Effects of dissolved oxygen on survival and immune responses of scallop (Chlamys farreri Jones et Preston)

This experiment investigated the effects of dissolved oxygen (DO) on the survival and immune responses of scallop Chlamys farreri. The scallops (initial mean dry weight of soft tissue 1.52+/-0.10 g) were cultivated in the seawater with different DO levels (8.5, 6.5, 4.5, and 2.5mg l(-1), respectively) for 21 d. Each treatment had triplicate groups of 35 animals. During the experimental period, the scallops were fed with Spirulina maxima, and water temperature ranged from 15.2 degrees C to 17.5 degrees C, salinity from 29.5 per thousand to 32.5 per thousand and pH from 7.5 to 8.2. Survival, specific growth rate (SGR) and total haemocyte count (THC) were examined at the end of the study, and superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (ALP), were examined at 12 h, 24 h, Day 7, Day 14 and Day 21 after being exposed to the graded DO levels. The lower DO levels (2.5 and 4.5mg l(-1))resulted in lower survivals of scallops, and the survival (81.7%) at 2.5mg 1(-1)DO was significantly lower than those (100.0%) at 8.5 and 6.5mg l(-1) DO. Similarly, the SGR and THC of scallop gradually reduced with decreasing DO levels, and reached significant levels at 2.5mg l(-1) DO (P<0.05). At higher DO levels (8.5 and 6.5mg l(-1)), the SOD activity maintained rather stable during the entire sampling period. At lower DO levels (4.5 and 2.5mg l(-1)), however, the SOD activity significantly increased at 12 h, and then significantly decreased to the levels below the normal. At the two lower DO levels, ACP activities had no significant changes before Day 7, and then declined to the levels that were significantly lower than the normal. Significantly higher ALP activity was only observed at 12 h in the treatment of 2.5mg l(-1) DO, but in all other treatments and sampling times it fluctuated in a narrow range. In conclusion, less than 4.5mg l(-1) DO reduced the survival and depressed the immune responses of C. farreri.     

An immersion of Gracilaria tenuistipitata extract improves the immunity and survival of white shrimp Litopenaeus vannamei challenged with white spot syndrome virus

The innate immunity and resistance against white spot syndrome virus (WSSV) in white shrimp Litopenaeus vannamei which received the Gracilaria tenuistipitata extract were examined. Shrimp immersed in seawater containing the extract at 0 (control), 400 and 600 mg L(-1) for 3 h were challenged with WSSV at 2 × 10(4) copies shrimp(-1). Shrimp not exposed to the extract and not received WSSV challenge served as unchallenged control. The survival rate of shrimp immersed in 400 mg L(-1) or 600 mg L(-1) extract was significantly higher than that of challenged control shrimp over 24-120 h. The haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, and lysozyme activity of shrimp immersed in 600 mg L(-1) extract were significantly higher than those of unchallenged control shrimp at 6, 6, 6, 6, and 6-24 h post-challenge. In another experiment, shrimp which had received 3 h immersion of 0, 400, 600 mg L(-1) extract were challenged with WSSV. The shrimp were then received a booster (3 h immersion in the same dose of the extract), and the immune parameters were examined at 12-120 h post-challenge. The immune parameters of shrimp immersed in 600 mg L(-1) extract, and then received a booster at 9, 21, and 45 h were significantly higher than those of unchallenged control shrimp at 12-48 h post-challenge. In conclusion, shrimp which had received the extract exhibited protection against WSSV as evidenced by the higher survival rate and higher values of immune parameters. Shrimp which had received the extract and infected by WSSV showed improved immunity when they received a booster at 9, 21, and 45 h post-WSSV challenge. The extract treatment caused less decrease in PO activity, and showed better performance of lysozyme activity and antioxidant response in WSSV-infected shrimp.     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

The modulation of catecholamines to the immune response against bacteria Vibrio anguillarum challenge in scallop Chlamys farreri

