A novel Kunitz protein with proposed dual function from Eudiplozoon nipponicum (Monogenea) impairs haemostasis and action of complement in vitro.

Serine peptidases are involved in many physiological processes including digestion, haemostasis and complement cascade. Parasites regulate activities of host serine peptidases to their own benefit, employing various inhibitors, many of which belong to the Kunitz-type protein family. In this study, we confirmed the presence of potential anticoagulants in protein extracts of the haematophagous monogenean Eudiplozoon nipponicum which parasitizes the common carp. We then focused on a Kunitz protein (EnKT1) discovered in the E. nipponicum transcriptome, which structurally resembles textilinin-1, an antihemorrhagic snake venom factor from Pseudonaja textilis. The protein was recombinantly expressed, purified and biochemically characterised. The recombinant EnKT1 did inhibit in vitro activity of Factor Xa of the coagulation cascade, but exhibited a higher activity against plasmin and plasma kallikrein, which participate in fibrinolysis, production of kinins, and complement activation. Anti-coagulation properties of EnKT1 based on the inhibition of Factor Xa were confirmed by thromboelastography, but no effect on fibrinolysis was observed. Moreover, we discovered that EnKT1 significantly impairs the function of fish complement, possibly by inhibiting plasmin or Factor Xa which can act as a C3 and C5 convertase. We localised Enkt1 transcripts and protein within haematin digestive cells of the parasite by RNA in situ hybridisation and immunohistochemistry, respectively. Based on these results, we suggest that the secretory Kunitz protein of E. nipponicum has a dual function. In particular, it impairs both haemostasis and complement activation in vitro, and thus might facilitate digestion of a host's blood and protect a parasite's gastrodermis from damage by the complement. This study presents, to our knowledge, the first characterisation of a Kunitz protein from monogeneans and the first example of a parasite Kunitz inhibitor that impairs the function of the complement.

Functional ultrastructure and cytochemistry of vitellogenesis and mature vitellocytes of the digenean Cainocreadium labracis (Dujardin, 1845), parasite of Dicentrarchus labrax (L., 1758).

Vitellogenesis and vitellocytes of Cainocreadium labracis were studied by transmission electron microscopy (TEM) and TEM cytochemistry. Four developmental stages were distinguished during vitellogenesis: (I) stem cell of high nucleo-cytoplasmic ratio; (II) early differentiation with chief activity focused on the beginning of protein synthesis and shell globule formation; (III) advanced differentiation with rapid intensification of protein synthesis, progressive fusion of single shell globules into large globule clusters, and formation of unsaturated lipid droplets surrounded by ß-glycogen particles; and (IV) mature vitellocyte. Early vitellogenesis with vitellocyte maturation consists of: (1) increase in cell volume; (2) increased development of large, parallel cisternae of GER with production of proteinaceous granules; (3) development of small Golgi complexes that package granules; and (4) within vacuoles, progressive enlargement of proteinaceous granules into shell globule clusters formed during vitellogenesis. Three types of inclusions accumulate in large amounts in mature vitelline cells: (1) shell globule clusters, important component in the formation of egg shell; (2) numerous unsaturated lipid droplets. Though fewer, there are also diphasic droplets consisting of saturated and unsaturated lipids in the same droplet, and (3) a relatively small amount of ß-glycogen particles, usually surround a few groups of lipid droplets. The ß-glycogen and lipid droplets are nutritive reserves for embryogenesis. General pattern and functional ultrastructure of vitellogenesis greatly resemble those observed in some lower cestodes, such as bothriocephalideans and diphyllobothrideans. Variations and differences in the amount of lipids and of glycogen during vitellogenesis in lower cestodes and other trematodes are compared and discussed.

Identification and partial characterization of a novel serpin from Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

BACKGROUND: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation. RESULTS: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed. CONCLUSION: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses.

Methods for quantifying eggs and oviposition rate of Dawestrema cycloancistrium (Monogenea: Dactylogyridae), monogenean parasite of Arapaima gigas (Teleostei: Osteoglossidae).

