Hemocyte Lysate Research Articles

First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine ( l-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of l-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.

Effects of temperature on cellular and biochemical parameters in the crab Carcinus aestuarii (Crustacea, Decapoda)

The effects of temperature on cellular and biochemical parameters of the crab Carcinus aestuarii were evaluated. Crabs were kept for 7 days at 4, 17 (reference value) and 30 °C (salinity of 35 psu), and total haemocyte count (THC), haemocyte volume, haemocyte proliferation, phenoloxidase (PO) activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH), CFH total protein and glucose levels, superoxide dismutase (SOD) and catalase (CAT) activities in both gills and digestive gland were evaluated. The lowest and the highest temperature significantly decreased THC, whereas haemocyte volume and haemolymph glucose concentration did not differ significantly among experimental conditions. Haemolymph protein concentration significantly reduced in crabs maintained at 30 °C, when compared with that of animals kept at 4 and 17 °C. Haemocyte proliferation increased significantly in crabs kept at 4 and 30 °C, when compared with that of crabs held at 17 °C. Likewise, a significantly higher PO activity was recorded in CFH from crabs kept at 4 and 30 °C, than in control crabs. Conversely, PO activity did not vary significantly in HL. With regard to antioxidant enzyme activities, a significant decrease in CAT activity was observed in gills from crabs kept at 4 °C, when compared to that of crabs kept at 17 °C and 30 °C. Results obtained demonstrated that the highest and lowest temperature tested influenced crab biological responses, and indicated that C. aestuarii modulated its cellular and biochemical parameters (mainly haemocyte proliferation, CFH protein concentrations and CFH PO activity) in order to cope with temperature.

Effect of Escherichia coli endotoxin on Archachatina marginata hemolymph coagulation system

Context: Archachatina marginata Swainson (Achatinidae) is found in Nigeria, West Africa. Its hemolymph is applied as a disinfectant to blades and fresh cuts of circumcision in Yorubaland. The hemolymph is also used in traditional medicine practice. Investigation into its anti-endotoxin response is being studied for the first time.Objective: This study determined whether endotoxin causes measurable and concentration-dependent protein coagulation in the separate hemolymph fractions and in hemocyte lysate (HL)/plasma mixtures.Materials and methods: Endotoxin was prepared by inoculating 5% w/v dextrose with locally isolated Escherichia coli cells and incubated for 48 h before sterilization. Pyrogenicity was determined by rabbit test method and use the of LAL kit. Hemolymph fractions were exposed to endotoxin while controls were exposed to endotoxin-free water (0.025 EU/ml). HL/plasma (1:1 v/v) was exposed to varied endotoxin concentrations.Results: Data indicated significantly higher protein coagulates induced by endotoxin in all the hemolymph fractions (P < 0.05). Maximum protein coagulation in mixture of HL/plasma 1:1 was recorded. Exposure of HL/plasma at optimal ratio to varied endotoxin caused linear protein coagulation up to 1.0 EU/ml, beyond which it dropped significantly and unresponsive to further increase in endotoxin doses.Discussion and conclusion: There was endotoxin-induced protein coagulation, which is endotoxin concentration-dependent. The optimal coagulation observed for 1:1 HL/plasma mixture suggests stronger interaction between the hemocytes and the plasma in response to endotoxin. There are LPS-binding proteins in the plasma and hemocytes of A. marginata. This finding may be employed in detection and quantification of endotoxin in future.

Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.

Combined effects of temperature and salinity on functional responses of haemocytes and survival in air of the clam Ruditapes philippinarum

The combined effects of temperature and salinity on both immune responses and survival in air of the clam, Ruditapes philippinarum, were evaluated for the first time. The animals were kept for 7 days at three differing temperature (5 °C, 15 °C, 30 °C) and salinity values (18 psu, 28 psu, 38 psu), and effects of the resulting 9 experimental conditions on total haemocyte count (THC), Neutral Red uptake (NRU), haemolymph protein concentration, and lysozyme activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. The survival-in-air test was also performed. Two-way ANOVA analysis revealed that temperature influenced significantly THC and NRU, whereas salinity and temperature/salinity interaction affected NRU only. Temperature and salinity did not influence significantly HL and CFH lysozyme activity, as well as haemolymph total protein content. Survival-in-air test is widely used to evaluate general stress conditions in clams. In the present study, temperature and salinity were shown to influence the resistance to air exposure of R. philippinarum. The highest LT 50 (air exposure time resulting in 50% mortality) value was recorded in clams kept at 18 psu and 15 °C, whereas the lowest value was observed in clams kept at 28 psu and 30 °C. Overall, results obtained demonstrated that temperature and salinity can affect some functional responses of haemocytes from R. philippinarum, and suggested a better physiological condition for animals kept at 15 °C temperature and 18 psu salinity.