Catecholamines are pivotal signal molecules in the neuroendocrine-immune regulatory network, and implicated in the modulation of immune response. In the present study, the activities of some immune-related enzymes and the concentration of catecholamines were determined in circulating haemolymph of scallops Chlamys farreri after bacteria Vibrio anguillarum challenge. The activities of superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ) increased significantly and reached 610 U mg(-1) at 12 h, 37.6 U mg(-1) at 6 h and 261.5 U mg(-1) at 6 h after bacteria challenge, respectively. The concentration of norepinephrine, epinephrine and dopamine also increased significantly and reached 114.9 ng mL(-1) at 12 h, 86.9 ng mL(-1) at 24 h and 480.4 pg mL(-1) at 12 h after bacteria challenge, respectively. Meanwhile, the activities of these immune-related enzymes in haemolymph were monitored in those scallops which were challenged by bacteria V. anguillarum and stimulated simultaneously with norepinephrine, epinephrine and adrenoceptor antagonist. The injection of norepinephrine and epinephrine repressed significantly the induction of bacteria challenge on the activities of immune-related enzymes, and they were reduced to about half of that in the control groups. The blocking of α and β-adrenoceptor by antagonist only repressed the increase of CAT and LYZ activities significantly, while no significant effect was observed on the increase of SOD activities. The collective results indicated that scallop catecholaminergic neuroendocrine system could be activated by bacteria challenge to release catecholamines after the immune response had been triggered, and the immune response against bacteria challenge could been negatively modulated by norepinephrine, epinephrine, and adrenoceptor antagonist. This information is helpful to further understand the immunomodulation of catecholamines in scallops.     

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 °C, 17 °C and 25 °C respectively. Thereafter, a recovery period of 24 h at 17 °C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immunomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 °C and 24 h of air exposure at 17 °C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 °C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 °C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 °C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops.     

Immunohistochemical Identification of Met-Enkephalin in Digestive System and Its Effect on Digestive Enzyme Activities of the Scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

The study was designed to determine whether methionine-enkephalin (M-ENK) was present in the digestive system of the scallop Chlamys farreri and investigate the effects of M-ENK on the activity of amylase, protease and lipase in the digestive system of C. farreri. The results indicated that M-ENK-like material was present in the epithelium and connective tissue of labial palps, mouth labia, stomach, intestine, rectum, and hepatopancreas of the scallop C. farreri. Moreover, it was also found that many isolated small cells showing M-ENK-like immunoreactivity were scattered in the epithelial layer of intestine, and many isolated big epithelial cells showing M-ENK-like immunoreactivity were scattered in tubules of hepatopancreas of the scallop. The activity levels of amylase and lipase in crystalline style, hepatopancreas and intestine were enhanced at 1 h after injection of exogenous M-ENK into adductor muscle of the scallops, whereas protease activity levels were significantly suppressed. Our report constitutes the first characterization of M-ENK in the digestive system of scallop C. farreri and investigates the effects of M-ENK on the activities of digestive enzymes of mollusk for the first time. The results suggest an involvement of M-ENK in the functional regulation of the digestive system of C. farreri.     

Action of cadmium on the elemental composition and cellular morphology of the hepatopancreas, kidneys and gonads of the mussel Mytilus edulis and scallop Mizuhopecten yessoensis

Studies have been made on the content and distribution of Cd, K, Mg, Ca, S and P in organs and cells of mussels and scallops before and after immersion in water containing cadmium chloride. In normal molluscs, the lowest content of Cd was found in the gonads; cadmium content of the hepatopancreas and kidney in the scallop is correspondingly 20 and 40 times higher than in the mussel. After immersion in water with cadmium (250 micrograms/l in summer experiments and 500 micrograms/l in spring ones), cadmium content of the hepatopancreas and kidney of the scallop was found to be also higher than that in the mussel, although the difference is less significant, being dependent on the season of year. The highest accumulation of cadmium (9.760 +/- 790 micrograms/g dry weight) was observed in the scallop kidney in summer period. After immersion in cadmium-containing water, the content of sulphur in the kidney and hepatopancreas increases, positive correlation being observed between the distribution of Cd and S. Exposure to cadmium results in the increase of Ca and Mg content in the gonads of scallops, as well as in the increase in Ca content of the gonads of mussels. Accumulation of cadmium in the gonads is rather small, maximum density of the material being observed histochemically in the gonadal wall. Morphological changes in the gonadal cells of mussels in summer period may be associated with the increase in cellular calcium.     