Dawestrema cycloancistrium is the main ectoparasite causing mortality in fingerlings of pirarucu (Arapaima gigas) in Amazonian aquaculture. Very little is known about the D. cycloancistrium reproductive index and appropriate methods of collecting eggs for investigation. This study aimed to determine the oviposition rate of D. cycloancistrium. To achieve this aim, two egg quantification methods were tested: the estimative method (ME) and the total counting method (MT). Compared with the MT, the ME overestimated the number of eggs counted, which were 2943.5 ± 2840.6 and 1041.5 ± 533 eggs, and the oviposition rate, which was 80.1 ± 58.7 and 31.4 ± 16.4 eggs/parasite/day, for ME and MT, respectively. These results show that for studies quantifying D. cycloancistrium eggs, the total eggs in the sample must be counted, as the estimates made using subsamples are not representative. Using the MT, the oviposition rate for D. cycloancistrium was determined to be 31.4 ± 16.4 eggs per adult parasite per day. The present study demonstrates the egg production capacity of a monogenean species parasite of A. gigas, providing basic biological data for D. cycloancistrium.

Lipid profiles in Himasthla elongata and their intermediate hosts, Littorina littorea and Mytilus edulis.

In this research, the lipid profiles in rediae and free-living larvae (cercariae) of the trematodes Himasthla elongata were studied. It was shown that the lipid profiles of cercariae and rediae reflect the selective accumulation of membrane phospholipids and polyunsaturated fatty acids. In addition, infected tissues' lipid compositions in the intermediate hosts Littorina littorea (whole organism) and Mytilus edulis (foot) were studied. Modifications in the molluscs' lipid compositions were mainly caused by the parasites' metabolic requirements, as the parasites are unable to engage in de novo lipid biosynthesis, and thus, they utilise the host tissues' metabolites. The assumption that changes in the lipid unsaturation of infected intermediate hosts may affect their temperature resistance was discussed.

Distribution of Tetrodotoxin in the New Zealand Clam, Paphies australis, Established Using Immunohistochemistry and Liquid Chromatography-Tandem Quadrupole Mass Spectrometry.

Tetrodotoxin (TTX) is one of the most potent neurotoxins known. It was originally thought to only occur in puffer fish but has now been identified in twelve different classes of freshwater and marine organisms, including bivalves. Despite being one of the world’s most studied biotoxins, its origin remains uncertain. There is contradictory evidence regarding the source of TTX and its pathway through food webs. To date, the distribution of TTX has not been examined in bivalves. In the present study, 48 Paphies australis, a TTX-containing clam species endemic to New Zealand, were collected. Thirty clams were dissected, and organs and tissues pooled into five categories (siphons, digestive gland, adductor muscles, and the ‘rest’) and analyzed for TTX using liquid chromatography-mass spectrometry (LC-MS). The micro-distribution of TTX was visualized in the remaining 18 individuals using an immunohistological technique incorporating a TTX-specific monoclonal antibody. The LC-MS analysis revealed that siphons contained the highest concentrations of TTX (mean 403.8 µg/kg). Immunohistochemistry analysis showed TTX in the outer cells of the siphons, but also in the digestive system, foot, and gill tissue. Observing TTX in organs involved in feeding provides initial evidence to support the hypothesis of an exogenous source in P. australis.

Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces.

We describe an atmospheric pressure matrix-assisted laser desorption-ionization mass spectrometry imaging system that uses long-distance laser triangulation on a micrometer scale to simultaneously obtain topographic and molecular information from 3D surfaces. We studied the topographic distribution of compounds on irregular 3D surfaces of plants and parasites, and we imaged nonplanar tissue sections with high lateral resolution, thereby eliminating height-related signal artifacts.

Threading: A novel insilico indagation method for genetic characterization of some diplostomoid metacercariae (Digenea:Diplostomidae Poirier, 1886).

