The health status of mussels, Mytilus spp., in Ireland and Wales with the molecular identification of a previously undescribed haplosporidian.

Both wild and cultured mussels (Mytilus edulis, Mytilus galloprovincialis and hybrids), are found along most of the Irish coastline. M. edulis is widespread along all Irish coasts and is the only mussel species present on both the east coast of Ireland and the Welsh coast in the Irish Sea. M. galloprovincialis and hybrids are found along the Irish coastline except for the east coast. Samples of Mytilus spp. were collected from twenty-four sites, encompassing all coasts of Ireland and the Welsh coast, at different times of the year and over several years (2008-2011). In total, 841 mussels were examined histologically to assess their health status and the presence of any parasites or commensals. Mussels from 14 of the 24 sites were screened using polymerase chain reaction (PCR) to determine which mytilid species were present. A range of parasites were observed, generally at low levels. The most diverse community of parasites was observed at a sheltered site with poor water quality. Of significance, a previously undescribed haplosporidian was detected in a single mussel sample in the Menai Strait, Wales, by PCR and was confirmed by direct sequencing and is most closely related to Minchina chitonis and a haplosporidian of the Florida marsh clam Cyrenoida floridana. While M. edulis were infected by a variety of micro- and macro-parasites, only trematodes were observed in M. galloprovincialis and hybrids. Habitat description and the environmental factors influencing the study sites, including water quality and exposure, were recorded.

Bacterial septicaemia in prerecruit edible crabs, Cancer pagurus L.

Juvenile edible crabs, Cancer pagurus L., were surveyed from Mumbles Head and Oxwich Bay in South Wales, UK, and the number of heterotrophic bacteria and vibrios in the hemolymph was determined. The percentage of crabs with hemolymph containing bacteria was variable over the survey with higher numbers of animals affected in summer than in winter. Post-moult crabs contained significantly higher numbers of heterotrophic bacteria in the hemolymph than pre- and intermoult animals. Crabs with cuticular damage to the gills also had significantly higher numbers of bacteria in the hemolymph. Crabs were found to have a high prevalence of infection by the dinoflagellate, Hematodinium. Such animals had significantly fewer bacteria in the blood in comparison with Hematodinium-free animals. Of the 463 crabs surveyed, only 3 individuals had hemolymph containing 2000 + CFU mL(-1). Based on 16S rRNA gene sequences, two of these crabs contained a Vibrio pectenicida-like isolate, while the other had a mixed assemblage of vibrios. Although 59% of the crabs surveyed had culturable bacteria in the hemolymph, the majority only had small numbers (<2000 CFU mL(-1) ), suggesting that such infections may be of limited importance to the sustainability of the crab fishery in this region.

An in vitro and in vivo assessment of the potential of Vibrio spp. as probiotics for the Pacific white shrimp, Litopenaeus vannamei.

AIMS: The objective of the work was to determine whether known strains of nonpathogenic vibrios can act as probiotics for the control of Vibrio infections in the Pacific white shrimp, Litopenaeus vannamei. METHODS AND RESULTS: Of the ten species tested, only Vibrio alginolyticus (NCIMB 1339) and Vibrio gazogenes (NCIMB 2250) showed antagonistic activity towards a panel of shrimp pathogenic vibrios. In the case of V. alginolyticus, this activity depended on the presence of live bacteria while in V. gazogenes both live and dead bacteria showed anti-Vibrio activity. Injection of shrimp with either V. alginolyticus or V. gazogenes at 3 × 10(7) or 3 × 10(5) total bacteria per shrimp resulted in mortality with higher levels in the case of V. alginolyticus (100% mortality 18 h postinjection of 3 × 10(7) bacteria). Juvenile shrimp were fed commercial diets top-coated with either chitin (an immune stimulant) or chitin + V. gazogenes. Both chitin and V. gazogenes caused a significant decline in the number of Vibrio-like bacteria in the fore and hind gut, and changes were also seen in the hepatosomatic index (a measure of digestive health) and the total number of blood cells in circulation. Analysis of mid/hindgut and faecal samples obtained using terminal restriction fragment length polymorphism showed that the gut microbiota of shrimp has limited bacterial diversity and that after 8 weeks exposure to the experimental diets there were significant changes in the microbial flora of the GI tract of shrimp as a result of the presence of V. gazogenes. CONCLUSIONS: Of the vibrios tested, V. gazogenes has potential as a probiotic for the control of bacterial diseases in shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, this study shows the promise of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.

