Mechanisms of the host immune response and helminth-induced pathology during Trichobilharzia regenti (Schistosomatidae) neuroinvasion in mice.

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Transcriptome analysis of goldfish (Carassius auratus) in response to Gyrodactylus kobayashii infection.

Gyrodactylid monogeneans are widespread parasites of teleost fishes, and infection with these parasites results in high host morbidity and mortality in aquaculture. To comprehensively elucidate the immune mechanisms against Gyrodactylus kobayashii, the transcriptome profiles of goldfish (Carassius auratus) skin after challenge with G. kobayashii were first investigated using next-generation sequencing. Approximately 21 million clean reads per library were obtained, and the average percentage of these clean reads mapped to the reference genome was 82.25%. A total of 556 differentially expressed genes (DEGs), including 344 upregulated and 212 downregulated genes, were identified, and 380 DEGs were successfully annotated and assigned to 95 signaling pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, 14 pathways associated with immune response were identified mainly including mTOR signaling pathway, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, toll-like receptor signaling pathway, and phagosome. Twelve genes were selected and validated using qRT-PCR. A similar trend of these genes between RNA-Seq and qRT-PCR was observed, indicating that RNA-Seq data was reliable. Besides, the ALP activity and NO content in serum were significantly higher in the infected goldfish compared with the non-infected goldfish. In summary, this study provides better understandings of immune defense mechanisms of goldfish against G. kobayashii, which will support future molecular research on gyrodactylids and facilitate the prevention and treatment of gyrodactylosis in aquaculture.

The evolution of TNF signaling in platyhelminths suggests the cooptation of TNF receptor in the host-parasite interplay.

BACKGROUND: The TNF signaling pathway is involved in the regulation of many cellular processes (such as apoptosis and cell proliferation). Previous reports indicated the effect of human TNF-α on metabolism, physiology, gene expression and protein phosphorylation of the human parasite Schistosoma mansoni and suggested that its TNF receptor was responsible for this response. The lack of an endogenous TNF ligand reinforced the idea of the use of an exogenous ligand, but also opens the possibility that the receptor actually binds a non-canonical ligand, as observed for NGFRs. METHODS: To obtain a more comprehensive view, we analyzed platyhelminth genomes deposited in the Wormbase ParaSite database to investigate the presence of TNF receptors and their respective ligands. Using different bioinformatics approaches, such as HMMer and BLAST search tools we identified and characterized the sequence of TNF receptors and ligand homologs. We also used bioinformatics resources for the identification of conserved protein domains and Bayesian inference for phylogenetic analysis. RESULTS: Our analyses indicate the presence of 31 TNF receptors in 30 platyhelminth species. All platyhelminths display a single TNF receptor, and all are structurally remarkably similar to NGFR. It suggests no events of duplication and diversification occurred in this phylum, with the exception of a single species-specific duplication. Interestingly, we also identified TNF ligand homologs in five species of free-living platyhelminths. CONCLUSIONS: These results suggest that the TNF receptor from platyhelminths may be able to bind canonical TNF ligands, thus strengthening the idea that these receptors are able to bind human TNF-α. This also raises the hypothesis that an endogenous ligand was substituted by the host ligand in parasitic platyhelminths. Moreover, our analysis indicates that death domains (DD) may be present in the intracellular region of most platyhelminth TNF receptors, thus pointing to a previously unreported apoptotic action of such receptors in platyhelminths. Our data highlight the idea that host-parasite crosstalk using the TNF pathway may be widespread in parasitic platyhelminths to mediate apoptotic responses. This opens up a new hypothesis to uncover what might be an important component to understand platyhelminth infections.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

Cytokine gene expression profiles in goldfish (Carassius auratus) during Gyrodactylus kobayashii infection.

Monogeneans of the genus Gyrodactylus are well-known pathogens causing huge mortalities in wild and cultured fish. Cytokine expression is one of most important host defense mechanisms against parasite infections. In this study, the expression pattern of the key pro-inflammatory (IL-1ß, IL-8, IFN-γ, TNF-α, IL-12 and iNOS) and anti-inflammatory cytokine genes (IL-10, TGFß and IL-4) of Gyrodactylus kobayashii infected goldfish (Carassius auratus) were determined by real-time quantitative PCR analysis. Our results showed that G. kobayashii infection caused increased expression of the pro-inflammatory cytokines including IFN-γ, TNF-α and iNOS in all detected tissues throughout the infection period. Among these genes, iNOS has the highest transcript level accompanied with increased nitric oxide (NO) concentration in the serum of all infected goldfish. The mRNA level of IL-1ß in the liver, spleen and head kidney was significantly up-regulated during the early stage of infection (days 2-8). While high expression level of IL-8 and IL-12 was observed during the elimination phase of infection (days 10-14). As for anti-inflammatory cytokines, the expression profiles of IL-10 were distinct from those of TGF-ß and IL-4. Specifically, the mRNA level of IL-10 did not increase in the spleen and head kidney during the early stage of infection, while increased expression of TGF-ß and IL-4 were likewise seen. Besides, all infected fish had significantly higher complement C3 but lower IgM levels than the non-infected fish. The results provide insights into the interaction between gyrodactylids and the fish host, and indicate that systemic cytokine responses are critical for controlling parasite infection in fish.

