Effects of insect host chemical secretions on the entomopathogenic nematode Steinernema carpocapsae.

Given the threat presented by parasites and pathogens, insects employ various defences to protect themselves against infection, including chemical secretions. The red flour beetle Tribolium castaneum releases a secretion containing the benzoquinones methyl-1,4-benzoquinone (MBQ) and ethyl-1,4-benzoquinone (EBQ) into the environment. These compounds have known antimicrobial effects; however, their role in defence against macroparasites is not known. Entomopathogenic nematodes, such as Steinernema carpocapsae, present a serious threat to insects, with successful infection leading to death. Thus, quinone-containing secretions may also aid in host defence. We tested how exposure to the individual components of this quinone secretion, as well as a mix at naturally-occurring proportions, affected the survival and thrashing behaviour of S. carpocapsae, as well as their virulence to a model host (Galleria mellonella). Exposure to high concentrations of MBQ and EBQ, as well as the quinone mix, significantly increased nematode death but did not consistently reduce thrashing, which would otherwise be expected given their toxicity. Rather, quinones may act as a host cue to S. carpocapsae by triggering increased activity. We found that exposure to quinones for 24 or 72 hours did not reduce nematode virulence, and surviving nematodes remained infective after non-lethal exposure. Our results indicate that quinone secretions likely serve as a defence against multiple infection threats by reducing S. carpocapsae survival, but further research is required to contextualize their roles by testing against other nematodes, as well as other helminths using insects as hosts.

Consumption of trematode parasite infectious stages: from conceptual synthesis to future research agenda.

Given their sheer cumulative biomass and ubiquitous presence, parasites are increasingly recognized as essential components of most food webs. Beyond their influence as consumers of host tissue, many parasites also have free-living infectious stages that may be ingested by non-host organisms, with implications for energy and nutrient transfer, as well as for pathogen transmission and infectious disease dynamics. This has been particularly well-documented for the cercaria free-living stage of digenean trematode parasites within the Phylum Platyhelminthes. Here, we aim to synthesize the current state of knowledge regarding cercariae consumption by examining: (a) approaches for studying cercariae consumption; (b) the range of consumers and trematode prey documented thus far; (c) factors influencing the likelihood of cercariae consumption; (d) consequences of cercariae consumption for individual predators (e.g. their viability as a food source); and (e) implications of cercariae consumption for entire communities and ecosystems (e.g. transmission, nutrient cycling and influences on other prey). We detected 121 unique consumer-by-cercaria combinations that spanned 60 species of consumer and 35 trematode species. Meaningful reductions in transmission were seen for 31 of 36 combinations that considered this; however, separate studies with the same cercaria and consumer sometimes showed different results. Along with addressing knowledge gaps and suggesting future research directions, we highlight how the conceptual and empirical approaches discussed here for consumption of cercariae are relevant for the infectious stages of other parasites and pathogens, illustrating the use of cercariae as a model system to help advance our knowledge regarding the general importance of parasite consumption.

Effects of road salt on a free-living trematode infectious stage.

Many temperate freshwater habitats are at risk for contamination by run-off associated with the application of road de-icing salts. Elevated salinity can have various detrimental effects on freshwater organisms, including greater susceptibility to infection by parasites and pathogens. However, to better understand the net effects of road salt exposure on host-parasite dynamics, it is necessary to consider the impacts on free-living parasite infectious stages, such as the motile aquatic cercariae of trematodes. Here, we examined the longevity and activity of cercariae from four different freshwater trematodes (Ribeiroia ondatrae, Echinostoma sp., Cephalogonimus sp. and an unidentified strigeid-type) that were exposed to road salt at five different environmentally relevant concentrations (160, 360, 560, 760 and 960 mg/ml of sodium chloride). Exposure to road salt had minimal detrimental effects, with cercariae activity and survival often greatest at intermediate concentrations. Only the cercariae of Cephalogonimus sp. showed reduced longevity at the highest salt concentration, with those of both R. ondatrae and the unidentified strigeid-type exhibiting diminished activity, indicating interspecific variation in response. Importantly, cercariae seem to be relatively unaffected by salt concentrations known to increase infection susceptibility in some of their hosts. More studies will be needed to examine this potential dichotomy in road salt effects between hosts and trematodes, including influences on parasite infectivity.

Prospective enzymes for omega-3 PUFA biosynthesis found in endoparasitic classes within the phylum Platyhelminthes.

The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

Effects of dietary antioxidants and environmental stressors on immune function and condition in Lithobates (Rana) sylvaticus.

