Littorina snails and Microphallus trematodes: Diverse consequences of the trematode-induced metabolic shifts.

The intricate relationships between parasites and hosts encompass a wide range of levels, from molecular interactions to population dynamics. Parasites influence not only the physiological processes in the host organism, but also the entire ecosystem, affecting mortality of individuals, the number of offspring through parasitic castration, and matter and energy cycles. Understanding the molecular mechanisms that govern host-parasite relationships and their impact on host physiology and environment remains challenging. In this study, we analyzed how infection with Microphallus trematodes affects the metabolome of two Littorina snail species inhabiting different intertidal zone shore levels. We applied non-targeted GC-MS-based metabolomics to analyze biochemical shifts induced by trematode infection in a host organism. We have identified changes in energy, amino acid, sugar, and lipid metabolism. In particular, we observed intensified amino acid catabolism and nitrogenous catabolites (glutamine, urea) production. These changes primarily correlated with infection and interspecies differences of the hosts rather than shore level. The changes detected in the host metabolism indicate that other aspects of life may have been affected, both within the host organism and at a supra-organismal level. Therefore, we explored changes in microbiota composition, deviations in the host molluscs behavior, and acetylcholinesterase activity (ACE, an enzyme involved in neuromuscular transmission) in relation to infection. Infected snails displayed changes in their microbiome composition. Decreased ACE activity in snails was associated with reduced mobility, but whether it is associated with trematode infection remains unclear. The authors suggest a connection between the identified biochemical changes and the deformation of the shell of molluscs, changes in their behavior, and the associated microbiome. The role of parasitic systems formed by microphallid trematodes and Littorina snails in the nitrogen cycle at the ecosystem level is also assumed.

Stable isotopes unravel the feeding mode-trophic position relationship in trematode parasites.

Stable isotopes have been sporadically used over the last two decades to characterise host-parasite trophic relationships. The main reason for this scarcity is the lack of an obvious pattern in the ratio of nitrogen stable isotope values (δ15 N) of parasites in comparison to their host tissues, which would be key to understand any host-parasite system dynamics. To circumvent this, we focused on a single snail host, Zeacumantus subcarinatus, and three of its trematode parasites. We used stable isotopes to investigate each host-trematode trophic relationship and shed light on the mechanisms utilised by the parasite to reroute its hosts' biomass. All our trematodes were found to be 15 N-enriched compared to their host, with their δ15 N values strongly related to their feeding behaviours: passive versus active. It was possible to 'rank' these parasite species and assess their 'relative' trophic position using δ15 N values. We also demonstrated that including a broader range of samples (e.g. host food and faeces, multiple parasite life stages) helped understand the metabolic mechanisms used by the various participants, and that using carbon stable isotope values and C:N ratios allowed to identify an important lipid requirement of these trematode parasites. Finally, we show how critical it is to not ignore parasitic infections as they can have a great influence on their host's trophic position. We have shown that by focussing on a single host species and a single taxonomic group of parasites, we can remove a certain amount of variation recorded by broader isotope studies. We hope that these data will ultimately improve our ability to place parasites in food webs, and thus improve our understanding of the connections and interactions that dictate food web dynamics.

The contributions of a trematode parasite infectious stage to carbon cycling in a model freshwater system.

Parasites remainunderstudied members of most ecosystems, especially free-living infectious stages, such as the aquatic cercariae of trematodes (flatworms). Recent studies are shedding more light on their roles, particularly as prey for a diverse array of aquatic predators, but the possible fates of cercariae remain unclear. While this is critical to elucidate because cercariae represent a large potential source of energy and nutrients, determining the fate of cercariae-derived organic matter involves many logistical challenges. Previous studies utilized elemental and stable isotope analysis when examining host-parasite interactions, but none has used such approaches to track the movement of cercariae biomass within food webs. Here we report that Plagiorchis sp. cercariae were effectively labelled with 13C by introducing this compound in the food of their snail host. We then added 13C-labelled cercariae as a potential food source to experimental mesocosms containing a simplified model freshwater food web represented by diving beetles (Dytiscidae sp.), dragonfly larvae (Leucorrhinia intacta), oligochaete worms (Lumbriculus variegatus), and a zooplankton community dominated by Daphnia pulex. The oligochaetes had the highest ratio of 13C to 12C, suggesting benthic detritivores are substantial, but previously unrecognized, consumers of cercariae biomass. In an experiment where L. variegatus were fed mass equivalents of dead D. pulex or cercariae, growth was greater with the latter diet, supporting the importance of cercariae as food source for benthic organisms. Given the substantial cercariae biomass possible in natural settings, understanding their contributions to energy flow and nutrient cycling is important, along with developing methods to do so.