The protein encoding zone of Mitochondrial DNA region (inherited from single lineage) seems most suitable and effective for taxonomic, systematic, ecological, evolutionary, DNA barcoding, cryptic species and population studies, exploiting nucleotide/amino acid datasets (1D/2D/3D conformational level). Nowadays, expeditious computerized methods are in trend for analyzing genetic material to demonstrate variations at various levels of protein structures. Structural proteomics have implemented here for genetic identification, differentiation and relationship of species from information rich data of mt COI gene of the family Diplostomidae with inclusion of molecular tools. Various aspects have been utilized herein for re-validation and infallible discrimination of Trematode diplostomoid metacercariae (Tetracotyle lucknowensis Pandey, 1971; T. xenentodoni Chakrabarti, 1970; T. fausti Rai and Pande, 1969; T. muscularius Chakrabarti, 1970 and Diplostomulum minutum Pandey, 1968), the infective stage in the life cycle, causing severe damage to fish host, whose adults are found mainly in fish eating birds and mammals.

Total mercury and selenium concentrations in Sarpa salpa and Balistes capriscus and in their respective Digenean endoparasites Robphildollfusium fractum and Neoapocreadium chabaudi from Tunisia.

The present study reports the levels of mercury and selenium in Sarpa salpa and Balistes capriscus collected along the coast of Mahdia and Sfax (Tunisia). The systems constituted by S. salpa and Robphildollfusium fractum and by B. capriscus and Neoapocreadium chabaudi were tested as potential bioindicators to monitor environmental Hg pollution in marine ecosystems. Mercury and selenium concentrations were assessed in kidney, liver and muscle of 51 S. salpa and of 45 B. capriscus as well as in their respective endoparasites R. fractum and N. chabaudi. The Se:Hg molar ratios were evaluated for both species across the study areas. Surprisingly, the Se:Hg molar ratio in B. capriscus muscle from Mahdia is significantly lower than in Sfax. Our results indicate that some parasites may also be implicated in the amount of Se and Hg available in tissues and therefore contribute to oscillations of the Se:Hg molar ratios. In the model involving the carnivorous species (B. capriscus), the 5.1-times higher levels of mercury in N. chabaudi than in B. capriscus muscle in Sfax enable this fluke to be a sensitive biomonitoring tool for Hg pollution. The present results confirm that the habitual consumption of S. salpa should not suppose any potential health risk for Tunisian people. On the other hand, the consumption of B. capriscus may be of concern and further monitoring is advisable, since the Hg average concentration in Mahdia was above the maximum allowed Hg concentration in the edible portion of fish fixed by the European Union.

Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.

The neuromuscular system in continuously swimming cercariae from Belarus. II Echinostomata, Gymnocephala and Amphistomata.

The neuromuscular system in cercariae of Moliniella anceps, Echinostoma revolutum, Cathaemasia hians, Psilochasmus oxyurus, Sphaeridiotrema globulus, Paramphistomum cervi and Diplodiscus subclavatus was studied with immunocytochemical methods and confocal scanning laser microscopy. The patterns of F-actin in the musculature, 5-HT immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR sensory receptors were investigated. The general patterns of musculature, 5-HT- and FMRFamide-IR neuronal elements in the 12 species studied here and in paper I are similar to those observed in other cercariae and reflect the morphology of the groups. The musculature of the tail shows variations which are related to the different strategies of host finding. In the Echinostomatoidea and Paramphistomoidea, the striated musculature of the tail is well developed compared to that in the Xiphidiocercariae. Specialized muscle fibres were found in S. globulus, which are able to change the shape of the tail. Nine of the species studied have seven paired 5-HT-IR neurons in the body, and two species have eight. No correlation between the body size and the number of 5-HT-IR neurons was observed. However, the size of the neurons followed the body size. The number of 5-HT-IR neurons in the brain ganglia increased from the primitive to the advanced forms. The number of FMRFamide-IR transverse commissures in the body correlates with the size of the cercariae. Regardless of the differences in the second intermediate host, the distribution of α-tubulin-IR sensory receptors shows a high degree of conformity in all species except in P. cervi, which encysts on plants.

Effects of ultraviolet radiation on an intertidal trematode parasite: an assessment of damage and protection.