Alternation of immune parameters and cellular energy allocation of Chlamys farreri under ammonia-N exposure and Vibrio anguillarum challenge

The complex interactions among host, pathogen and environment are believed to be the main causes for the mass mortality of cultured scallops. In the present study, the temporal variations of immune parameters and cellular energy allocation (CEA) of Chlamys farreri under ammonia-N, Vibrio anguillarum as well as their combined treatment were investigated to better understand the energetic mechanisms of scallop in immune defense. After 1 d exposure to ammonia-N, V. anguillarum and their combination, the superoxide anion level and superoxide dismutase (SOD) activity in the serum of scallops increased substantially. At 24 d post exposure, the mRNA expression levels of isocitrate dehydrogenase (IDH), heat shock protein 70 (HSP 70), HSP 90 and glutamine synthetase (GS), as well as the malondialdehyde content remarkably increased, while SOD activity was depressed significantly (P < 0.05). The glycogen reserved in the tissues from scallops exposed to the combined stress for 1 d, 12 d and 24 d were significantly lower than those in the control (P < 0.05). The CEA values in all the examined tissues including gonad, gill, hepatopancreas and adductor muscle were significantly lower than those of control (P < 0.05) when exposure to ammonia-N, V. anguillarum and their combined treatment for 12 and 24 d. Furthermore, the combined stress also had a significant impact upon CEA in all the examined tissues in scallops post 1 d exposure (P < 0.05). The above results demonstrated that SOD, IDH, HSPs and GS in hemolymph of treated scallops are necessary, but not sufficient to the complete protection against stress-induced cellular damage along with the treatment duration. Immune defense against the combination of pathogen invasion and environmental stress can impose greater costs on scallop's energy expenditure than a single stressor, and the combined treatment preferentially consumed more available glycogen in scallops for immune defense. Hence, in addition to be used in immunological evaluation, CEA is also a powerful tool to provide valuable insights into possible mechanisms of mass mortalities in cultured scallops.     

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.     

Monitoring cellular accumulation of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite (FLT-MP) by LC-MS/MS as a measure of cell proliferation in vitro

Accurate measurement of in vitro cell growth is critical for oncology drug development, but cell counting and the most accurate indirect proliferation assays are impractical. Here, we describe a robust alternative method that monitors proliferating cell thymidine kinase 1 (TK1) activity via LC-MS/MS quantification of 3'-deoxy-3'-fluorothymidine (FLT) and its monophosphate metabolite FLT-MP. LNCaP prostate cancer cells were cultured at four densities (20,000; 10,000; 5000; and 500 cells/well) and incubated with 2000 ng/mL FLT in multi-well plates. Internal standards were FLT-d3 for FLT and d4-thymidine for FLT-MP. In culture medium, peak area ratios of FLT to FLT-d3 and FLT-MP to d4-thymidine were linear over the range 0.25-100 ng/mL (r(2)≥0.998). Accuracy for quality controls was between -7.3% and 6.3% for FLT, and from -3.3% to 1.7% for FLT-MP. Quality control precision was from 2.4% to 5.7% for FLT and 3.2% to 7.5% for FLT-MP. The limit of quantification was 0.25 ng/mL, with good control results (precision of 9.6% for FLT and 14.8% for FLT-MP). FLT-MP formation was linearly proportional to cell number from 500 to 20,000 cells/well 1 h after FLT addition. FLT-MP and ATP generation were comparable in LNCaP cells exposed to cell cycle inhibitor drugs (Spearman r=0.925, p<0.0001), demonstrating assay suitability for drug screening. This fit for purpose method is amenable to analysis of tumor tissue extracts, and should enable direct assessment of in vitro-in vivo relationships in animal models of cancer.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels

White shrimp Litopenaeus vannamei held in 25 per thousand seawater were injected with TSB-grown Vibrio alginolyticus (1 x 10(4) cfu shrimp(-1)), and then transferred to 5, 15, 25 (control) and 35 per thousand. Over 24-96 h, the mortality of V. alginolyticus-injected shrimp held in 5 per thousand and 15 per thousand was significantly higher than that of shrimp held in 25 per thousand and 35 per thousand, and the mortality of V. alginolyticus-injected shrimp held in 5 per thousand was the highest. Shrimp held in 25 per thousand and then transferred to 5, 15, 25 (control) and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus after 12-72 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 and 15 per thousand after 12 h. It is concluded that the shrimp transferred from 25 per thousand to low salinity levels (5 and 15 per thousand) had reduced immune ability and decreased resistance against V. alginolyticus infection.     