Worm expulsion of Gymnophalloides seoi from C57BL/6 mice: role of metacercarial exosomes in upregulating TLR2 and MUC2 expression in intestinal tissues.

Gymnophalloides seoi worms were rapidly expelled from C57BL/6 mice within days 3-6 post-infection probably due to operation of mucosal innate immunity. To understand better the mucosal immunity related to worm expulsion from the host, we isolated exosomes of G. seoi metacercariae and investigated their role in induction of mRNA and protein expression of several Toll-like receptors and mucin-related factors in vitro. G. seoi-secreted exosomes were collected using differential ultracentrifugation, and cellular internalization of the exosomes into HT-29 intestinal epithelial cells was visualized by confocal microscopy. The expression of TLR2 and MUC2 in HT-29 cells was up-regulated in stimulation with the exosomes. We suggest that G. seoi-secreted exosomes offer a new point of view in the mechanism of worm expulsion from the host through enhancement of TLR2 and MUC2 expression.

Helminth.net: expansions to Nematode.net and an introduction to Trematode.net.

Helminth.net (http://www.helminth.net) is the new moniker for a collection of databases: Nematode.net and Trematode.net. Within this collection we provide services and resources for parasitic roundworms (nematodes) and flatworms (trematodes), collectively known as helminths. For over a decade we have provided resources for studying nematodes via our veteran site Nematode.net (http://nematode.net). In this article, (i) we provide an update on the expansions of Nematode.net that hosts omics data from 84 species and provides advanced search tools to the broad scientific community so that data can be mined in a useful and user-friendly manner and (ii) we introduce Trematode.net, a site dedicated to the dissemination of data from flukes, flatworm parasites of the class Trematoda, phylum Platyhelminthes. Trematode.net is an independent component of Helminth.net and currently hosts data from 16 species, with information ranging from genomic, functional genomic data, enzymatic pathway utilization to microbiome changes associated with helminth infections. The databases' interface, with a sophisticated query engine as a backbone, is intended to allow users to search for multi-factorial combinations of species' omics properties. This report describes updates to Nematode.net since its last description in NAR, 2012, and also introduces and presents its new sibling site, Trematode.net.

Parasite infection alters nitrogen cycling at the ecosystem scale.

Despite growing evidence that parasites often alter nutrient flows through their hosts and can comprise a substantial amount of biomass in many systems, whether endemic parasites influence ecosystem nutrient cycling, and which nutrient pathways may be important, remains conjectural. A framework to evaluate how endemic parasites alter nutrient cycling across varied ecosystems requires an understanding of the following: (i) parasite effects on host nutrient excretion; (ii) ecosystem nutrient limitation; (iii) effects of parasite abundance, host density, host functional role and host excretion rate on nutrient flows; and (iv) how this infection-induced nutrient flux compares to other pools and fluxes. Pathogens that significantly increase the availability of a limiting nutrient within an ecosystem should produce a measurable ecosystem-scale response. Here, we combined field-derived estimates of trematode parasite infections in aquatic snails with measurements of snail excretion and tissue stoichiometry to show that parasites are capable of altering nutrient excretion in their intermediate host snails (dominant grazers). We integrated laboratory measurements of host nitrogen excretion with field-based estimates of infection in an ecosystem model and compared these fluxes to other pools and fluxes of nitrogen as measured in the field. Eighteen nitrogen-limited ponds were examined to determine whether infection had a measurable effect on ecosystem-scale nitrogen cycling. Because of their low nitrogen content and high demand for host carbon, parasites accelerated the rate at which infected hosts excreted nitrogen to the water column in a dose-response manner, thereby shifting nutrient stoichiometry and availability at the ecosystem scale. Infection-enhanced fluxes of dissolved inorganic nitrogen were similar to other commonly important environmental sources of bioavailable nitrogen to the system. Additional field measurements within nitrogen-limited ponds indicated that nitrogen flux rates from the periphyton to the water column in high-snail density/high-infection ponds were up to 50% higher than low-infection ponds. By altering host nutrient assimilation/excretion flexibility, parasites could play a widespread, but currently unrecognized, role in ecosystem nutrient cycling, especially when parasite and host abundances are high and hosts play a central role in ecosystem nutrient cycling.