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.

Cattle naturally infected by Eurytrema coelomaticum: Relation between adenosine deaminase activity and zinc levels.

The enzyme adenosine deaminase (ADA) is critical for modulating the immune system, and in the presence of zinc, its activity is catalyzed. The aim of this study was to evaluate the ADA activity in pancreas of cattle naturally infected by Eurytrema coelomaticum in relation to the results of zinc levels, pathological findings and parasite load. For this study 51 slaughtered cattle were used. The animals were divided into two groups: Group A consisting of animals naturally infected by E. coelomaticum (n=33) and Group B of uninfected animals (n=18). Blood and pancreas were collected of each animal for analysis of zinc and ADA, respectively. Infected cattle showed a reduction on seric levels of zinc, and decreased ADA activity in the pancreas (P>0.05). A positive correlation between zinc levels and ADA activity was observed. Thus, high parasite load and severity of histopathologic lesions affect the ADA activity in pancreas, as well as the zinc levels in serum of infected animals (negative correlation between these variables). Therefore, we can conclude that cattle infected by E. coelomaticum have low ADA activity in pancreas, which can be directly related to zinc reduction, responsible for ADA activation and catalyzes.

Modulation of innate immune responses and induction of oxidative stress biomarkers in Pangasianodon hypophthalmus following an experimental infection with dactylogyrid monogeneans.

Modulation of innate immune activity and oxidative stress response of Pangasianodon hypophthalmus through experimental infection with (Thaparocleidus sp.) dactylogyrid monogenean was studied. A standard cohabitation method was used to infect healthy experimental fish. After 14 days, dactylogyrid (gill monogenean) infested fish were sampled and categorised into three different infected groups namely (T1) low (<10 mean dactylogyrid per gill arch per fish), (T2) moderate (10-49 mean dactylogyrid per gill arch per fish) and (T3) high (>50 mean dactylogyrid per gill arch per fish) along with a control group T0 (un-infested fish). Serum and tissues (gills and liver) were collected from experimental fish and analyzed for markers of innate immune and oxidative stress, respectively. The results showed that respiratory burst activity, myeloperoxidase level, serum lysozyme, α-2 macroglobulin and total serum immunoglobulin level were significantly (p < 0.05) elevated in fish with different degrees of parasite infestation compared to the control (un-infested group). Similarly, cellular oxidative biomarkers superoxide dismutase, catalase, glutathione-S-transferase and Na+-K+-ATPase activities of gills and liver were significantly (p < 0.05) elevated in dactylogyrid infested fish in comparison to the control. However, significantly decreased level of albumin, albumin to globulin ratio, total serum antiprotease and ceruloplasmin were observed in fish infested with low degree of dactylogyrids, while no significant differences in these parameters were observed between moderately infested and the control groups. The results suggested that varying degree of gill monogenean dactylogyrid infestation not only modulated the innate immune response of P. hypophthalmus by lowering albumin, total serum antiprotease and ceruloplasmin and inducing respiratory burst activity, phagocytic activity, myeloperoxidase, lysozyme, α-2 macroglobulin and total immunoglobulins, but also the oxidative stress biomarkers. The baseline data obtained in the present study will be valuable in understanding the host-parasite relationship and the dynamics of innate, oxidative stress responses and susceptibility of P. hypophthalmus to different degrees of parasitosis.

Comparative transcriptomics of stickleback immune gene responses upon infection by two helminth parasites, Diplostomum pseudospathaceum and Schistocephalus solidus.