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.     

The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress

White shrimp Litopenaeus vannamei (also known as Penaeus vannamei) held in 34 per thousand seawater at pH 8.2 were injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 8 x 10(5) colony-forming units (cfu) shrimp(-1), and then transferred to tanks at pH 6.5, 8.2 (control) and 10.1, respectively. After 24-168 h, the mortality of V. alginolyticus-injected shrimp that were transferred to pH 6.5 and pH 10.1 tanks was significantly higher than that of V. alginolyticus-injected shrimp held at pH 8.2. In another experiment, L. vannamei held at pH 8.2 following transfer to pH 6.5, 8.2 (control) and 10.1 for 6, 12, 24, 72 and 120 h were examined for immune parameters, phagocytic activity, and the clearance efficiency of shrimp against V. alginolyticus. The results indicated that the shrimp that were transferred to pH 6.5 and 10.1 showed significantly decreased phenoloxidase (PO) activity, respiratory burst, phagocytic activity, and clearance efficiency against V. alginolyticus over 6-72 h; significantly decreased superoxide dismutase (SOD) activity over 6-24h; and decreased total haemocyte count (THC) over 12-72 h. Shrimp transferred to pH 10.1 showed significantly decreased granular cell counts, and THC after 6h, and decreased SOD activity after 72 h. The immune parameters of shrimp transferred to pH 6.5 and 10.1 returned to the original values after 120 h. However, shrimp transferred to pH 6.5 still maintained lower phagocytic activity, and clearance efficiency against V. alginolyticus, and shrimp transferred to pH 10.1 still maintained lower clearance efficiency against V. alginolyticus. It was therefore concluded that low pH and high pH stress decrease the resistance of white shrimp L. vannamei against V. alginolyticus and decrease several parameters of the immune response.     

Superoxide radical production in response to environmental hypoxia in cultured shrimp

Markers of oxidative stress in response to hypoxia and reoxygenation were assessed in Pacific white shrimp (Litopenaeus vannamei). Adult shrimp were either exposed to hypoxia (1 mg O(2)/L) for 6, 12, or 24 h followed by 1-h reoxygenation, or exposed to hypoxia for 24 h followed by 1- to 6-h reoxygenation. In all cases, shrimp maintained at constant normoxia were used as controls. Spectrophotometric techniques were applied to analyze lactate concentration, superoxide radical (O(2)(*-)) production, lipid peroxidation (TBARS), and antioxidant capacity status in muscle, hepatopancreas, and gill samples. Results indicate differences among tissues, even under control conditions. O(2)(*-) production and TBARS levels were higher in hepatopancreas than in gill or muscle. No effect of exposure to hypoxia was found. However, reoxygenation following exposure to hypoxia was found to affect the oxidative metabolism of muscle and hepatopancreas from cultured shrimp. Lactate concentration and O(2)(*-) production increased while antioxidant capacity decreased in hepatopancreas and muscle in the first hours of reoxygenation. This could translate into tissue damage, which may significantly jeopardize the commercial aquaculture product.     

Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus

Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0-20 mg l(-1). White shrimp Litopenaeus vannamei (7-12 g in the intermolt stage) were challenged with V. alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5. 10 and 20 mg l(-1)). There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l(-1)ammonia-N after 120 h of challenge. The shrimps were challenged with V. alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l(-1)(control), 1.10, 5.24, 11.10 and 21.60 mg l(-1). Mortality of shrimp in 5.24, 11.10 and 21.60 mg l(-1)was significantly higher than those in the control solution (0.01 mg l(-1)) after 48-168 h. Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l(-1)ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V. alginolyticus. No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations. Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l(-1)ammonia-N and greater after 7 days. The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l(-1)ammonia-N. With shrimps exposed to 11.21 and 21.22 mg l(-1)ammonia-N for 7 days, phagocytic activity and clearance efficiency to V. alginolyticus significantly decreased. It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L. vannamei from the V. alginolyticus infection.     

