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We show that the globally invasive, human-infectious flatworm, Haplorchis pumilio , possesses the most physically specialized soldier caste yet documented in trematodes. Soldiers occur in colonies infecting the first intermediate host, the freshwater snail Melanoides tuberculata , and are readily distinguishable from immature and mature reproductive worms. Soldiers possess a pharynx five times absolutely larger than those of immature and mature reproductives, lack a germinal mass, and have a different developmental trajectory than reproductives, indicating that H. pumilio soldiers constitute a reproductively sterile physical caste. Neither immature nor mature reproductives showed aggression in in vitro trials, but soldiers readily attacked heterospecific trematodes that coinfect their host. Ecologically, we calculate that H. pumilio caused ~94% of the competitive deaths in the guild of trematodes infecting its host snail in its invasive range in southern California. Despite being a dominant competitor, H. pumilio soldiers did not attack conspecifics from other colonies. All prior reports documenting division of labor and a trematode soldier caste have involved soldiers that may be able to metamorphose to the reproductive stage and have been from nonhuman-infectious marine species; this study provides clear evidence for an obligately sterile trematode soldier, while extending the phenomenon of a trematode soldier caste to freshwater and to an invasive species of global public health concern., Competing Interests: Competing interests statement:The authors declare no competing interest.

Australapatemon spp. are cosmopolitan trematodes that infect freshwater snails, aquatic leeches, and birds. Despite their broad geographic distribution, relatively little is known about interactions between Australapatemon spp. and their leech hosts, particularly under experimental conditions and in natural settings. We used experimental exposures to determine how Australapatemon burti cercariae dosage (number administered to leech hosts, Erpobdella microstoma) affected infection success (fraction to encyst as metacercariae), infection abundance, host survival, and host size over the 100 days following exposure. Interestingly, infection success was strongly density-dependent, such that there were no differences in metacercariae load even among hosts exposed to a 30-fold difference in cercariae. This relationship suggests that local processes (e.g., resource availability, interference competition, or host defenses) may play a strong role in parasite transmission. Our results also indicated that metacercariae did not become evident until ~4 weeks post exposure, with average load climbing until approximately 13 weeks. There was no evidence of metacercariae death or clearance over the census period. Parasite exposure had no detectable effects on leech size or survival, even with nearly 1,000 cercariae. Complementary surveys of leeches in California revealed that 11 of 14 ponds supported infection by A. burti (based on morphology and molecular sequencing), with an average prevalence of 32% and similar metacercariae intensity as in our experimental exposures. The extended development time and extreme density dependence of A. burti has implications for studying naturally occurring host populations, for which detected infections may represent only a fraction of cercariae to which animals have been exposed. Future investigation of these underlying mechanisms would be benefical in understanding host-parasite relationships., (Published by Elsevier Inc.)

Parasites can have strong effects on invertebrate host behaviour, fecundity and survival in marine ecosystems. However, parasites are often poorly documented and still rarely integrated into marine ecological modelling; comprehensive surveys of infection in marine invertebrates are sporadic at best. For example, rock crabs are an important part of Californian coastal ecosystems, both as regulators of mussel populations and non-native species, and as prey items for predators like sea otters, but their parasite communities and potential effects on crab population dynamics are seldom studied or understood. Here, we present the first report of infection by the trematode Helicometrina nimia in the economically and ecologically important red rock crab (Cancer productus) and Pacific rock crab (Romaleon antennarium). As intermediate hosts, they are a missing link for infection by H. nimia in Californian fish that was unreported until now. Based on these findings, we advocate for further research into parasite diversity and their potential effects on ecologically and commercially important species.

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.

