Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increas... more Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased ‘leakiness’ in rat models, and is used routinely as an assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (1 – 7.5 μg/larva) toG. mellonellavia intrahaemocoelic injection and gavage (force-feeding) and monitored larval survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4 to 24-hours by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accom...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 1999
The role of prostanoids and their precursor fatty acids in the aggregatory response of thrombocyt... more 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 TXB2/3by 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 μM), with the exception of indomethacin at 20 and 40 μM. 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 polyunsatu...
Epizootiologists recurrently encounter symbionts and pathobionts in the haemolymph (blood equival... more Epizootiologists recurrently encounter symbionts and pathobionts in the haemolymph (blood equivalent) of shellfish. One such group is the dinoflagellate genus Hematodinium, which contains several species that cause debilitating disease in decapod crustaceans. The shore crab Carcinus maenas acts as a mobile reservoir of microparasites, including Hematodinium sp., thereby posing a risk to other co-located commercially important species, e.g. velvet crabs (Necora puber). Despite the widespread prevalence and documented seasonality of Hematodinium infection dynamics, there is a knowledge gap regarding host-pathogen antibiosis, namely, how Hematodinium avoids the host's immune defences. Herein, we interrogated the haemolymph of Hematodinium-positive and Hematodinium-negative crabs for extracellular vesicle (EV) profiles (a proxy for cellular communication), alongside proteomic signatures for post-translational citrullination/deimination performed by arginine deiminases, which can infer a pathologic state. Circulating EV numbers in parasitized crab haemolymph were reduced significantly, accompanied by smaller EV modal size profiles (albeit non-significantly) when compared to Hematodinium-negative controls. Differences were observed for citrullinated/deiminated target proteins in the haemolymph between the parasitized and control crabs, with fewer hits identified overall in the former. Three deiminated proteins specific to parasitized crab haemolymph were actin, Down syndrome cell adhesion molecule (DSCAM), and nitric oxide synthase-factors that contribute to innate immunity. We report, for the first time, Hematodinium sp. could interfere with EV biogenesis, and that protein deimination is a putative mechanism of immune-modulation in crustacean-Hematodinium interactions.
Comparative Biochemistry and Physiology B, Aug 1, 2007
Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatt... more Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography-mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE2 or PGD2, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0-25 microM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.
A large knowledge gap exists regarding the disease profile and pathologic condition of the invasi... more A large knowledge gap exists regarding the disease profile and pathologic condition of the invasive, non-native, slipper limpet Crepidula fornicata. To help address this, we performed a yearlong health survey across two sites in South Wales UK – subtidal Swansea Bay and intertidal Milford Haven. In total, 1,800 limpets were screened systematically for haemolymph bacterial burdens using both general and vibrio-selective growth media (TSA +2% NaCl and TCBS, respectively), haemolymph (blood) inspection using microscopy, a PCR-based assay targeting Vibrio spp., and multi-tissue histology. Over 99% of haemolymph samples contained cultivable bacterial colony forming units, and 83% of limpets tested positive for the presence of vibrios via PCR (confirmed via Sanger sequencing). Vibrio presence did not vary greatly across sites, yet a strong temporal (seasonal) effect was observed – significantly higher bacterial loads during the summer. Binomial logistic regression models revealed larger (...
Marine ecosystems are affected by multiple, well-known stressors like fishing and climate change,... more Marine ecosystems are affected by multiple, well-known stressors like fishing and climate change, but a less documented concern is disease. Marine reserves have been successful in replenishing stocks and aiding recruitment but studies have shown that high population abundances in marine reserves may lead to unwanted secondary effects such as increase in predators and competition, altering trophic webs, and disease. Here, we investigate factors contributing to disease prevalence in a brown crab (Cancer pagurus) population around Lundy Island (the UK's first MPA) after 7 years of no-take protection. Population parameters (size, sex, and abundance), disease (shell disease, Hematodinium spp. infection) and injury presence (a known precursor to some disease conditions) were assessed over two years in both fished and unfished areas of the MPA. We found no significant difference in prevalence between the disease prevalence in fished and unfished areas, however overall, the number of in...
While most crab production for human consumption worldwide comes from capture fisheries, there is... more While most crab production for human consumption worldwide comes from capture fisheries, there is increasing production of selected species using aquaculture-based methods. This is both for the purpose of stock replacement and direct yield for human consumption. Disease has limited the ability to produce larval crabs in commercial hatcheries and this together with suitable feeds, are major hurdles in the sustainable development of cultivation methods. Juvenile and adult crabs are also subject to a range of diseases that can cause severe economic loss. Emerging pathogens/parasites are of major importance to crab aquaculture as they can cause high levels of mortality and are difficult to control. Diseases caused by viruses and bacteria receive considerable attention but the dinoflagellate parasites,Hematodiniumspp., also warrant concern because of their wide host range and lack of control methods to limit their spread. This concise review examines the emerging diseases in several crab...