Trematode parasites are integral components of intertidal ecosystems which experience high levels of ultraviolet radiation. Although these parasites mostly live within hosts, their life cycle involves free-living larval transmission stages such as cercariae which are directly exposed to ambient conditions. UV has previously been shown to considerably reduce the survival of cercariae. Here, we investigated potential mechanisms of protection and damage related to UV in the intertidal trematode Maritrema novaezealandensis. Firstly, the presence of sunscreen compounds (i.e. mycosporine-like amino acids) was quantified in the parasite tissue producing cercariae within a snail host, as well as in the free-swimming cercariae themselves. Secondly, levels of oxidative stress in cercariae after exposure to UV were investigated (i.e.protein carbonyls, catalase and superoxide dismutase). Thirdly, the DNA damage (i.e. cyclobutane­pyrimidine dimers) was compared between cercariae exposed and not exposed to UV. Lastly, functional aspects(survival and infectivity) of cercariae were assessed, comparing cercariae under light conditions versus dark after exposure to UV. We confirmed the presence of my cosporine-like amino acids in cercariae-producing tissue from within snail hosts, but were unable to do so in cercariae directly. Results further suggested that exposure to UV induced high levels of oxidative stress in cercariae which was accompanied by a reduction in the levels of protective antioxidant enzymes present. We also identified higher levels of DNA damage in cercariae exposed to UV, compared with those not exposed. Moreover, no clear effect of light condition was found on survival and infectivity of cercariae after exposure to UV. We concluded that cercariae are highly susceptible to UV damage and that they have very little scope for protection against or repair of UV-induced damage.

Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures.

Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.

Proteomics of foodborne trematodes.

Food-borne trematodiases are among the most neglected tropical diseases, not only in terms of research funding, but also in the public media. The Trematoda class contains several species identified as the causal agents of these diseases whose biological cycle, geographical distribution and epidemiology have been well characterised. The diagnosis of these diseases is based on parasitological techniques and only a limited number of drugs are currently available for treatments, most of which are unspecific. Therefore, in-depth studies to identify new and specific targets for both effective diagnosis and treatments are urgently needed. Currently, little molecular information is available regarding the host-parasite interaction. In this regard, proteomic studies have the potential to identify diagnostic biomarkers for the early detection of the diseases, as well as new vaccine targets. In this review, a description of the biology, clinical features and current diagnostic tools of the main groups of trematodes and the corresponding diseases they cause is followed by a discussion of the available studies using proteomic techniques to identify key parasite proteins involved in the pathogenesis of food-borne trematodiases.

Estimating isotope fractionation between cercariae and host snail with the use of isotope measurement designed for very small organisms.

Most studies have reported negative carbon and nitrogen isotope fractionations between hosts and parasites, but isotope values have not yet been determined for many parasite species, such as trematodes, due to their relatively small body sizes. We investigated the carbon and nitrogen isotope values of freshwater snails and trematode parasites by using a method for organisms with very small body sizes. We found negative isotope fractionation values between host snails and trematode parasites, similar to published values for other parasite groups with larger body sizes, which suggest that the mechanisms for determining isotope fractionations between hosts and parasites provide similar results.

Variation in the morphometrics of diplostomid metacercariae (Digenea: Trematoda) infecting the catfish, Clarias gariepinus in Tanzania.

Diplostomum species are economically important worldwide due to their metacercariae which parasitize the eyes of fish, in both natural and aquaculture systems. However, their striking morphological similarity, especially at the metacercarial stage, makes species separation difficult. Three closely related diplostomid metacercariae, namely Diplostomum mashonense (type 3), Tylodelphys sp. 1 and 2 (type 1 and 2, respectively), coexist in the cranial cavity of the catfish, Clarias gariepinus from Mindu dam, Lake Victoria, Msimbazi, Ruvu and Kilombero rivers. The morphometrics of these three species were analysed by discriminant function analysis to investigate the degree of variation among the populations from the five localities sampled. The first canonical functions for all visual examination plots accounted for over 50% of the between-group variability. The observed differences were mainly from measurements associated with length, indicating that these measurements are important in the description of population characteristics. Visual examination of the samples along the canonical functions showed a clear between-population differentiation. The overall random assignment of individuals into their original groups was high (97%). These extensive morphometric variations introduce doubt about the reliability of measurements in the determination of species in these trematodes, as they may lead to misidentifications.

Histopathological effects of the intramolluscan stages of Zygocotyle lunata, Echinostoma trivolvis, and Ribeiroia ondatrae on Helisoma trivolvis and observations on keratin in the trematode larvae.