A histological study of shell disease syndrome in the edible crab Cancer pagurus.

Shell disease syndrome is characterised by the external manifestation of black spot lesions in the exoskeletons of crustaceans. In the present study, gills, hepatopancreas and hearts from healthy (<0.05% black spot coverage) and diseased (5 to 15% coverage) edible crabs, Cancer pagurus, were examined histologically to determine whether this disease can cause internal damage to such crabs. There was clear evidence of cuticular damage in the gills of diseased crabs leading to the formation of haemocyte plugs termed nodules. Nephrocytes found within the branchial septa of the gills showed an increase in the accumulation of dark material in their vacuoles in response to disease. In the hepatopancreas, various stages of tubular degradation were apparent that correlated with the severity of external disease. Similarly, there was a positive correlation between the number of viable bacteria in the haemolymph and the degree of shell disease severity. Approximately 21% of the haemolymph-isolated bacteria displayed chitinolytic activity. Overall, these findings suggest that shell disease syndrome should not be considered as a disease of the cuticle alone. Furthermore, it shows that in wild populations of crabs shell perforations may lead to limited septicaemia potentially resulting in damage of internal tissues. Whether such natural infections lead to significant fatalities in crabs is still uncertain.

Differences in fuel utilization between trout and human thrombocytes in physiological media.

Cell culture preparations now play a significant and essential role in physiological and biochemical studies of cell biology. However, the fuels offered in cell culture media are only glucose and glutamine, plus whatever might be in the added sera. It is currently difficult to find a rational way forward on this problem, as there are few data on what fuels cells use in vivo or even in an in vitro physiological situation. A recent study on human platelets redressed the situation somewhat by finding that 75% of ATP turnover could be accounted for by aerobic glycolysis, and by the oxidation of glucose, hydroxybutyrate, acetate, glutamine, palmitate and oleate. In the present study we used a similar strategy to investigate fuel choices by trout thymocytes, cells with a similar function but from a different phylogenetic group. When these cells were presented with a physiological medium, we found that aerobic glycolysis accounted for 9% of total ATP turnover, glucose and glutamine oxidation made a combined contribution of 2.3%, oleate and palmitate oxidation accounted for 15%, and 74% was unaccounted for. These patterns of fuel use are very different from that in human platelets. They demonstrate the cell- and animal-specific nature of cellular metabolism and again expose the inadequacy of the fuel component in culture media.

Identification, purification and properties of a beta-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions.

A lectin specific for laminarin, a beta-1,3-glucan, agglutinating baker's yeast and enhancing prophenoloxidase activation by laminarin, has been purified from the cockroach, Blaberus discoidalis, serum. Purification involved gel filtration with Bio-gel P300 and affinity chromatography on blue Sepharose CL-6B and laminarin-Sepharose 4B. The purified lectin has a molecular mass estimate of 520 kDa determined by gel filtration, and approximately 80 and 82 kDa by SDS-PAGE, under non-reducing and reducing conditions, respectively. After isoelectric focusing the lectin focused as a single band at pH 4.9. The purified lectin was stained by the periodic acid/Schiff's reagent showing that it is a glycoprotein, and was deglycosylated by endo-beta-N-acetylglu-cosaminidase F. Amino acid composition analysis showed the protein is similar to previously purified beta-1,3-glucan binding proteins from other invertebrates. In electron micrographs by negative staining, the protein formed large aggregates with 'Y'-shaped 'structural units' ca. 79 x 65 nm. Immunological tests confirmed that this lectin is not related to any other lectins previously purified from the same insect. This protein appears to be part of the hexamerin family of proteins. This is one of the first reports of a hexamerin-like molecule with lectin activity.