Additional file 1: Table S1. Full model used in order to predict response variable of presence of... more Additional file 1: Table S1. Full model used in order to predict response variable of presence of Hematodinium sp. before reduction. Asterisk denotes significance (P ≤ 0.05). Table S2. Full model used in order to predict response variable of presence of Hematodinium sp. in the Dock location before reduction. Asterisk denotes significance (P ≤ 0.05). Table S3. Full model used in order to predict response variable of presence of Hematodinium sp. in the Pier location before reduction. Asterisk denotes significance (P ≤ 0.05). Table S4. Accession numbers, deposited in GenBank, and corresponding sampling numbers for all Hematodinium-positive animals successfully sequenced from study, and used in the phylogenetic tree (Fig. 7).
The involvement of a putative integrin-like fibrinogen receptor in the aggregatory and phagocytic... more 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 attac...
Eicosanoids are a group of oxygenated fatty-acid derivatives formed from C20 polyunsaturated fatt... more Eicosanoids are a group of oxygenated fatty-acid derivatives formed from C20 polyunsaturated fatty acids including arachidonic and eicosapentaenoic acids. In mammals, these compounds have been shown to be key molecules in several physiological processes including regulation of the vascular system. This study determined whether eicosanoids or their precursors are involved in the regulation of heart rate in the sea squirt Ciona intestinalis. Eicosanoid generation by both heart and blood cells was measured. The major lipoxygenase products formed were both derivatives of eicosapentaenoic acid, namely 8- and 12-hydroxyeicosapentaenoic acids (8-HEPE and 12-HEPE). Smaller amounts of 8,15-dihydroxyeicosapentaenoic acid (8,15-diHEPE) were also formed. The cyclo-oxygenase product prostaglandin E was also found in small amounts in the heart. Isolated hearts were exposed either to these fatty acid precursors or to 8-HEPE, 12-HEPE or prostaglandin E3, and the effect on heart rate was recorded. B...
Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increas... more Indomethacin is a non-steroidal anti-inflammatory drug that causes gastric ulceration and increased ‘leakiness’ in rat models, and is used routinely as an assay to screen novel compounds for repair and restitution properties. We set out to establish conditions for indomethacin-induced gut damage in wax-moth (Galleria mellonella) larvae with a view to reducing the need for rodents in such experimentation. We administered indomethacin (1 – 7.5 μg/larva) toG. mellonellavia intrahaemocoelic injection and gavage (force-feeding) and monitored larval survival and development, blood cell (haemocyte) numbers, and changes in gut permeability. Increased levels of gut leakiness were observed within the first 4 to 24-hours by tracking fluorescent microspheres in the faeces and haemolymph (blood equivalent). Additionally, we recorded varying levels of tissue damage in histological sections of the insect midgut, including epithelial sloughing and cell necrosis. Degeneration of the midgut was accom...
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 1999
The role of prostanoids and their precursor fatty acids in the aggregatory response of thrombocyt... more 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 TXB2/3by 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 μM), with the exception of indomethacin at 20 and 40 μM. 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 polyunsatu...
Epizootiologists recurrently encounter symbionts and pathobionts in the haemolymph (blood equival... more Epizootiologists recurrently encounter symbionts and pathobionts in the haemolymph (blood equivalent) of shellfish. One such group is the dinoflagellate genus Hematodinium, which contains several species that cause debilitating disease in decapod crustaceans. The shore crab Carcinus maenas acts as a mobile reservoir of microparasites, including Hematodinium sp., thereby posing a risk to other co-located commercially important species, e.g. velvet crabs (Necora puber). Despite the widespread prevalence and documented seasonality of Hematodinium infection dynamics, there is a knowledge gap regarding host-pathogen antibiosis, namely, how Hematodinium avoids the host's immune defences. Herein, we interrogated the haemolymph of Hematodinium-positive and Hematodinium-negative crabs for extracellular vesicle (EV) profiles (a proxy for cellular communication), alongside proteomic signatures for post-translational citrullination/deimination performed by arginine deiminases, which can infer a pathologic state. Circulating EV numbers in parasitized crab haemolymph were reduced significantly, accompanied by smaller EV modal size profiles (albeit non-significantly) when compared to Hematodinium-negative controls. Differences were observed for citrullinated/deiminated target proteins in the haemolymph between the parasitized and control crabs, with fewer hits identified overall in the former. Three deiminated proteins specific to parasitized crab haemolymph were actin, Down syndrome cell adhesion molecule (DSCAM), and nitric oxide synthase-factors that contribute to innate immunity. We report, for the first time, Hematodinium sp. could interfere with EV biogenesis, and that protein deimination is a putative mechanism of immune-modulation in crustacean-Hematodinium interactions.