Competition - colonization tradeoffs occur in many systems, and theory predicts that they can strongly promote species coexistence. However, there is little empirical evidence that observed competition - colonization tradeoffs are strong enough to maintain diversity in natural systems. This is due in part to a mismatch between theoretical assumptions and biological reality in some systems. We tested whether a competition - colonization tradeoff explains how a diverse trematode guild coexists in California horn snail populations, a system that meets the requisite criteria for the tradeoff to promote coexistence. A field experiment showed that subordinate trematode species tended to have higher colonization rates than dominant species. This tradeoff promoted coexistence in parameterized models but did not fully explain trematode diversity and abundance, suggesting a role of additional diversity maintenance mechanisms. Spatial heterogeneity is an alternative way to promote coexistence if it isolates competing species. We used scale transition theory to expand the competition - colonization tradeoff model to include spatial variation. The parameterized model showed that spatial variation in trematode prevalence did not isolate most species sufficiently to explain the overall high diversity, but could benefit some rare species. Together, the results suggest that several mechanisms combine to maintain diversity, even when a competition - colonization tradeoff occurs.

This study validated a technique for non-invasive hormone measurements in California killifish Fundulus parvipinnis, and looked for associations between cortisol (a stress hormone) and 11-ketotestosterone (KT, an androgen) release rates and the density or intensity of the trematode parasites Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB) in wild-caught, naturally infected F. parvipinnis. In experiment 1, F. parvipinnis were exposed to an acute stressor by lowering water levels to dorsal-fin height and repeatedly handling the fish over the course of an hour. Neither parasite was found to influence cortisol release rates in response to this acute stressor. In experiment 2, different F. parvipinnis were exposed on four consecutive days to the procedure for collecting water-borne hormone levels and release rates of 11-KT and cortisol were quantified. This design examined whether F. parvipinnis perceived the water-borne collection procedure to be a stressor, while also exploring how parasites influenced hormone release rates under conditions less stressful than those in experiment 1. No association was found between RENB and hormone release rates, or between EUHA and 11-KT release rates. The interaction between EUHA density and handling time, however, was an important predictor of cortisol release rates. The relationship between handling time and cortisol release rates was negative for F. parvipinnis harbouring low or intermediate density infections, and became positive for fish harbouring high densities of EUHA., (© 2016 The Fisheries Society of the British Isles.)

Infection heterogeneity is one of the most fundamental patterns in disease ecology, yet surprisingly few studies have experimentally explored its underlying drivers. Here, we used large-scale field assessments to evaluate the degree of parasite aggregation within amphibian host populations followed by a novel experimental approach to assess the potential influence of host size, behaviour and immunity in reproducing such heterogeneity. Among 227 wetlands, 2468 hosts and seven parasite species, infections were consistently aggregated among host individuals within populations of the Pacific chorus frog (Pseudacris regilla). For each parasite species, the relationship between the log-mean and log-variance of infection load was strongly linear (R(2): 0·91-0·98) with a slope between 1·37 and 1·67, indicative of aggregation relative to the expected Poisson slope of unity. In laboratory trials with P. regilla and the most virulent trematode (Ribeiroia ondatrae), experimental reductions in either host immunity (through corticosterone exposure) or antiparasite behaviours (through anaesthesia exposure) increased parasite infection loads in isolated hosts by 62-102% relative to unmanipulated individuals. In a second experiment designed to test how variation in host immunity, behaviour and body size affected variation in infection load within small groups (dyads), a reduction in immune function or behaviour of one host significantly amplified infection heterogeneity within the group, effectively doubling the variance-to-mean ratio. However, immunity affected aggregation only in the absence of behavioural manipulation, and changing the size distribution of hosts did not appreciably affect aggregation. Using Taylor's Power Law to integrate field and laboratory data, we found that only treatments involving behavioural reductions achieved aggregation levels comparable to natural host populations. Thus, despite their short duration, our treatments generated heterogeneity in infection loads similar to natural observations. These results emphasize how, alongside extrinsic variation in parasite exposure risk, individual host attributes generally and behaviour in particular have the potential to influence infection success and parasite aggregation. Continued integration of infection heterogeneity research across space, among host species, and over time has important implications for understanding and managing human and wildlife diseases., (© 2014 The Authors. Journal of Animal Ecology © 2014 British Ecological Society.)