[Serotonin and neuropeptide FMRFamide in the nervous system of Opisthioglyphe ranae (Trematoda: Plagiorchiidae). an immunocytochemical study].

The presence and localization of the serotoninergic and FMRFamidergic structures in the nervous system of the trematode Opisthioglyphe ranae, the marsh frog intestinal parasite, was studied using immunocytochemistry. The serotonin-immunoreactive nerve cells and fibers were revealed in the head ganglia, circular commissure, longitudinal nerve cords and their connective commissures, as well as around the oral and ventral suckers, oesophagus and genital pore. FMRF-specific immunoreactivity was observed in the head ganglia, longitudinal nerve cords and terminal parts of the reproductive system. The results obtained are discussed in light of the available data on the presence and functional significance of the above-mentioned neurotransmitters in trematodes.

The study of the sporocyst broodsacs coloring in Leucochloridium paradoxum (Trematoda: Brachylaemidae).

The secretory cells were found in the subtegument of the sporocysts Leucochloridium paradoxum by histological assay. Pigment granules are formed by these cells. The movement of granules from secretory cells to the tegument external layer was observed. These pigment granules provide the yellow color of sporocysts broodsacs and the brown color of protuberant spots in the terminal part of broodsacs. It was shown that the pigment granules did not contain proteins, nucleotides, lipids and carbohydrates. The positive result was received while staining on bile pigments. The question on the nature of the green pigment remains open. The paletot on the surface of sporocyst formed by spreading hemocytes was observed. This structure was not described before in brachylaemid parthenites.

Is overwintering mortality driving enigmatic declines? Evaluating the impacts of trematodes and the amphibian chytrid fungus on an anuran from hatching through overwintering.

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species' conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering-an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).

A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.

Sterol profile of Neobenedenia melleni, a marine ectoparasite fish.

Neobenedenia melleni, a marine fish ectoparasite, is responsible for considerable losses in the mariculture industry. In maintaining the parasite's homeostasis, sterols are structural and functional lipids that perform vital functions. Thus, understanding the mechanisms of biosynthesis and the uptake of sterols can reveal potential pharmacological targets. The objective of this work was thereby to characterize the N. melleni sterols. The most abundant sterol found was cholesterol in either its free (47.48 ± 15.93 %) or esterified form. However, its precursors, squalene (3.53 ± 0.92 %) and desmosterol (0.25 ± 0.03 %), were also found, suggesting the uptake of these intermediates from hosts or an unusual active pathway of sterol biosynthesis, which can be further explored as pharmacological targets.

Hypoxic and nitrosative stress conditions modulate expression of myoglobin genes in a carcinogenic hepatobiliary trematode, Clonorchis sinensis.