Fish macrophages express a cyclo-oxygenase-2 homologue after activation.

In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.

The eicosanoid generating capacity of isolated cell populations from the gills of the rainbow trout, Oncorhynchus mykiss.

Rainbow trout gill filaments generated a wide range of eicosanoid products following calcium ionophore challenge. The putative lipoxygenase products were separated by reverse phase high performance liquid chromatography (RP-HPLC), while prostanoids were quantified by enzyme immunoassay. Three main monohydroxy compounds containing conjugated dienes were observed after RP-HPLC namely 12-(S) hydroxyeicosatetraenoic acid (12-HETE), 12-(S) hydroxyeicosapentaenoic acid (12-HEPE) and 14-(S) hydroxydocosahexaenoic acid (14-HDHE), derived from endogenous arachidonic, eicosapentaenoic and docosahexaenoic acids, respectively. Their identification was confirmed by mass spectrometry. A further five compounds containing conjugated trienes were also observed but in lesser amounts. One of these products was identified as 8,15-dihydroxyeicosatetraenoic acid (8,15-DiHETE) based on its UV spectrum, co-elution with authentic standard on RP-HPLC and mass spectrometry. Overall, the generation of these products suggests the presence of 12- and possibly 15-lipoxygenase activities in trout gill acting on endogenous sources of fatty acid. To determine if the various cell types in trout gill had differing eicosanoid generating potential, gills were disrupted and the resultant cell suspensions separated by density gradient centrifugation. Following this three bands were formed on the gradients and the cell populations from these were characterised using periodic acid Schiff's (PAS) reactivity for mucosubstances, haematoxylin and eosin staining, and immunoreactivity with both monoclonal and polyclonal antibodies. The first band consisted of polygonal cells and other more minor cell types, the second cell band contained mainly polygonal and PAS-positive goblet epithelial cells, while the third band consisted of mainly erythrocytes. There were significant differences in the eicosanoid generating potential of the isolated cells, with cells from the second band generating significantly more 12-HETE and 8,15-DiHETE than those from both the first band and unfractionated populations. The eicosanoid generating activity of the trout gill epithelial cell line, RTG-W1, was also elucidated. It proved to be a modest generator of eicosanoids in that only low levels of thromboxane B2 and prostaglandin E2 were detected while no lipoxygenase products were observed.

Effect of prostanoids and their precursors on the aggregation of rainbow trout thrombocytes.

The role of prostanoids and their precursor fatty acids in the aggregatory response of thrombocytes (platelet equivalents of fish) from the rainbow trout, Oncorhynchus mykiss, was studied. Aggregation of these cells was induced by the thromboxane mimetic U-46619 or arachidonic acid (AA) in the presence of human or trout fibrinogen. The production of TXB2/3 by thrombocytes in response to stimulation with AA was inhibited by aspirin, ibuprofen, and indomethacin. However, thrombocyte aggregation in response to AA stimulation was not significantly altered by these agents at the concentrations tested (10-100 microM), with the exception of indomethacin at 20 and 40 microM. Effects on cytosolic calcium concentration have been suggested as an alternative mechanism for the inhibitory action of indomethacin on human platelet aggregation. The present study, however, failed to identify this as a mechanism for the inhibition of U-46619-induced trout thrombocyte aggregation by indomethacin. The polyunsaturated fatty acids docosahexaenoic acid and eicosapentaenoic acid both exhibited an inhibitory effect on U-46619-induced thrombocyte aggregation similar to that observed with mammalian platelets. Unlike the case in mammalian hemostasis, prostacyclin inhibited thrombocyte aggregation only at high concentrations (>5 microM). Prostaglandin E2, however, inhibited thrombocyte aggregation at much lower concentrations (>0.01 microM), suggesting that it may be the major inhibitory eicosanoid in trout.