Comparative Biochemistry and Physiology B, Aug 1, 2007
Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatt... more Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography-mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE2 or PGD2, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0-25 microM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.
A large knowledge gap exists regarding the disease profile and pathologic condition of the invasi... more A large knowledge gap exists regarding the disease profile and pathologic condition of the invasive, non-native, slipper limpet Crepidula fornicata. To help address this, we performed a yearlong health survey across two sites in South Wales UK – subtidal Swansea Bay and intertidal Milford Haven. In total, 1,800 limpets were screened systematically for haemolymph bacterial burdens using both general and vibrio-selective growth media (TSA +2% NaCl and TCBS, respectively), haemolymph (blood) inspection using microscopy, a PCR-based assay targeting Vibrio spp., and multi-tissue histology. Over 99% of haemolymph samples contained cultivable bacterial colony forming units, and 83% of limpets tested positive for the presence of vibrios via PCR (confirmed via Sanger sequencing). Vibrio presence did not vary greatly across sites, yet a strong temporal (seasonal) effect was observed – significantly higher bacterial loads during the summer. Binomial logistic regression models revealed larger (...
Marine ecosystems are affected by multiple, well-known stressors like fishing and climate change,... more Marine ecosystems are affected by multiple, well-known stressors like fishing and climate change, but a less documented concern is disease. Marine reserves have been successful in replenishing stocks and aiding recruitment but studies have shown that high population abundances in marine reserves may lead to unwanted secondary effects such as increase in predators and competition, altering trophic webs, and disease. Here, we investigate factors contributing to disease prevalence in a brown crab (Cancer pagurus) population around Lundy Island (the UK's first MPA) after 7 years of no-take protection. Population parameters (size, sex, and abundance), disease (shell disease, Hematodinium spp. infection) and injury presence (a known precursor to some disease conditions) were assessed over two years in both fished and unfished areas of the MPA. We found no significant difference in prevalence between the disease prevalence in fished and unfished areas, however overall, the number of in...
While most crab production for human consumption worldwide comes from capture fisheries, there is... more While most crab production for human consumption worldwide comes from capture fisheries, there is increasing production of selected species using aquaculture-based methods. This is both for the purpose of stock replacement and direct yield for human consumption. Disease has limited the ability to produce larval crabs in commercial hatcheries and this together with suitable feeds, are major hurdles in the sustainable development of cultivation methods. Juvenile and adult crabs are also subject to a range of diseases that can cause severe economic loss. Emerging pathogens/parasites are of major importance to crab aquaculture as they can cause high levels of mortality and are difficult to control. Diseases caused by viruses and bacteria receive considerable attention but the dinoflagellate parasites,Hematodiniumspp., also warrant concern because of their wide host range and lack of control methods to limit their spread. This concise review examines the emerging diseases in several crab...
Additional file 1: Table S1. Full model used in order to predict response variable of presence of... more Additional file 1: Table S1. Full model used in order to predict response variable of presence of Hematodinium sp. before reduction. Asterisk denotes significance (P ≤ 0.05). Table S2. Full model used in order to predict response variable of presence of Hematodinium sp. in the Dock location before reduction. Asterisk denotes significance (P ≤ 0.05). Table S3. Full model used in order to predict response variable of presence of Hematodinium sp. in the Pier location before reduction. Asterisk denotes significance (P ≤ 0.05). Table S4. Accession numbers, deposited in GenBank, and corresponding sampling numbers for all Hematodinium-positive animals successfully sequenced from study, and used in the phylogenetic tree (Fig. 7).
The involvement of a putative integrin-like fibrinogen receptor in the aggregatory and phagocytic... more 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 attac...
Eicosanoids are a group of oxygenated fatty-acid derivatives formed from C20 polyunsaturated fatt... more Eicosanoids are a group of oxygenated fatty-acid derivatives formed from C20 polyunsaturated fatty acids including arachidonic and eicosapentaenoic acids. In mammals, these compounds have been shown to be key molecules in several physiological processes including regulation of the vascular system. This study determined whether eicosanoids or their precursors are involved in the regulation of heart rate in the sea squirt Ciona intestinalis. Eicosanoid generation by both heart and blood cells was measured. The major lipoxygenase products formed were both derivatives of eicosapentaenoic acid, namely 8- and 12-hydroxyeicosapentaenoic acids (8-HEPE and 12-HEPE). Smaller amounts of 8,15-dihydroxyeicosapentaenoic acid (8,15-diHEPE) were also formed. The cyclo-oxygenase product prostaglandin E was also found in small amounts in the heart. Isolated hearts were exposed either to these fatty acid precursors or to 8-HEPE, 12-HEPE or prostaglandin E3, and the effect on heart rate was recorded. B...
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