A lytic mechanism based on soluble phospholypases A2 (sPLA2) and β-galactoside specific lectins is exerted by Ciona intestinalis (ascidian) unilocular refractile hemocytes against K562 cell line and mammalian erythrocytes

Hemocytes from the ascidian Ciona intestinalis exert in vitro Ca 2+-dependent cytotoxic activity toward mammalian erythrocytes and K562 cells. To examine the lytic mechanism, hemocyte populations were separated (B1–B6 bands) through a Percoll discontinuous density gradient, the hemocyte cytotoxic activity (HCA) and the lytic activity of the hemocyte lysate supernatant (HLS) were assayed. In addition the separated hemocytes were cultured and the cell-free culture medium (CFM) assayed after 3 h culture. Results support that unilocular refractile hemocytes (URGs), enriched in B5, are cytotoxic. The B5-HLS contains lysins and the activity of B5-CFM shows that lysins can be released into a culture medium. The B5 activity was blocked by d-Galactose, α-Lactose, Lactulose, LacNAc, thiodigalactoside (TDG), l-Fucose, d-Mannose, d-Glucose, sphingomyelin (SM), and soluble phospholipase A2 (sPLA2) inhibitors (dibucain, quinacrine). Accordingly, HLS chemico-physical properties (alkaline medium, high thermostability, Ca 2+-dependence, trypsin treatment, protease inhibitors) and SEM observations of the affected targets suggested that sPLA2 could be responsible for changes and large alterations of the target cell membrane. An apoptotic activity, as recorded by a caspase 3, 7 assay, was found by treating K562 cells with very diluted HLS. A lytic mechanism involving sPLA2 and lectins promptly released by URGs and morula cells respectively is suggested, whereas target cell membrane SM could be a modulator of the enzyme activity.

Can starvation influence cellular and biochemical parameters in the crab Carcinus aestuarii?

Crustacea experience periods of starvation during moulting or when limited food availability occurs. The effects of starvation on Crustacea physiological responses have been demonstrated, whereas the effects of starvation on Crustacea immune parameters remain to be more fully studied. In the present study the effects of starvation on immune parameters and antioxidant enzyme activities of the crab Carcinus aestuarii were evaluated for the first time. Treated crabs were starved for 7 days, whereas control crabs were fed daily with mussels. Total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, cell-free haemolymph (CFH) glucose and total protein levels, and phenoloxidase (PO) activity in both haemocyte lysate (HL) and CFH were measured in crabs. In addition, superoxide dismutase (SOD) and catalase (CAT) activities were evaluated in both gills and digestive gland from crabs, in order to evaluate whether starvation induced oxidative stress in C. aestuarii. THC increased significantly in starved crabs, with respect to controls, whereas no significant variations were observed in haemocyte diameter, volume and proliferation. In CFH of starved animals glucose concentration significantly increased, whereas total protein concentration significantly reduced. A significantly higher PO activity was recorded in HL from starved crabs, than in control crabs. Conversely, PO activity did not vary significantly in CFH. Starvation did not cause significant alterations in antioxidant enzyme activities in both gills and digestive gland. Results obtained demonstrated that starvation influenced crab immune parameters, but did not induce oxidative stress. Results also indicated that C. aestuarii can modulate its cellular and biochemical parameters in order to cope with starvation.

Cellular and biochemical parameters in the crab Carcinus aestuarii after experimentally-induced stress: Effects of bacterial injection, leg ablation and bacterial injection/leg ablation combination

The effects of bacterial injection, leg ablation and bacterial injection/leg ablation combination on cellular and biochemical parameters of the crab Carcinus aestuarii were evaluated. Total haemocyte count (THC), haemocyte volume, haemocyte proliferation, phenoloxidase (PO) activity in both haemocyte lysate (HL) and cell-free haemolymph (CFH) and CFH glucose levels were used as biomarkers of stress in crabs. After 24h following bacterial injection, no significant variation in THC were observed, whereas haemocyte diameter and volume decreased significantly in injected crabs with respect to controls. Haemocyte proliferation and glucose levels increased significantly in injected crabs, whereas no significant variations in PO activity was recorded between control and injected crabs. THC increased significantly one-day after leg ablation, whereas it decreased significantly after 3 and 7days. Haemocyte diameter and volume did not vary significantly between control and de-clawed crabs at each sampling times. In de-clawed crabs, haemocyte proliferation was significantly higher after 7days, whereas glucose and PO activity increased significantly in CFH after 1 and 3days. The bacterial injection/leg ablation combination caused a statistically significant decrease in both THC and haemocyte diameter and volume, whereas increased significantly glucose levels in CFH. Overall, results obtained demonstrated that stress conditions investigated in the present study affected both cellular and biochemical parameters in C. aestuarii. Results also indicated that crabs can cope with differing stressful conditions modulating their immune parameters depending on the kind of stress undergone. Although preliminary, our results may explain – partially at least – the great adaptability and widespread of C. aestuarii out of its native area.