Immune systems of vertebrates are much more diverse than previously thought, in particular at the base of the vertebrate clade. RNA-seq was used to describe in detail the transcriptomic response of stickleback hosts to infection by two helminth parasites, the trematode Diplostomum pseudospathaceum (2 genotypes plus a genotype mix) and the cestode Schistocephalus solidus. Based on a global transcription profiling, we present immune genes that are active during chronic or multiple repeated infection. We found that the transcription profiles of D. pseudospathaceum genotypes were as divergent as those of the two parasite species. When comparing the host immune response, only 5 immune genes were consistently upregulated upon infection by both species. These genes indicated a role for enhanced toll like receptor (TLR) activity (CTSK, CYP27B1) and an associated positive regulation of macrophages (CYP27B1, THBS1) for general helminth defense. We interpret the largely differentiated gene expression response among parasite species as general redundancy of the vertebrate immune system, which was also visible in genotype-specific responses among the different D. pseudospathaceum infections. The present study provides the first evidence that IL4-mediated activation of T-helper lymphocyte cells is also important in anti-helminthic immune responses of teleost fish.

Relationship Between Pathological Findings and Cholinesterase Activity and Nitric Oxide Levels in Cattle Infected Naturally by Eurytrema coelomaticum.

The aim of this study was to evaluate the role of butyrylcholinesterase (BChE) (in the serum and pancreas), acetylcholinesterase (AChE) (in the whole blood and pancreas) and nitric oxide (NO) (in the serum and pancreas) in cattle infected naturally by Eurytrema coelomaticum. Fifty-one cattle were studied, including 33 infected by E. coelomaticum and 18 uninfected animals. Significantly greater AChE activity was found in the pancreas of infected animals (P <0.01); however, these cattle had lower AChE activity in whole blood. BChE activity was greater in the sera of infected animals (P = 0.05), but was less in pancreatic samples. NO levels were significantly higher in the sera (P <0.05) and pancreas (P <0.001) of infected cattle compared with uninfected animals. A positive correlation was found between AChE activity in the pancreas and parasite load, but there was negative correlation between pancreatic BChE activity and parasitic load. Expression of AChE, BChE and NO is therefore linked to the inflammation caused by E. coelomaticum in cattle.

Immunohistochemical study and mRNA cytokine profile of the local immune response in cattle naturally infected with Calicophoron daubneyi.

In order to recognize the local immune response of the definitive host to Calicophoron daubneyi natural infection, an immunohistochemical study was carried out in the reticulum and rumen in 49 naturally infected cattle. The role of cytokines (IL-4 and IL-10 interleukins and IFN-γ) in the activation of specific defence mechanisms was evaluated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays to study cytokine mRNA expression. In all infected animals, CD3+ T lymphocytes seemed to be the main element of the inflammatory infiltrate in the reticular and ruminal lamina propria at the point of the parasite adhesion. Intraepithelial globule leukocytes also showed immunolabelling for CD3. Most CD3+ cells also expressed CD4 (T cell helper) antigen although sporadic CD8+-cytotoxic lymphocytes were observed. Local expression of IFN-γ was observed in damaged papillae at the site of parasite attachment and in scattered cells in the lamina propria. B cells (CD79αcy+, CD45+ and IgG+) were found constantly in relation to lymphoid aggregates. MAC387 was expressed in squamous epithelium and in macrophages of the lamina propria of affected papillae. Macrophages in this location also stained positively for CD163 and CD68. Intraepithelial Langerhans cells and macrophages located in the lamina propria showed immunopositivity for MHCII in the affected areas. RT-qPCR analysis confirmed a statistical significant increase of IFN-γ, and IL-10 expression (p<0.01) in the rumen associated with the presence of flukes. These findings suggest a predominant Th1 polarized local immune response with the probable involvement of Th regulatory cells in cattle C. daubneyi natural infection.

Thermodynamics of trematode infectivity.

Temperature is an important factor influencing the biology of organisms and is intrinsically linked to climate change. The establishment of trematodes in target hosts is potentially susceptible to temperature changes effecting parasite infectivity or host susceptibility, and therefore in order to develop predictive frameworks of host-parasite dynamics under climate change large-scale analyses are required. The present study analyses the thermodynamics of the infectivity of larval trematodes including miracidia, cercariae and metacercariae from experimental data contained in the scientific literature using the Arrhenius critical incremental energy of activation (E*), an accurate measure of temperature-driven reaction rates. For miracidia and cercariae, infectivity increases as the temperature rises reaching a plateau over optimal thermal ranges before declining at higher temperatures. In contrast, metacercarial infectivity is at its greatest at low temperatures, declining with increasing temperature.

Depressed neuronal growth associated protein (GAP)-43 expression in the small intestines of mice experimentally infected with Neodiplostomum seoulense.

