are unlikely to function as hosts. These results underscore the importance of studying host-parasite interactions in complex communities and of broadly linking research on predation, biodiversity loss, and infectious diseases.

Tetrodotoxin does not protect red-spotted newts, Notophthalmus viridescens, from intestinal parasites.

Tetrodotoxin (TTX) and its analogue 6-epiTTX had been detected in the red-spotted newt, Notophthalmus viridescens. Thirty specimens of a population from Pennsylvania, USA were histologically examined for the presence of intestinal parasites. More than 50% were found to be infected with nematodes (Trichocephalidae), trematodes or cestodes (Pseudophyllidae). The mean values of TTX and 6-epiTTX in parasitized and in non-parasitized newts were not significantly different. Using a monoclonal antibody-based immunoenzymatic technique, TTX was localized in the intestinal tissue as well as in the parasites indicating that they accumulate the toxin and suggesting that TTX and 6-epiTTX are not providing protection from parasites to the newts.

Dermocystid infection and associated skin lesions in free-living palmate newts (Lissotriton helveticus) from Southern France.

Since the early 1900s, mesomycetozoan parasites have been reported in both European anuran and caudate species. These reports have primarily been descriptive, which has made assessing the impact of these parasites on host populations difficult. Anecdotal reports of dermocystidium-like parasites are becoming widespread across Europe, possibly indicating that these mesomycetozoan parasites are increasing in distribution and/or abundance. This highlights the need for further investigations into the occurrence, pathogenesis and effects on host health of these parasitic infections for free-living amphibian populations, particularly those which are already stressed or threatened by other factors. Here we report the results of pathological, microbiological and molecular investigations used to characterize unidentified skin lesions in palmate newts (Lissotriton helveticus) from Larzac, France. We confirm that the lesions are the result of infection with a novel dermocystidium-like parasite, which is related to Amphybiocystidium ranae. We also show that the same parasite is distributed across several newt breeding sites. The lesions that result from infection with this parasite range from single or few vesicular or nodular cutaneous lesions to multiple coalescing skin ulcers with extensive hemorrhages. The latter have not been previously described in amphibians due to mesomycetozoan parasitic infection. Dermocystid DNA was detected only in newts that showed lesions, providing comparative evidence of the parasite's pathogenicity. We discuss the potential significance of the presence of this pathogen in the context of the population health of palmate newts.

A new species of Hysterothylacium (Nematoda: Anisakidae) from the stomach of the red-spotted newt, Notophthalmus viridescens, from Pennsylvania fishless ponds.

Species of Hysterothylacium Ward & Magath, 1917 (Nematoda: Anisakidae) have previously been reported only from marine and freshwater fishes. Here, we describe a new species that infects red-spotted newts (Notophthalmus viridescens), a North American amphibian species with fully aquatic adults. Aside from the unique characteristic of infecting an amphibian host, the new species differs from congeners by the presence of lateral alae, the length of intestinal cecum (0.54­0.73 mm, 39.67­49.09% of esophageal length), the size of the spicules (0.33­0.39 mm, 2.75­3.25% of body length), and the absence of tail tip ornamentation. The absence of fish in the ponds from which these specimens were obtained suggests that newts are the normal definitive host for this species. We suggest that this species might have diverged from a Hysterothylacium parasite of the freshwater fishes which usually live in close proximity with newts.

Helminth fauna of Lycisalamandra billae (Franzen & Klewen) (Luschan salamander) collected from Antalya.

Fifty (26 males, 21 female and 3 juvenile) Lyciasalamandra luschani (Luschan's salamander) collected in Antalya, were examined for helminths during 1997-2001. In this study, 3 helminth species, Aplectana acuminata, Cosmocerca longicauda and Angiostoma aspersae (Nematoda) were found. This study is the first helminthological research of Luschan's salamander in the world. This is the first time that Luschan's salamander has been shown to be the host of these parasites.

Three rarely reported digeneans inhabiting amphibians from Vancouver Island, British Columbia, Canada.

Three rarely reported species of digeneans were collected in amphibian hosts from Diversion Reservoir, Sooke, Vancouver Island, British Columbia, Canada. The endangered anuran Rana aurora hosted Glypthelmins californiensis, Gorgoderina multilobata, and Megalodiscus microphagus. In addition, the anuran Pseudacris regilla and the caudatan Taricha granulosa hosted Megalodiscus microphagus. Gorgoderina multilobata has been reported only once since 1936 and has never been reported outside of California. We note several characters not previously reported for Gorgoderina multilobata and G. aurora.

Composition and structure of helminth communities of the salamanders, Aneides lugubris, Batrachoseps nigriventris, Ensatina eschscholtzii (Plethodontidae), and Taricha torosa (Salamandridae) from California.

Component helminth communities of Aneides lugubris (N=31), Batrachoseps nigriventris (N=63), Ensatina eschscholtzii (N=30), and Taricha torosa (N=68) from California were determined. The compound helminth community consisted of 1 species of cestode, Bitegmen gerrhonoti and 6 species of nematodes, Batracholandros salamandrae, Cosmocercoides variabilis, Falcaustra pretiosa, Megalobatrachonema gigantica, Oswaldocruzia pipiens, and Rhabdias joaquinensis. The least diverse infracommunities were associated with Batrachoseps nigriventris while T. torosa had the most diverse fauna. Only B. salamandrae occurred in each of the 4 component communities. Each of the other helminth species was harbored by a single host species. New host records for B. salamandrae in B. nigriventris and T. torosa; O. pipiens in A. lugubris; C. variabilis in E. eschscholtzii; F. pretiosa and M. gigantica in T. torosa are established.

Longitudinal patterns of trypanosome infections in red-spotted newts.

Longitudinal data on Trypanosoma diemyctyli infections in individual red-spotted newts, Notophthalmus viridescens, were collected over a 5-yr period. Many newts (37%) retained infections throughout their adult lives and only 4.5% appeared to lose infections following their initial autumn sample. Individual infection levels were higher at their first sample as compared to their second sample. Newts of known age were transplanted between leech-free and leech-infested ponds. The time course of infection between previously exposed and unexposed individuals was similar when both were caged in a leech-infested pond. Previously infected individuals maintained stationary chronic levels for 3 mo in both leech-infected and leech-free ponds. The trends observed in these longitudinal data suggested that transmission by leeches is necessary for infection, that continued transmission does not significantly alter the dynamics of infrapopulation growth and stasis, and that constraints on trypanosome population growth occurred at the host individual level.