For trophically transmitted parasites that manipulate the phenotype of their hosts, whether the parasites do or do not experience resource competition depends on such factors as the size of the parasites relative to their hosts, the intensity of infection, the extent to which parasites share the cost of defending against the host's immune system or manipulating their host, and the extent to which parasites share transmission goals. Despite theoretical expectations for situations in which either no, or positive, or negative density-dependence should be observed, most studies document only negative density-dependence for trophically transmitted parasites. However, this trend may be an artifact of most studies having focused on systems in which parasites are large relative to their hosts. Yet, systems are common where parasites are small relative to their hosts, and these trophically transmitted parasites may be less likely to experience resource limitation. We looked for signs of density-dependence in Euhaplorchis californiensis (EUHA) and Renicola buchanani (RENB), two manipulative trematode parasites infecting wild-caught California killifish (Fundulus parvipinnis). These parasites are small relative to killifish (suggesting resources are not limiting), and are associated with changes in killifish behavior that are dependent on parasite-intensity and that increase predation rates by the parasites' shared final host (indicating the possibility for cost sharing). We did not observe negative density-dependence in either species, indicating that resources are not limiting. In fact, observed patterns indicate possible mild positive density-dependence for EUHA. Although experimental confirmation is required, our findings suggest that some behavior-manipulating parasites suffer no reduction in size, and may even benefit when "crowded" by conspecifics., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

1. Ecologists often measure the biomass and productivity of organisms to understand the importance of populations and communities in the flow of energy through ecosystems. Despite the central role of such studies in the advancement of freshwater ecology, there has been little effort to incorporate parasites into studies of freshwater energy flow. This omission is particularly important considering the roles that parasites sometimes play in shaping community structure and ecosystem processes. 2. Using quantitative surveys and dissections of over 1600 aquatic invertebrate and amphibian hosts, we calculated the ecosystem-level biomass and productivity of trematode parasites alongside the biomass of free-living aquatic organisms in three freshwater ponds in California, USA. 3. Snails and amphibian larvae, which are both important intermediate trematode hosts, dominated the dry biomass of free-living organisms across ponds (snails = 3.2 g m(-2); amphibians = 3.1 g m(-2)). An average of 33.5% of mature snails were infected with one of six trematode taxa, amounting to a density of 13 infected snails m(-2) of pond substrate. Between 18% and 33% of the combined host and parasite biomass within each infected snail consisted of larval trematode tissue, which collectively accounted for 87% of the total trematode biomass within the three ponds. Mid-summer trematode dry biomass averaged 0.10 g m(-2), which was equal to or greater than that of the most abundant insect orders (coleoptera = 0.10 g m(-2), odonata = 0.08 g m(-2), hemiptera = 0.07 g m(-2) and ephemeroptera = 0.03 g m(-2)). 4. On average, each trematode taxon produced between 14 and 1660 free-swimming larvae (cercariae) infected snail(-1) 24 h(-1) in mid-summer. Given that infected snails release cercariae for 3-4 months a year, the pond trematode communities produced an average of 153 mg m(-2) yr(-1) of dry cercarial biomass (range = 70-220 mg m(-2) yr(-1)). 5. Our results suggest that a significant amount of energy moves through trematode parasites in freshwater pond ecosystems, and that their contributions to ecosystem energetics may exceed those of many free-living taxa known to play key roles in structuring aquatic communities., (© 2013 The Authors. Journal of Animal Ecology © 2013 British Ecological Society.)

The ability of free-swimming larval parasites to control emergence from their hosts can be critical in increasing the chances of successful infection transmission. For a group of estuarine trematodes, emergence of cercariae from their snail hosts is known to match favorable temperature, tidal activity, and light intensity. How the larvae time this behavior is not well understood, but the pathway that the larvae take through their host may play a role. Through video and histological analysis, we were able to identify the snail's anus as the emergence point and the peri-intestinal sinus dorsal to the intestines as the route by which they reach that point. By moving through this open sinus, the larvae have an energetically efficient pathway to reach their emergence point while minimizing damage to the host. Most importantly, it allows control over emergence to be maintained by the parasite, not the host, thus increasing the chances of the larva successfully reaching its intended destination.