Trout thrombocytes contain 12- but not 5-lipoxygenase activity.

Fish thrombocytes are thought to be the evolutionary forerunners of mammalian platelets. Thrombocyte preparations made by conventional methods, such as density gradient centrifugation, contain other cell types such as neutrophilic granulocytes and lymphocytes that could interfere with subsequent experiments. In this study, rainbow trout thrombocytes were separated by density gradient centrifugation and further purified by magnetic cell sorting (MACS) using the thrombocyte specific monoclonal antibody, 30D8. Thrombocyte purity was assessed by reactivity to 30D8 using flow cytometry and immunocytochemistry. Following purification by density gradient centrifugation, thrombocytes were 66.9+/-9.2% (mean value+/-S.E.M., n=3) pure. Further purification by MACS significantly increased thrombocyte purity to 97.3+/-0.6%, whereas only 1.4% of the MACS -ve fraction were identified as these cells. Incubation of thrombocytes isolated by density gradient alone with calcium ionophore, A23187, generated a range of eicosanoids derived from arachidonic or eicosapentaenoic acids, namely, leukotriene (LT)B4, LTB5, lipoxin (LX)A4, LXA5, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE). A similar eicosanoid generation profile was observed for cells in the MACS -ve fraction; however, MACS +ve cells (thrombocytes) generated no 4 or 5 series LT or LX but did generate significant amounts of the 12-lipoxygenase (LO) products, 12-HETE and 12-HEPE. These results indicate that trout thrombocytes contain no demonstrable 5-LO activity and like their mammalian counterparts possess 12-LO activity.

Eicosanoid biosynthesis in an advanced deuterostomate invertebrate, the sea squirt (Ciona intestinalis).

The eicosanoid generating potential of tunic, branchial basket, intestine, ovary and tadpole larvae from the sea squirt, Ciona intestinalis, was examined using a combination of reverse phase high performance liquid chromatography, gas chromatography-mass spectrometry and enzyme immunoassay. All organs examined synthesized the lipoxygenase products 12-hydroxyeicosapentaenoic acid (12-HEPE) and 8-HEPE implying that both 8- and 12-lipoxygenase activity are widely distributed in this species. In addition, tunic and branchial basket generated significant amounts of 8,15-diHEPE and smaller amounts of 8,15-dihydroxyeicosatetraenoic acid (8,15-diHETE), while tunic alone generated small amounts of conjugated tetraene-containing material with a UV chromophore and mass ion characteristic of a lipoxin-like compound. The broad range lipoxygenase inhibitors, esculetin and nordihydroguaiaretic acid, both caused a significant dose dependent inhibition of 12-HEPE and 8,15-diHEPE biosynthesis in tunic, while the specific 5-lipoxygenase inhibitor, REV-5901, and the specific 5-lipoxygenase activating protein inhibitor, MK-866, had no observable effect on the lipoxygenase profile of this tissue. Tunic, branchial basket, intestine and ovary all generated significant amounts of prostaglandin (PG) E and PGF immunoreactive material and smaller amounts of thromboxane B immunoreactive material as measured by enzyme immunoassay. The non-specific cyclooxygenase (COX) inhibitor, indomethacin, the selective COX-1 inhibitors, resveratrol and valerylsalicylate, and the specific COX-2 inhibitors, NS-398, etolodac and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone) all caused a significant dose dependent inhibition of the biosynthesis of PGE immunoreactive material. However, the specific COX-2 inhibitors were most effective, perhaps implying that a COX-2-like enzyme may be present in this species.

Adhesion-dependent release of elastase from human neutrophils in a novel, flow-based model: specificity of different chemotactic agents.