Neodiplostomum seoulense (Digenea: Neodiplostomidae) is an intestinal trematode that can cause severe mucosal pathology in the small intestines of mice and even mortality of the infected mice within 28 days after infection. We observed neuronal growth associated protein-43 (GAP-43) expression in the myenteric plexus of the small intestinal wall of N. seoulense-infected mice until day 35 post-infection (PI). BALB/c mice were infected with 200 or 500 N. seoulense metacercariae isolated from naturally infected snakes and were killed every 7 days for immunohistochemical demonstration of GAP-43 in the small intestines. N. seoulense-infected mice showed remarkable dilatation of intestinal loops compared with control mice through days 7-28 PI. Conversely, GAP-43 expression in the mucosal myenteric plexus was markedly (P<0.05) reduced in the small intestines of N. seoulense-infected mice during days 7-28 PI and was slightly normalized at day 35 PI. From this study, it is evident that neuronal damage occurs in the intestinal mucosa of N. seoulense-infected mice. However, the correlation between intestinal pathology, including the loop dilatation, and depressed GAP-43 expression remains to be elucidated.

Worms to the rescue: can worm glycans protect from autoimmune diseases?

Autoimmune and autoinflammatory diseases represent a significant health burden, especially in Western societies. For the majority of these diseases, no cure exists. Recently, research on parasitic worms (helminths) has demonstrated great potential for whole worms, their eggs or their excretory/secretory proteins in down-regulating inflammatory responses both in vitro and in vivo, in various disease models and, in some cases, even in clinical trials. The worms are thought to induce Th2 and regulatory T cells, interfere with Toll-like receptor (TLR) signaling and to down-regulate Th17 and Th1 responses. The molecular mechanisms underlying the worms' ability to modulate the host immune response are not well understood, and many hypotheses have been proposed to explain the observed immune modulation. Increasing evidence suggests that carbohydrate structures (glycans), for example, phosphorylcholine-modified glycans or Galbeta1-4(Fucalpha1-3)GlcNAc- (Lewis X, Le(X)) containing glycans, expressed by the worms contribute to these modulating properties by their interaction with antigen presenting cells. Helminths express a broad variety of protein- and lipid-linked glycans on their surface and on secretory products. These glycans differ in amount and composition and several of these structures are species specific. However, worms also express glycan antigens that are found in a wide variety of different species. Some of these "common" worm glycans are particularly interesting with regard to regulating host responses, because they have the potential to interact with C-type lectins on dendritic cells and thereby may interfere with T-cell polarization. Helminths and helminth-derived molecules form a novel and promising group of therapeutics for autoinflammatory diseases. However, much has to be learned about the molecular mechanisms behind the helminth-mediated antiinflammatory properties. This review will describe some of the emerging evidence in selected disease areas as well as discuss the putative role of glycans in helminth-mediated immunosuppression.

[Increased accumulation of some trace elements in peripheral blood and bile of patients infested with Opisthorchis felineus (Rivolta, 1884) and Metorchis bilis (Braun, 1890)].

The invasion with Opisthorchis felineus (Rivolta, 1884) is known to be common in the Ob River region, West Siberia. These trematodes parasitize biliary tract of devinitive host (man or some species of animals). Other opisthorchiid species occurring in West Siberia, Metorchis bilis (Braun, 1890), has also been recorded recently as human parasite. Life cycles of both these trematodes include fish-eating mammal hosts. Eggs of O. felineus and M. bilis are very similar morphologically and can hardly be indentified. Chronic invasion with the helminthes is found to be a cause of disbolism of trace elements or high accumulation of some essential and toxic elements in the organism of definitive host. The aim of the present study was to determine concentrations of some essential and toxic elements in samples of peripheral blood and bile obtained from patients infested with Opisthorchis and/or Metorchis using instrumental neuron-activation technique. At first, all patients with microscopically confirmed opisthorchiasis (by microscopic examination of faeces and bile for the helminth eggs) were examined with serological method (ELISA) for specific anti-Opisthorchis and anti-Metorchis antibodies. Among 139 examined patients, 56.1% had specific antibodies against both Opisthorchis and Metorchis, 41.7% showed anti-Opisthorchis antibodies only, and 2.1% turn out to be seropositive for anti-Metorchis antibodies only. Of 31 elements detected in the samples of peripheral blood and bile, the concentration of nine essential and toxic elements (mercury, chromium, cesium, rubidium, lanthanum, bromine, selenium, zinc, and cobalt) in the patients with the mixt-infection of Opisthorchis and Metorchis (78 individuals) and with the Opisthorchis mono-infection (58 individuals) were significantly higher than those in healthy uninfested individuals. Among these elements, mercury and chromium showed the highest concentrations in infested patients. We suggest that the significant increase in concentrations of some toxic elements in blood and bile of patients may be associated with increased bioaccumulation of these elements in environment, especially in the local river water and fish. Moreover, concentrations of such elements as chromium, mercury, cesium, lanthanum, selenium, and zinc in blood and/or bile of the patients with mixt-infection of Opisthorchis and Metorchis were significantly higher than those in the patients with mono-infection of Opisthorchis. Accumulation of these elements in the organisms of patients was positively correlated with the number of parasites and the duration of helminth infection.