Within most free-living species exists a cryptic community of interacting parasites. By combining multiscale field data with manipulative experiments, we evaluated patterns of parasite coinfection in amphibian hosts and their underlying mechanisms. Surveys of 86 wetlands and 1273 hosts revealed positive correlations between two pathogenic trematodes (Ribeiroia ondatrae and Echinostoma trivolvis) both between wetlands and within individual hosts. In infection and coinfection experiments, Ribeiroia caused greater pathology than Echinostoma, including high host mortality (24%) and severe limb malformations (75%). No interactive effects were noted for host pathology, but both parasites decreased the per capita persistence of one another by 17-36%. Thus, in spite of consistently positive associations from field data, these parasites negatively affected the persistence of one another, likely via cross immunity (apparent competition). These findings underscore the danger of inferring parasite interactions from coinfection patterns and emphasize the potential disconnect between within-host processes (e.g., competition) and between-host processes (e.g., exposure and transmission). Here, correlated coinfections likely resulted from similarities in the parasites' host requirements and heterogeneity in host susceptibility or exposure. Understanding complex interactions among parasites depends critically on the scale under consideration, highlighting the importance of combining coinfection field studies with mechanistic experiments.

We describe the distribution and abundance of the brain-encysting trematode Euhaplorchis californiensis and its second intermediate host, the California killifish (Fundulus parvipinnis), in 3 estuaries in southern California and Baja California. We quantified the density of fish and metacercariae at 13-14 sites per estuary and dissected 375 killifish. Density (numbers and biomass) was examined at 3 spatial scales, i.e., small replicate sites, habitats, and entire estuaries. At those same scales, factors that might influence metacercaria prevalence, abundance, and aggregation in host individuals and populations were also examined. Metacercaria prevalence was 94-100% among the estuaries. Most fish were infected with 100s to 1,000s of E. californiensis metacercariae, with mean abundance generally increasing with host size. Although body condition of fish did not vary among sites or estuaries, the abundance of metacercariae varied significantly among sites, habitats, estuaries, and substantially with host size and gender. Metacercariae were modestly aggregated in killifish (k > 1), with aggregation decreasing in larger hosts. Across the 3 estuaries, the total populations of killifish ranged from 9,000-12,000 individuals/ha and from 7-43 kg/ha. The component populations of E. californiensis metacercariae ranged from 78-200 million individuals/ha and from 0.1-0.3 kg/ha. Biomass of E. californiensis metacercariae constituted 0.5-1.7% of the killifish biomass in the estuaries. Our findings, in conjunction with previously documented effects of E. californiensis, suggest a strong influence of this parasite on the size, distribution, biomass, and abundance of its killifish host.

Parasites can have strong impacts but are thought to contribute little biomass to ecosystems. We quantified the biomass of free-living and parasitic species in three estuaries on the Pacific coast of California and Baja California. Here we show that parasites have substantial biomass in these ecosystems. We found that parasite biomass exceeded that of top predators. The biomass of trematodes was particularly high, being comparable to that of the abundant birds, fishes, burrowing shrimps and polychaetes. Trophically transmitted parasites and parasitic castrators subsumed more biomass than did other parasitic functional groups. The extended phenotype biomass controlled by parasitic castrators sometimes exceeded that of their uninfected hosts. The annual production of free-swimming trematode transmission stages was greater than the combined biomass of all quantified parasites and was also greater than bird biomass. This biomass and productivity of parasites implies a profound role for infectious processes in these estuaries.

Measuring biodiversity is difficult. This has led to efforts to seek taxa whose species richness correlates with the species richness of other taxa. Such indicator taxa could then reduce the time and cost of assessing the biodiversity of the more extensive community. The search for species richness correlations has yielded mixed results, however. This may be primarily because of the lack of functional relationships between the taxa studied. Trematode parasites are highly promising bioindicators. Diverse assemblages of larval trematode parasites are easily sampled in intermediate host snails. Through their life cycles these parasites are functionally coupled with the surrounding free-living diversity of vertebrate and invertebrate animals. It has been shown that larval trematodes in snails correlate positively with bird diversity and abundance. Here, we explore whether trematodes also correlate with standard measures of fishes, and large and small benthos, for 32 sites in three wetlands. We found associations between trematodes and benthic communities that were not consistent across wetlands. The associations were, however, consistently positive for large benthic species richness and density. Some of the contrasting associations between trematode and benthos may be explained by negative associations between large and small benthos. We found no associations with fish communities (probably because of the inadequacy of standard "snapshot" sampling methods for highly mobile fishes). The results support further exploration of trematodes as bioindicators of diversity and abundance of animal communities.