Neutrophils must adhere to the vessel wall, migrate, and degranulate in an ordered manner to perform their protective function. Disruption of these processes may be pathogenic. Current knowledge of the degranulation process is derived almost exclusively from studies on neutrophils in suspension, in which priming with the nonphysiological agent cytochalasin B is necessary to obtain elastase release in response to activating agents. To avoid this, we have adopted a different approach. Using a novel flow-based adhesion system, we have been able to quantify the release of elastase from the primary granules of activated neutrophils adherent to immobilized platelets or purified receptors without priming. Comparing stimuli, formyl tripeptide (fMLP), interleukin-8 (IL-8), activated complement fragment C5a, and platelet-activating factor (PAF) all induced rapid conversion to CD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused over neutrophils already rolling on platelet monolayers or purified P-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced release of elastase from the adherent cells in minutes. Neutrophils stimulated in suspension showed little degranulation. Treatment of neutrophils with an inhibitor of 5-lipoxygenase-activating protein (MK886) and thus synthesis of leukotrienes (LTs) or with an antagonist of the LTB4 receptor (LY223982) blocked the release of elastase. This indicated that endogenous synthesis of 5-lipoxygenase products such as LTs and autocrine activation of neutrophils was required for fMLP-driven elastase release. We hypothesize that the differential ability of PAF and fMLP to induce elastase release from surface-adherent neutrophils could arise from differential ability to generate leukotrienes, such as LTB4, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents are not released until activated neutrophils have migrated into the extravascular tissues.

Studies on the eosinophilic granule cells in the gills of the rainbow trout, Oncorhynchus mykiss.

Eosinophilic granule cells (EGCs) found in the gills, skin and alimentary canals of fish have been likened to mammalian mast cells in terms of their structure and function. To investigate this situation further, gill explant cultures from the rainbow trout, Oncorhynchus mykiss, were set-up and incubated with either lipopolysaccharide (LPS; 5 micrograms ml-1) or human recombinant tumour necrosis factor-alpha (TNF-alpha; 25 iu ml-1) alone or in combination for 7 days. Examination of histological sections of these gill explants after this incubation showed a significant increase in the number of EGCs in those explants incubated with a combination of LPS and TNF-alpha compared with the control. Similarly, exposure of trout to short-term (> 6 h) handling and confinement stress resulted in a significant increase in the number of EGCs in the gills, while longer term stress (> 6 days) was without significant effect. The EGCs in the gills were shown to contain granules that reacted with both basic dyes, such as methylene blue, and eosin but failed to react with periodic acid Schiff's reagent. Of particular interest was the finding that only some of the EGCs reacted with the leucocyte-specific monoclonal antibody, 21G6, suggesting some heterogeneity within this cell type in the gill.

Are integrins involved in the aggregatory and phagocytic behaviour of fish haemostatic cells?

The involvement of a putative integrin-like fibrinogen receptor in the aggregatory and phagocytic behaviour of thrombocytes (platelet equivalents of fish) from the rainbow trout Oncorhynchus mykiss was studied. Aggregation of trout thrombocytes was induced by the thromboxane mimetic U-46619 in the presence of trout fibrinogen. Thrombocyte aggregation was inhibited by the tetrapeptide RGDS, but not by RGES or fibrinogen binding inhibitor peptide (HHLGGAKQAGDV). A range of monoclonal antibodies against the human platelet integrin alphaIIbbeta3 (anti-CD41a, anti-beta3 and LK7r) showed no reactivity with trout thrombocytes. Subsequently, a panel of monoclonal antibodies was raised against thrombocyte membrane preparations in an attempt to obtain an antibody against the putative integrin fibrinogen receptor. Of these monoclonal antibodies, four were found to inhibit thrombocyte aggregation, namely 12G2, 30D8, 32F8 and 32H10. The antibody 32H10 was shown significantly to inhibit the attachment of thrombocytes to immobilised trout fibrinogen, suggesting that it and the other antibodies recognise the putative fibrinogen receptor on trout thrombocytes. FITC-labelled Bacillus cereus were employed as test particles to prove that thrombocytes internalise bacteria via an active process and not simply by passive sequestration into the open canalicular system. Preincubation of bacteria with trout fibrinogen resulted in a significant increase in the number of thrombocytes exhibiting phagocytosis. This enhancement of phagocytosis by preincubation of B. cereus with trout fibrinogen could be inhibited by the tetrapeptide RGDS, but not by RGES, hence implicating the putative fibrinogen receptor in the internalisation of microorganisms. The relevance of these findings to the possible existence of an integrin-like receptor on trout thrombocytes is discussed.