Comparison of antibacterial activity in the hemolymph of marine bivalves from Galicia (NW Spain)

A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative).

Edible Mushroom (Flammulina velutipes) Extract Inhibits Melanosis in Kuruma Shrimp (Marsupenaeus japonicus)

This study compared the potential of an aqueous extract of an edible mushroom (Flammulina velutipes) to prevent melanosis in cultured Kuruma shrimp (Marsupenaeus japonicus) with other antimelanosic compounds in vivo. The mushroom extract contained 9.1 mg/mL ergothioneine (ESH). Immersion of live full-grown shrimp in a 0.5% w/v solution of mushroom extract significantly reduced PPO activity in shrimp hemolymph. In addition, expression of the prophenoloxidase (proPO) gene decreased in hemocytes, suggesting that the extract blocked the activation of the proPO cascade. Consequently, the development of melanosis in the treated shrimp was significantly suppressed during ice storage. Treatment with a 0.05% w/v solution of sodium ascorbate and 4-hexyl-1,3-benzenediol had the same effect. In vitro experiments showed that ESH effectively inhibited PPO activity and activation of the proPO cascade in hemocyte lysate supernatant. This study suggests that in vivo application of F. velutipes mushroom extract is an effective natural alternative to synthetic antimelanosic agents to inhibit postmortem melanosis in shrimp. Practical Application: The extract of an edible mushroom (F. velutipes) containing ergothioneine can be a promising natural alternative to synthetic antimelanosic agents used to prevent postharvest melanosis in shrimp and other crustaceans. Furthermore, utilization of the mushroom trimmings could also help address the growing concerns on the disposal of such agricultural wastes and instead use it into a novel purpose as a source of antimelanosic and antioxidants for food and industrial application.

Upregulation in response to infection and antibacterial activity of oyster histone H4

Several histones and histone-derived peptides have been shown to have antimicrobial activity and a potential role in innate immune defenses. A histone H4 sequence was identified in a subtractive suppression library containing genes upregulated in American cupped oysters, Crassostrea virginica, in response to challenge with the protozoan parasite Perkinsus marinus. Oyster histone H4 protein levels significantly increased in hemocyte lysates and cell free hemolymph of oysters experimentally challenged with P. marinus. The complete histone H4 coding sequence of C. virginica was cloned into a Saccharomyces cerevisiae yeast expression system and recombinant expression was confirmed using SDS-PAGE analysis and western blot. Delivery of yeast cells expressing recombinant oyster histone H4 into the gut of brine shrimp, Artemia salinas, challenged with a streptomycin resistant strain of Vibrio anguillarum resulted in a significant and dose-dependent decrease in the load of V. anguillarum. Purified recombinant histone H4 showed antimicrobial activity against V. anguillarum and Escherichia coli at micromolar concentrations, but did not affect the viability of P. marinus in culture. These results support the role of histone H4 in the defense of oysters against bacterial infection and validate the use of a novel oyster antimicrobial H4 in a yeast feed-based delivery system for the treatment of bacterial infections in aquaculture applications.

Effect of Escherichia coli endotoxin on Archachatina marginata haemolymph coagulation system

AbstractThe effect of E. coli endotoxin on the heamolymph coagulation response of Archachatina marginata was studied. Heamocyte Lysate(HL), Haemocyte Lysate Supernatant(HLS) and Haemocyte Lysate Debris (HLD) were exposed to Escherichia coli endotoxin. Controls were prepared with endotoxin-free water(&amp;lt;0.025 EU/ml). The differential protein coagulation was estimated in each mixture. Fractions of the haemolymph exposed to endotoxin produced higher protein coagulates than endotoxin-free fractions when incubated at 37 °C for 1 h (p&amp;lt;0.05). The results showed significantly higher (p&amp;lt;0.05) concentrations of protein coagulated when HL/plasma mixture were used than when either fraction was used. At a ratio 1:1 of HL:Plasma, highest protein coagulation was recorded. This study revealed that maximum protein coagulation in response to endotoxin was elicited by a synergy between plasma and haemocyte lysate(HL). From this research haemolymph fractions of Archachatina marginata may provide an alternative test material for endotoxin in medical preparations in the future.

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 μM, while the titer of tyrosine, a substrate for the PO cascade, was 141 μM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 μM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-releated mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.