An unappreciated facet of biodiversity is that rich communities and high abundance may foster parasitism. For parasites that sequentially use different host species throughout complex life cycles, parasite diversity and abundance in 'downstream' hosts should logically increase with the diversity and abundance of 'upstream' hosts (which carry the preceding stages of parasites). Surprisingly, this logical assumption has little empirical support, especially regarding metazoan parasites. Few studies have attempted direct tests of this idea and most have lacked the appropriate scale of investigation. In two different studies, we used time-lapse videography to quantify birds at fine spatial scales, and then related bird communities to larval trematode communities in snail populations sampled at the same small spatial scales. Species richness, species heterogeneity and abundance of final host birds were positively correlated with species richness, species heterogeneity and abundance of trematodes in host snails. Such community-level interactions have rarely been demonstrated and have implications for community theory, epidemiological theory and ecosystem management.

Of the 18 trematode species that use the horn snail, Cerithidea californica, as a first intermediate host, 6 have the potential to use raccoons as a final host. The presence of raccoon latrines in Carpinteria Salt Marsh, California, allowed us to investigate associations between raccoons and trematodes in snails. Two trematode species, Probolocoryphe uca and Stictodora hancocki, occurred at higher prevalences in snails near raccoon latrines than in snails away from latrines, suggesting that raccoons may serve as final hosts for these species. Fecal remains indicated that raccoons fed on shore crabs, the second intermediate host for P. uca, and fish, the second intermediate host for S. hancocki. The increase in raccoon populations in the suburban areas surrounding west coast salt marshes could increase their importance as final hosts for trematodes in this system.

Trematode parasites in intertidal estuaries experience constantly varying conditions, with the presence or absence of water potentially limiting larval transport between hosts. Given the short life spans (< or =24 h) of cercariae, emergence timing should be optimized to enhance the probability of successful transmission. In the present study, field measurements and laboratory experiments identified processes that regulate the emergence of cercariae from their first intermediate snail hosts in an intertidal marsh. Larvae emerged over species-specific temperature ranges, exclusively during daylight hours, and only when snails were submerged. The three factors operate over different temporal scales: temperature monthly, light diurnally (24-h period), and water depth tidally (12-h period). Each stimulus creates a necessary condition for the next, forming a hierarchy of environmental cues. Emergence as the tide floods would favor transport within the estuary, and light may trigger direct (downward or upward) swimming toward host habitats. Abbreviated dispersal would retain asexually reproduced cercariae within the marsh, and local mixing would diversify the gene pool of larvae encysting on subsequent hosts. In contrast to the timing of cercarial release, emergence duration was under endogenous control. Duration of emergence decreased from sunrise to sunset, perhaps in response to the diminishing lighted interval as the day progresses. Circadian rhythms that control cercarial emergence of freshwater species (including schistosomes) are often set by the activity patterns of subsequent hosts. In this estuary, however, the synchronizing agent is the tides. Together, exogenous and endogenous factors control emergence of trematode cercariae, mitigating the vagaries of an intertidal environment.

The causes of amphibian deformities and their role in widespread amphibian declines remain conjectural. Severe limb abnormalities were induced at high frequencies in Pacific treefrogs (Hyla regilla) exposed to cercariae of a trematode parasite (Ribeiroia sp.). The abnormalities closely matched those observed at field sites, and an increase in parasite density caused an increase in abnormality frequency and a decline in tadpole survivorship. These findings call for further investigation of parasite infection as a cause of amphibian deformities in other sites and species.