Generation and characterisation of monoclonal antibodies against rainbow trout, Oncorhynchus mykiss, leucocytes.

Monoclonal antibodies were raised against head kidney macrophages of the rainbow trout, Oncorhynchus mykiss. Despite the establishment of a significant number of different hybridoma clones, none of these released antibody specific for determinants only found on macrophages. Instead, all the monoclonal antibodies generated reacted with lymphocytes, granulocytes, and monocytes/macrophages, although thrombocytes (the platelet equivalents in fish) and erythrocytes were not recognized by these antibodies. Western blotting of solubilised macrophages revealed that two of the hybridoma lines, designated 21G6 and 21F11, reacted with at least five proteins of 80, 104, 110, 140, and > 170 kDa. Immunocytochemistry was performed on histological sections of trout alimentary canal, gill, liver, spleen, and haemopoietic head kidney using antibodies from several of the hybridoma lines, and all of these showed a similar pattern of reactivity in each tissue. In the alimentary canal, for example, immunoreactive material was found in the eosinophilic granular cells, blood vessel margins, mucus in the lumen, and in the columnar epithelial cells. In the gills, epithelial cells and blood vessels also showed intense immunoreactive products, while in the liver, such reactivity was localised in the sinusoids and adherent macrophages. Both the spleen and head kidney had largely homogenous immunoreactivity.

Characterisation of the binding of leukotriene B4 to macrophages of the rainbow trout Oncorhynchus mykiss.

The binding of leukotriene B4 (LTB4) to macrophages from the head kidney of the rainbow trout Oncorhynchus mykiss was measured. Binding of [3H]LTB4 achieved a steady state after approximately 30 min of incubation and was 30% reversible in the presence of a minimum of 1000-fold excess of LTB4. Scatchard analysis of the kinetics of LTB4 binding over a range of [3H]LTB4 concentrations indicated the existence of only a single class of receptor with a dissociation constant, KD, of 0.14 nmol l-1 and a maximum receptor density, Bmax, of approximately 17,800 sites per macrophage. The LTB4 receptor antagonist LY223982 was ineffective in inhibiting the binding of [3H]LTB4 to trout macrophages, although another receptor antagonist, LTB4-dimethylamide, displaced a maximum of 25% of the total binding. LTB5 was equally effective as LTB4 at displacing [3H]LTB4, while other eicosanoids tested were without significant effect. It is suggested that the putative receptors for LTB4 on trout macrophages are similar to the high-affinity receptors for this compound reported to occur on mammalian granulocytes, although any structural similarities of the binding sites await further investigation.

The evolution of inflammatory mediators.

Invertebrates do not display the level of sophistication in immune reactivity characteristic of mammals and other 'higher' vertebrates. Their great number and diversity of forms, however, reflect their evolutionary success and hence they must have effective mechanisms of defence to deal with parasites and pathogens and altered self tissues. Inflammation appears to be an important first line defence in all invertebrates and vertebrates. This brief review deals with the inflammatory responses of invertebrates and fish concentrating on the cell types involved and the mediators of inflammation, in particular, eicosanoids, cytokines and adhesion molecules.

The thromboxane mimetic, U-46619, induces the aggregation of fish thrombocytes.