Antioxidant enzymes from the crab Scylla paramamosain: Gene cloning and gene/protein expression profiles against LPS challenge

Recent studies revealed that antioxidant enzymes play important roles in antioxidant responses caused by metabolic process or pathogen invasion. Catalase is one of these key enzymes which has been characterized and highly conserved from invertebrates to vertebrates. In the present study, a full-length cDNA sequence of catalase was cloned from the hemocyte suppression subtractive hybridization library of the crab Scylla paramamosain. The Sp-catalase ( Sp-CAT) cDNA sequence contained 2551 bp with an open reading frame of 1551 bp encoding 517 amino acid residues. The conserved catalytic active residues His-71, Asn-144 and Tyr-354 were predicted in the amino acid sequence of Sp-CAT. The deduced Sp-CAT protein had a calculated molecular mass of 59 kDa with an estimated isoelectric point of 6.4. Multiple alignment analysis revealed that the deduced amino acid sequence of Sp-CAT shared high identity (75.4%) with those of other species. The Sp-CAT mRNA transcripts were demonstrated in multiple tissues of normal S. paramamosain. After LPS challenge, the expression level of Sp-CAT gene was increased significantly in hemocyte at 3 and 6 h, and in hepatopancreas at 6 h, respectively, determined by quantitative real-time PCR. Furthermore, the activities of CAT and SOD were also measured in different tissues and serum after LPS challenge. The CAT activity was significantly increased at 3, 6, 24 and 48 h in hemocyte lysate, at 3 h in serum, and at 24 and 48 h in hepatopancreas after LPS challenge. In addition, the SOD activity was significantly induced at 3 and 6 h in hemocyte lysate, 3 and 12 h in serum, 12 and 48 h in hepatopancreas post LPS stimulation, indicating a tissue and time-dependent antioxidant response in the crab. Taken together, these data demonstrated that a strong antioxidant response occurred in the LPS-challenged crab, which might be involved in the protection of host against microbial infections.

First evidence of gender-related differences in immune parameters of the clam Ruditapes philippinarum (Mollusca, Bivalvia)

For the first time, gender-related differences in some important functional parameters of haemocytes from the clam Ruditapes philippinarum have been studied. Clams were collected during the pre-spawning phase, when sex can be distinguished by microscopic observation of gonadal smears, in order to ascertain whether the two sexes reach the stressful spawning period with different degrees of immunosurveillance. Total haemocyte count (THC), haemocyte size frequency distribution, capability of haemocytes to assume the vital dye neutral red (NR, indicative of endocytotic activity), lysozyme-like and acid phosphatase activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH), and superoxide dismutase (SOD) and catalase (CAT) activities in HL were all evaluated. No differences in THC values were found between the two sexes, but differing haemocyte size frequency distributions were observed: the fraction of larger haemocytes (7–9 μm diameter, 200 fl volume) was markedly increased in females and that of smaller haemocytes (<5 μm diameter, <200 fl volume) markedly increased in males. Interestingly, when haemocytes were observed under the light microscope, a significantly higher percentage of granulocytes was found in haemolymph from females, compared with that of males. Significantly increased NR uptake was recorded in female haemocytes. No significant variation in lysozyme-like activity was observed in HL, whereas in CFH, enzyme activity was significantly higher in females. HL acid phosphatase activity was significantly higher in males with respect to females; no significant variations in enzyme activity were observed in CFH. HL SOD and CAT activities were significantly higher in females than in males. Overall, on the basis of the cell parameters studied here, gender-related differences in immune parameters were found in R. philippinarum, indicating that females have more active haemocytes than males during the pre-spawning period.

First evidence of laccase activity in the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.

Effects of Ergothioneine from Mushrooms (Flammulina velutipes) on Melanosis and Lipid Oxidation of Kuruma Shrimp (Marsupenaeus japonicus)

The antimelanosic and antioxidative properties of a hot water extract prepared from the fruiting body of the edible mushroom (Flammulina velutipes) were evaluated by dietary supplementation in Kuruma shrimp (Marsupenaeus japonicus) for possible aquaculture application. The extract contained ergothioneine (ERT) at a level of 2.05 mg/mL. A commercial standard of l-ergothioneine (l-ERT) and the mushroom extract showed inhibitory activity against mushroom polyphenoloxidase (PPO). Feeding of the extract had no adverse effects on the immune systems of the shrimp under the present experimental conditions. Supplementation of the extract in the diet significantly suppressed PPO activities in the hemolymphs of the shrimp. Expression of the prophenoloxidase (proPO) gene decreased in the hemocyte of the Kuruma shrimp fed with the mushroom extract. Consequently, development of melanosis was significantly suppressed in the supplement fed shrimp during ice storage. Lipid oxidation was also effectively controlled in the supplement fed group throughout the storage period. In vitro experiments showed that l-ERT effectively inhibited the activation of proPO in the hemocyte lysate supernatant (HLS). The transcript of the proPO gene in the hemocyte showed lower expression in the l-ERT-treated HLS. It was concluded that dietary supplementation of the mushroom extract in shrimp could be a promising approach to control post mortem development of melanosis and lipid oxidation in shrimp muscles.

The role of haemocytes from the crab Carcinus aestuarii (Crustacea, Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10 6) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.