The histopathological effects of Zygocotyle lunata, Echinostoma trivolvis, and Ribeiroia ondatrae in naturally infected Helisoma trivolvis were studied in hematoxylin and eosin sections of infected digestive glands fixed in 10% neutral buffered formalin. The larval stages of all three trematodes damaged the snail digestive gland. Most notable histopathology included disrupted digestive gland tubules, lysed cells, compressed tubules, and edematous spaces between tubules. Considerable damage was done by rediae ingesting digestive cells. There was a detectable hemocytic response by H. trivolvis in response to the rediae and cercariae of Z. lunata. Histochemical studies on sectioned material stained with the Ayoub-Shklar method for keratin detected the presence of this protein in the rediae and cercariae of Z. lunata and R. ondatrae. The presence of keratin is probably related to its role in cercarial encystment.

Field preservation of monogenean parasites for molecular and morphological analyses.

In the present work we examined the efficacy of three different chemical solutions (EtOH 70%, DMSO-NaCl solution, and Longmire buffer) in field preservation of fish gills to be subsequently screened for monogenean specimens destined to morphological and molecular analyses. Degree of difficulty in collecting monogeneans from gills, morphological state of parasites, integrity of their DNA and reliability of sequence reading were observed and qualitatively compared to those of gills and parasites stored in 5% formalin and 99% ethanol. Data were collected over a period of 2 months. Storage in Longmire buffer resulted in dissociation of gills and parasites, while both DMSO and 70% ethanol provided a fine physical and molecular preservation of gills and monogeneans, allowing rapid collection of parasites from lamellae, and easy extraction, amplification and sequencing of parasitic DNA.

Lipid content and fatty acid composition of the monogenean Neobenedenia girellae and comparison between the parasite and host fish species.

Neobenedenia girellae, a capsalid monogenean, is a destructive fish parasite. We studied the lipid content and fatty acid composition of N. girellae and the skin and cutaneous mucus of a host fish, the amberjack Seriola dumerili (Carangidae). The lipid content of adult N. girellae was less than one-fourth that of both the skin and cutaneous mucus of its host. Adult N. girellae, S. dumerili skin and mucus had a relatively high weight-percentage of C16:0, C18:1(n-9), C18:0 and C22:6(n-3) fatty acids. When S. dumerili were fed a diet supplemented with [13C] fatty acids, [13C] fatty acids were detected in S. dumerili skin and adult N. girellae on S. dumerili, but no [13C] fatty acids were detected in the S. dumerili cutaneous mucus. In addition, the epidermis of S. dumerili, attached with N. girellae, was markedly thin. These results suggest that N. girellae feeds primarily on host epithelial cells. We then infected 2 host fishes, S. dumerili and the spotted halibut Verasper variegatus (Pleuronectidae; a host less susceptible to N. girellae infection), and compared the fatty acid composition of N. girellae with that of the skin and cutaneous mucus of the hosts. The fatty acid profiles from all samples were qualitatively and quantitatively similar. Thus, the fatty acid composition of the host may not contribute to the difference in susceptibility between S. dumerili and V. variegatus. These results may serve to develop new strategies for the control of N. girellae infections.

The use of fluorescent fatty acid analogs as labels in trematode experimental infections.

We examined the utility of fluorescent fatty acid analog dyes for labeling larval trematodes to use in experimental infections. Our goals were to identify two dyes that label larval trematodes belonging to the species Maritrema novaezealandensis and Coitocaecum parvum, determine if the dyes influence survival and infectivity of larval trematodes and/or host mortality, and if larval trematodes labeled with alternative dyes could be distinguished post-infection. The two dyes tested, BODIPY FL C(12) and BODIPY 558/568 C(12), successfully labeled all treated larval trematodes, did not influence cercariae survival or infectivity, and did not influence host mortality in either host-parasite system. All larval parasites were fluorescent and distinguishable after 5 days in amphipod intermediate hosts. In addition, larval Acanthoparyphium sp. were strongly fluorescent with both dyes after 5 weeks within cockle hosts. This method should be extremely useful for experimental studies using trematode-host systems as models for addressing a range of ecological and evolutionary questions.