The negative effects of abiotic and biotic stressors on animal condition, physiology, behaviour, and fitness have become increasingly recognized, making it critical to understand how these may be mitigated, such as through diet. When consumed, dietary antioxidants (secondary metabolites produced by plants) provide protection from oxidative damage that can result from exposure to stressors. We examined the effects of dietary antioxidants (ß-carotene and vitamin E) on the ability of Lithobates sylvaticus (wood frog) tadpoles to overcome the detrimental effects imposed by two common environmental stressors - exposure to the herbicide atrazine or infection by a pathogenic helminth parasite (Echinostoma trivolvis). Tadpoles which were fed high concentrations of either vitamin E or ß-carotene had a greater immune response compared to those given trace diets of either compound as measured by circulating white blood cells and a phytohemaglutination assay (PHA). Similarly, tadpoles in the herbicide and parasitism treatments that were given trace antioxidant diets had significant weight loss, whereas those consuming high levels of vitamin E or ß-carotene did not. Our results suggest that dietary antioxidants, specifically ß-carotene and vitamin E, have a positive effect on tadpole immune systems and overall condition that likely allows them to better cope with natural stressors, with potential implications for their foraging behaviour.

Sub-chronic exposure to a neonicotinoid does not affect susceptibility of larval leopard frogs to infection by trematode parasites, via either depressed cercarial performance or host immunity.

Little information is available on the effects of neonicotinoid insecticides on vertebrates. Previous work using amphibians found chronic exposure to some neonicotinoids had no detrimental effects on fitness-relevant traits. However, there is some evidence of more subtle effects of neonicotinoids on immune traits and evidence that other pesticides can suppress tadpole immunity resulting in elevated levels of parasitism in the exposed tadpoles. The objective of our study was to assess whether neonicotinoid exposure affected tadpole immunometrics and susceptibility to parasitic helminths. We assessed northern leopard frog tadpole (Lithobates pipiens) levels of parasitism and leukocyte profiles following exposure to environmentally relevant concentrations of clothianidin and free-living infective cercariae of a helminth parasite, an Echinostoma sp. trematode. When comparing tadpoles from controls to either 1 or 100 µg/L clothianidin treatments, we found similar measures of parasitism (i.e. prevalence, abundance and intensity of echinostome cysts) and similar leukocyte profiles. We also confirmed that clothianidin was not lethal for cercariae; however, slight reductions in swimming activity were detected at the lowest exposure concentration of 0.23 µg/L. Our results show that exposure to clothianidin during the larval amphibian stage does not affect leukocyte profiles or susceptibility to parasitism by larval trematodes in northern leopard frogs although other aspects such as length of host exposure require further study.

Development and application of an eDNA method to detect and quantify a pathogenic parasite in aquatic ecosystems.

Approaches based on organismal DNA found in the environment (eDNA) have become increasingly utilized for ecological studies and biodiversity inventories as an alternative to traditional field survey methods. Such DNA-based techniques have largely been used to establish the presence of free-living organisms, but have much potential for detecting and quantifying infectious agents in the environment, which is necessary to evaluate disease risk. We developed an eDNA method to examine the distribution and abundance of the trematode Ribeiroia ondatrae, a pathogenic parasite known to cause malformations in North American amphibians. In addition to comparing this eDNA approach to classical host necropsy, we examined the detectability of R. ondatrae in water samples subject to different degradation conditions (time and temperature). Our test exhibited high specificity and sensitivity to R. ondatrae, capable of detecting as little as 14 fg (femtograms) of this parasite's DNA (1/2500th of a single infectious stage) from field water samples. Compared to our results from amphibian host necropsy, quantitative PCR was -90% concordant with respect to R. ondatrae detection from 15 field sites and was also a significant predictor of host infection abundance. DNA was still detectable in lab samples after 21 days at 25°C, indicating that our method is robust to field conditions. By comparing the advantages and disadvantages of eDNA vs. traditional survey methods for determining pathogen presence and abundance in the field, we found that the lower cost and effort associated with eDNA approaches provide many advantages. The development of alternative tools is critical for disease ecology, as wildlife management and conservation efforts require reliable establishment and monitoring of pathogens.

Benefits of fidelity: does host specialization impact nematode parasite life history and fecundity?

The range of hosts used by a parasite is influenced by macro-evolutionary processes (host switching, host-parasite co-evolution), as well as 'encounter filters' and 'compatibility filters' at the micro-evolutionary level driven by host/parasite ecology and physiology. Host specialization is hypothesized to result in trade-offs with aspects of parasite life history (e.g. reproductive output), but these have not been well studied. We used previously published data to create models examining general relationships among host specificity and important aspects of life history and reproduction for nematodes parasitizing animals. Our results indicate no general trade-off between host specificity and the average pre-patent period (time to first reproduction), female size, egg size, or fecundity of these nematodes. However, female size was positively related to egg size, fecundity, and pre-patent period. Host compatibility may thus not be the primary determinant of specificity in these parasitic nematodes if there are few apparent trade-offs with reproduction, but rather, the encounter opportunities for new host species at the micro-evolutionary level, and other processes at the macro-evolutionary level (i.e. phylogeny). Because host specificity is recognized as a key factor determining the spread of parasitic diseases understanding factors limiting host use are essential to predict future changes in parasite range and occurrence.