Despite recent evidence suggesting that adult trematodes require oxygen for the generation of bioenergy and eggshells, information on the molecular mechanism by which the parasites acquire oxygen remains largely elusive. In this study, the structural and expressional features of globin genes identified in Clonorchis sinensis, a carcinogenic trematode parasite that invades the hypoxic biliary tracts of mammalian hosts, were investigated to gain insight into the molecules that enable oxygen metabolism. The number of globin paralogs substantially differed among parasitic platyhelminths, ranging from one to five genes, and the C. sinensis genome encoded at least five globin genes. The expression of these Clonorchis genes, named CsMb (CsMb1-CsMb3), CsNgb, and CsGbX, according to their preferential similarity patterns toward respective globin subfamilies, exponentially increased in the worms coinciding with their sexual maturation, after being downregulated in early juveniles compared to those in metacercariae. The CsMb1 protein was detected throughout the parenchymal region of adult worms as well as in excretory-secretory products, whereas the other proteins were localized exclusively in the sexual organs and intrauterine eggs. Stimuli generated by exogenous oxygen, nitric oxide (NO), and nitrite as well as co-incubation with human cholangiocytes variously affected globin gene expression in live C. sinensis adults. Together with the specific histological distributions, these hypoxia-induced patterns may suggest that oxygen molecules transported by CsMb1 from host environments are provided to cells in the parenchyma and intrauterine eggs/sex organs of the worms for energy metabolism and/or, more importantly, eggshell formation by CsMb1 and CsMb3, respectively. Other globin homologs are likely to perform non-respiratory functions. Based on the responsive expression profile against nitrosative stress, an oxygenated form of secreted CsMb1 is suggested to play a pivotal role in parasite survival by scavenging NO generated by host immune cells via its NO dioxygenase activity.

Proteomic analysis of Ascocotyle longa (Trematoda: Heterophyidae) metacercariae.

Ascocotyle longa is parasitic trematode with wide distribution throughout America, Europe, Africa, and Middle East. Despite the fact that this fish-borne pathogen has been considered an agent of human heterophyiasis in Brazil, the molecules involved in the host-parasite interaction remain unknown. The present study reports the proteome profile of A. longa metacercariae collected from the fish Mugil liza from Brazil. This infective stage for humans, mammals and birds was analyzed using nLC-MS/MS approach. We identified a large repertoire of proteins, which are mainly involved in energy metabolism and cell structure. Peptidases and immunogenic proteins were also identified, which might play roles in host-parasite interface. Our data provided unprecedented insights into the biology of A. longa and represent a first step to understand the natural host-parasite interaction. Moreover, as the first proteome characterized in this trematode, it will provide an important resource for future studies.

Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro.

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

The pattern of serotonin and FMRFamide in cercaria from different taxonomic groups--a preliminary study.

Serotoninergic and FMRFamidergic components of the nervous system were examined in cercaria from different types using immunocytochemical techniques interfaced with confocal scanning laser microscopy. Cercariae from 9 families were studied - Opisthorchis felineus, Parafasciolopsis fasciolaemorpha, Echinochasmus coaxatus, Echinoparyphium aconiatum, Notocotylus attenuatus, Psilotrema tuberculata, Plagiorchis sp., Cyathocotyle bithyniae, Diplostomum chromatophorum. The results show that 5-HT-IR and FMRFamide-IR occur in all types of cercariae, regardless of what morphological, taxonomic and biological group they belong to. Small differences in the patterns of 5-HT-IR and FMRFamide-IR were observed.

A Multi-Omics Database for Parasitic Nematodes and Trematodes.

Helminth.net ( www.helminth.net ) is a web-based resource that was launched in 2000 as simply " Nematode.net " to host and investigate gene sequences from nematode genomes. Over the years it has evolved to become the moniker for a collection of databases: Nematode.net and Trematode.net . These databases host information for 73 nematode (roundworms) and 17 trematode (flatworms) species and serve as backbone for a number of tools that allow users to query slices of the data for multifactorial combinations of species-omics properties. Recent focus has been on inclusion of gene and protein expression data, population genomics and cross-kingdom interactions (metagenomics datasets). This chapter describes the website, the available tools and some of the new features.

Interactive effects of contamination and trematode infection in cockles biochemical performance.