Fish blood contains nucleated cells termed thrombocytes which are thought to be functionally analogous to mammalian platelets. The present study was undertaken to assess the aggregatory response of peripheral blood thrombocytes from the rainbow trout, Oncorhynchus mykiss. Incubation of Percoll density gradient purified thrombocytes with the thromboxane A2 mimetic, U-46619 (0.03-10 microM), alone or in the presence of human or trout fibrinogen, elicited a dose-dependent aggregatory response. A greater amount of aggregation was observed in the presence of fish, rather than human, fibrinogen (e.g. c 55% compared with 15% maximal aggregation respectively). This response was inhibited by pre-incubation of thrombocytes with the specific thromboxane A2 receptor antagonist, GR32191 (0.01-10 microM) with an IC50 value of 5.7 microM. Ultrastructural and aggregometry studies of thrombocyte aggregation at various time periods (1-12 min) after incubation with U-46619 (0.5 microM) revealed clear differences between the amount and dynamics of thrombocyte clumping in the presence of human, compared with trout, fibrinogen (final concentration 400 micrograms ml-1). Thrombocytes rapidly underwent shape change and aggregation after only 1 min incubation without the initial involvement of any other cell types. The maximum degree of aggregation was achieved after 4-8 min with larger aggregates formed in the presence of trout compared than human fibrinogen. Ultrastructurally, the thrombocytes in these aggregates displayed a number of changes compared with non-stimulated cells, including increased pseudopodial activity, a more pronounced canalicular system and condensation of the nuclear heterochromatin. After 12 min incubation the clumps of thrombocytes showed progressive disaggregatory behaviour, with some cells reverting back to their normal in vivo appearance. Neutrophilic granulocytes, present as contaminants in the thrombocyte suspensions, were occasionally found attached to the thrombocyte aggregates, perhaps suggesting a specific interaction similar to that occurring in mammals. Finally, and also of some significance, was the finding that trout thrombocytes exhibit phagocytic activity in vitro towards cell debris and contaminating bacteria, indicating that this cell type may possess both haemostatic and immunological capacities.

Immunoregulatory activities of eicosanoids in the rainbow trout (Oncorhynchus mykiss).

Eicosanoids such as prostaglandins (PG), leukotrienes (LT) and lipoxins (LX) have been shown to be potent immunoregulatory molecules in mammals. To determine if they have similar roles in 'lower' animals, rainbow trout (Oncorhynchus mykiss) were immunized with either sheep erythrocytes or Aeromonas salmonicida in the presence or absence of the stable analogue of PGE2, 16,16-dimethyl-PGE2, and the number of plaque-forming cells (PFC) or specific antibody levels determined. The higher dose of 16,16-dimethyl-PGE2 (200 micrograms/kg body weight) caused a significant reduction in both PFC number and antibody titre compared with the control. The effect of PGE2, PGE3, 16,16-dimethyl-PGE2, LTB4, LTB5, LXA4, 12-HETE and 12-HEPE on PFC generation following the in vitro challenge of trout splenocytes with sheep erythrocytes was also determined. All of the prostaglandins tested showed a dose-dependent inhibition of PFC after 11 days in culture, while of the remaining eicosanoids only LXA4 had any effect on PFC number, with a dose-dependent stimulatory effect. The cyclo-oxygenase inhibitor, indomethacin, also caused a stimulation in the number of PFC generated, with a maximal effect at c. 25 microM, while the lipooxygenase inhibitors, esculetin and nordihydroguaiaretic acid (5-100 microM), had no significant effect on PFC generation at all concentrations tested. The present results show that, as in mammals, prostaglandins and the cyclo-oxygenase pathway are also important in the regulation of the piscine humoral immune response. Of the lipoxygenase products tested, however, only LXA4 had any significant effect on PFC generation, suggesting that these compounds have only a limited role to play in immune regulation in this organism. Overall this work shows that eicosanoids have a long evolutionary history in immunoregulation, probably dating back at least to the appearance of bony fish some 400 million years ago.