Interactions of environmental stressors impact survival and development of parasitized larval amphibians.

Infected hosts are exposed to many environmental stressors that must be taken into account in order to determine the importance of disease, as various combinations can interact in unpredictable ways. Here, northern leopard frog (Rana pipiens) tadpoles, a species in decline, were exposed to stressors singly or in combination. Stressors included infection by Echinostoma trivolvis (a trematode parasite), exposure to predator chemical cues (larval dragonflies), and exposure to varying concentrations of the herbicide atrazine. Parasitism decreased survival only in combination with exposure to 3 microg/L atrazine, with a negative interaction observed for mass as well. Similarly, a negative interaction of parasitism and predation on survival occurred. However, atrazine exposure alone negatively affected the survival, mass, and developmental stage of tadpoles. These results indicate that certain stressor combinations are particularly deleterious for young parasitized tadpoles. Notably, very common low-intensity parasite infection can be particularly harmful in certain situations. Such negative impacts on larval amphibians in certain scenarios may contribute to ongoing amphibian population declines, emphasizing that the combination of environmental stressors must be considered when evaluating the general role of disease in species extinctions.

Interspecific and intraspecific variation in cercariae release.

Given the importance of temperature for the shedding of trematode infective stages (cercariae) from gastropod first intermediate hosts, we investigated the response to temperature increases of trematodes of the intertidal snail Zeacumantus subcarinatus collected from different latitudes (differing in mean annual summer temperature) on the South Island of New Zealand. We investigated whether shedding of cercariae at elevated temperatures differed both between species (interspecific variation) and among populations of each trematode species (intraspecific variation). The shedding of one trematode species, Maritrema novaezealandensis (Microphallidae), appears to decrease with increased temperature and differed among locations. In contrast, the shedding of a second species, Acanthoparyphium sp. (Echinostomatidae), increased at warmer temperatures. In particular, Acanthoparyphium sp.-infected snails from 1 location showed the greatest increase in the shedding of cercariae in response to elevated temperature. Our results demonstrate that different trematode species and populations may be dissimilarly impacted by temperature changes resulting from global warming. In addition, both interspecific and intraspecific variation may result in different impacts of trematodes on ecosystems at different locales.

Effects of temperature and salinity on emergence of Gynaecotyla adunca cercariae from the intertidal gastropod Ilyanassa obsoleta.

Fluctuating abiotic conditions within intertidal zones have been shown to affect the emergence of free-swimming trematode infectious stages (cercariae) from their gastropod first intermediate hosts, likely reflecting adaptations to maximize transmission in this marine environment. We investigated the influences of temperature (17 and 22 C) and salinity (25, 30, and 35 ppt) on the emergence of marine cercariae (Gynaecotyla adunca) from their mud snail first intermediate host ( Ilyanassa obsoleta ). Cercariae emerged in greater numbers at 22 C and the 2 lowest salinities, with a sharp decrease at the 35 ppt level, but there was no interactive effect. We discuss these patterns of G. adunca emergence as possible adaptations to facilitate transmission to its amphipod second intermediate host ( Corophium volutator ) in conditions common to the Upper Bay of Fundy.

Larval amphibian growth and development under varying density: are parasitized individuals poor competitors?

Population density and infection with parasites often are important factors affecting the growth and development of individuals. How these factors co-occur and interact in nature should have important consequences for individual fitness and higher-order phenomena, such as population dynamics of hosts and their interactions with other species. However, few studies have examined the joint effects of density and parasitism on host growth and development. We examined the co-influences of rearing density and parasitism, by the trematode Echinostoma trivolvis, on the growth and development of larval frogs, Rana (=Lithobates) pipiens. We also examined the potential role of parasite-mediated intraspecific competition by observing how unparasitized individuals performed when housed with other unparasitized tadpoles, versus housing with a combination of unparasitized and parasitized hosts. Mean mass and mean developmental stage were reduced under high rearing densities. The presence of parasitized conspecifics had no significant effect, but there was a significant interaction of density and parasitism presence on host mass, due to the fact that parasitized conspecifics grew poorly at high densities. Unparasitized individuals reared with parasitized and unparasitized conspecifics fared no better than unparasitized individuals reared only with one another. This result indicates that infected hosts compete as much as uninfected hosts for resources, even though infected individuals have reduced mass under high-density conditions. Resource acquisition and resource allocation are different processes, and parasitism, if it only affects the latter, might not have a discernible impact on competitive interactions.