Anthropogenic activities, especially those involving substances that pollute the environment can interfere with bivalve populations, as well as parasitism, a fundamental ecological interaction often neglected. In marine environments, organisms are concomitantly exposed to pollutants and parasites, a combination with synergistic, antagonistic or additive effects representing a potential threat to aquatic communities sustainability. In the present study, Cerastoderma edule (the edible cockle)-Himasthla elongata (trematode) was used as host-parasite model. Cockles are worldwide recognized as good sentinel and bioindicator species and can be infected by several trematodes, the most abundant macroparasites in coastal waters. Tested hypotheses were: 1) cockles exposed to increasing parasite pressure will present greater stress response; 2) cockles exposure to arsenic (single concentration test: 5.2 µg L-1) will change parasite infection success and cockles stress response to infection. Arsenic was used for being one of the most common pollutants in the world and stress response assessed using biochemical markers of glycogen content, metabolism, antioxidant activity and cellular damage. Results showed that intensity of parasite pressure was positively correlated to biochemical response, mainly represented by higher metabolic requirements. Contamination did not affect parasite infection success. Compared to arsenic, trematode infection alone exerted a stronger impact: higher glycogen storage, metabolism and cellular damage and antioxidant activity inhibition. In interaction, parasitism and arsenic reduced hosts metabolism and cellular damage. Therefore, to a certain extent and in a contamination scenario, cockles may benefit from trematode infection, working as a protection for the pollutant accumulation in the organisms, reducing overall ROS production, which can consequently led to less toxic effects. These findings highlighted the deleterious effects of trematode infection in their hosts and showed the importance of including parasitology in ecotoxicological studies.

Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus.

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

[Changes produced by trematode larvae in the phospholipid fatty acid composition of digestive gland of the mollusc Littorina saxatilis].

Lipids of the digestive gland of the mollusc Littorina saxatilis from the White and Barents Seas were studied. Changes of its biochemical composition are discussed in the connection with different temperature of the habitat and with infestation with trematode larvae. Comparative analysis of the fatty acid (FA) composition of each of phospholipids in intact molluscs has revealed essential differences. Phosphatidylcholine and monophosphatidylinositol (MPI) FA did not differ in the omega 3/omega 6 ratio, which is due to their tolerance to the temperature factor, whereas more unsaturated phospholipids--phosphatidylethanolamine (FEA), its plasmalogen form (pFEA), and phosphatidylserine--differed 1.5-2 times in the studied molluscs. Predominance of omega 3 acids in the Borents Sea molluscs undoubtedly is due to the lower habitat temperatures, as it provides a higher fluidity of membrane phospholipids. Infestation affected to the greatest degree the quantitative FA composition in pFEA and MPI. At infestation, out of all considered phospholipids, only in MPI there was revealed a threefold decrease of the content of eikosenoic acid C20 : 1, whereas in all other phospholipids, in the contrary, it increased. Monophosphatidylinositols also differed essentially from other phospholipids by the saturated FA amount, which changed the unsaturation index of these phospholipids. Since the functional significance of this minor phospholipid is determined by its participation in the so-called phosphatidylinositol system of the hormonal signal transduction, it seems interesting to elucidate whether an increase of this membrane phospholipid saturation at invasion affects the reflex connection between signals from receptors located in a parasite and enzymatic processes.

Trace element and metal sequestration in vitellaria and sclerites, and reactive oxygen intermediates in a freshwater monogenean, Paradiplozoon ichthyoxanthon.

Exposure to metals and other trace elements negatively affects infection dynamics of monogeneans, including diplozoids, but, physiological mechanisms linked to exposure have yet to be documented. In this study sequestration of trace elements and reactive oxygen intermediate production in the monogenean, Paradiplozoon ichthyoxanthon, was demonstrated. During dissection of host fish, Labeobarbus aeneus, the gills were excised and assessed for P. ichthyoxanthon, which were removed and frozen for fluorescence microscopy or fixed for transmission electron microscopy. Trace elements were sequestered in the vitellaria and sclerites in P. ichthyoxanthon, and the presence of reactive oxygen intermediates was observed predominantly in the tegument of the parasite. Trace elements and metals identified and ranked according to weight percentages (wt%) in the vitellaria were Cu > C > Au > O > Cr > Fe > Si while for the sclerites C > Cu > O > Au > Fe > Cr > Si were identified. For most element detected, readings were higher in the vitellaria than the sclerites, except for C and O which were higher in sclerites. Specifically for metals, all levels detected in the vitellaria were greater than in sclerites. Based on the proportion of trace elements present in the vitellaria and sclerites it appears that most trace elements including metals were sequestered in the vitellaria. The results of reactive oxygen intermediate production in the tegument of the parasite suggests either trace element accumulation takes place across the tegument or results from the action of the host's immune response on the parasite. The results serve as the first demonstration of trace element sequestration and reactive oxygen intermediates in a freshwater monogenean parasite.