Characterization of fucose-binding lectins in rock- and mud-dwelling snails inhabiting Kuwait Bay.

Two novel lectins that bind selectively to trematode-associated fucosylated determinants have been characterized and purified from the hemolymph of wild-type rock-dwelling Turbo coronatus and mud-dwelling Cerithidea cingulata snails inhabiting Kuwait Bay. Both lectins were separately purified from the hemolymph of T. coronatus (designated Tc lectin) and C. cingulata (designated Cc lectin) by affinity chromatography on immobilized L-fucose-agarose columns and elution with a gradient of 0.1-1 M L-fucose. Assessments of the structural characteristics, by one- and two-dimensional polyacrylamide gel electrophoresis, indicated that Tc and Cc lectins were structurally distinct, and exist in their native forms as multimers of non-covalently associated subunits of different sizes and pIs. Removal of N-linked glycans by endo-beta-N-acetylglucosaminidase F resolved the heterodisperse pattern of Tc lectin subunits into 31 and 27 kDa components (each with a pI of 7.8) as well as an 8 kDa component with a pI of 7.2, and collapsed the size microheterogeneity of the Cc lectin subunits into a single spot of 33 kDa (pI 5.4). Each of these subunits is subjected to differential post-translational N-linked glycosylations, which involve variable acidic complex-type units in Tc lectin and neutral high-mannose-type units in Cc lectin. Based on trematode glycoprotein binding and inhibition assays, the two lectins exhibited optimum binding at a similar pH range, but were distinct in terms of their temperature stability as well as binding affinities towards the fucose moiety constituting the fucosylated target.

Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis.

California killifish (Fundulus parvipinnis) infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours rendering them more susceptible to predation by avian final hosts. Heavily infected killifish grow and reproduce normally, despite having thousands of cysts inside their braincases. This suggests that E. californiensis affects only specific locomotory behaviours. We hypothesised that changes in the serotonin and dopamine metabolism, essential for controlling locomotion and arousal may underlie this behaviour modification. We employed micropunch dissection and HPLC to analyse monoamine and monoamine metabolite concentrations in the brain regions of uninfected and experimentally infected fish. The parasites exerted density-dependent changes in monoaminergic activity distinct from those exhibited by fish subjected to stress. Specifically, E. californiensis inhibited a normally occurring, stress-induced elevation of serotonergic metabolism in the raphae nuclei. This effect was particularly evident in the experimentally infected fish, whose low-density infections were concentrated on the brainstem. Furthermore, high E. californiensis density was associated with increased dopaminergic activity in the hypothalamus and decreased serotonergic activity in the hippocampus. In conclusion, the altered monoaminergic metabolism may explain behavioural differences leading to increased predation of the infected killifish by their final host predators.

Response of rainbow trout (Oncorhynchus mykiss) in skin and fin tissue during infection with a variant of Gyrodactylus salaris (Monogenea: Gyrodactylidae).

Response mechanisms of rainbow trout Oncorhynchus mykiss (Walbaum), experimentally infected with a Danish strain of Gyrodactylus salaris Malmberg, 1957 were investigated using molecular tools (qPCR) and immunohistochemistry. Expression of ten immune-relevant genes and reactivity with five different antibodies in the epidermis of skin and fin tissue were analysed in susceptible but responding rainbow trout. Rainbow trout were susceptible with regard to the parasite strain which initially colonised fins but relocated to the body region as infection progressed. The ten investigated genes encoding the cytokines IL-1beta, TNF-alpha, IFN-gamma, IL-10 and markers for adaptive immune activity, such as CD-4, CD-8, TCR-alpha, IgM, IgT and MHC II, were not found significantly regulated during the course of infection although IFN-gamma showed a slight up-regulation. Immunohistochemical analyses showed positive reactivity with antibodies against CD3, B-lymphocytes, neutrophilic granulocytes and collectin but not with mAb against IgM. No staining differences between infected and non-infected skin and fin tissue were detected.

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.

[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.