Shrimp hepatopancreatic deoxyribonuclease--purification and characterization as well as comparison with bovine pancreatic deoxyribonuclease

Deoxyribonuclease (DNase), isolated from shrimp hepatopancreas by chromatography on DEAE-cellulose, Sephadex G-100, phenyl-Sepharose and hydroxyapatite, is homogeneous as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The metal ion requirements and the pH-activity optima of shrimp DNase are very similar to those of bovine DNase. Both shrimp and bovine DNases are sensitive to iodoacetate inactivation under the same condition. The active shrimp DNase molecule is a monomer of Mr 44,000, approx. 13,000 larger than the Mr of bovine DNase. Shrimp DNase is rich in glutamic acid, glycine and half-cystine. The single polypeptide chain of shrimp DNase is highly cross-linked by 18 disulfides as compared to only two disulfides in bovine DNase. In contrast to bovine DNase, shrimp DNase is not a glycoprotein, is devoid of the activity against p-nitrophenyl phenylphosphonate (a synthetic substrate for bovine DNase), and resists to inactivation by beta-mercaptoethanol or trypsin under the Ca2(+)-free condition at pH 8. Shrimp DNase shows an isoelectric point of 4.06 on the thin-layer isoelectric focusing and rapidly loses its activity at pH below 5.     

Prey selection and the functional response of sea stars (Asterias vulgaris Verrill) and rock crabs (Cancer irroratus Say) preying on juvenile sea scallops (Placopecten magellanicus (Gmelin)) and blue mussels (Mytilus edulis Linnaeus)

Predators in nature include an array of prey types in their diet, and often select certain types over others. We examined (i) prey selection by sea stars (Asterias vulgaris) and rock crabs (Cancer irroratus) when offered two prey types, juvenile sea scallops (Placopecten magellanicus) and blue mussels (Mytilus edulis), and (ii) the effect of prey density on predation, prey selection, and component behaviours. We quantified predation rates, behavioural components (proportion of time spent searching for prey, encounter probabilities) and various prey characteristics (shell strength, energy content per prey, handling time per prey) to identify mechanisms underlying predation patterns and to assess the contribution of active and passive prey selection to observed selection of prey. Sea stars strongly selected mussels over scallops, resulting from both active and passive selection. Active selection was associated with the probability of attack upon encounter; it was higher on mussels than on scallops. The probability of capture upon attack, associated with passive selection, was higher for mussels than for scallops, since mussels can not swim to escape predators. Sea stars consumed few scallops when mussels were present, and so did not have a functional response on scallops (the target prey). Rock crabs exhibited prey switching: they selected mussels when scallop density was very low, did not select a certain prey type when scallop density was intermediate, and selected scallops when scallop density was high relative to mussel density. The interplay between encounter rate (associated with passive selection) and probability of consumption upon capture (associated with both active and passive selection) explained observed selection by crabs. Scallops were encountered by crabs relatively more often and/or mussels less often than expected from random movements of animals at all scallop densities. However, the probability of consumption varied with scallop density: it was lower for scallops than mussels at low and intermediate scallop densities, but tended to be higher for scallops than mussels at high scallop densities. When mussels were absent, crabs did not have a functional response on scallops, but rather were at the plateau of the response. When mussels were present with scallops at relatively low density, crabs exhibited a type II functional response on scallops. Our results have implications for the provision of protective refuges for species of interest (i.e., scallops) released onto the sea bed, such as in population enhancement operations and bottom aquaculture.     

Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis--II. Subcellular distribution of metals and metal-binding proteins

Cd accumulation in the hepatopancreas and gills of the scallop resulted in an increase of metal concentration in the cytoplasm, while in control animals the general amount of Cd in the hepatopancreas was concentrated in the microsomes and cytoplasm. Cd appears to be equally distributed among high molecular weight proteins and metallothioneins in control animals. It was shown that during the experiment the metal was bound mainly to high molecular weight proteins. After 30 days of exposure of scallops to flowing water Cd was redistributed to metallothionein (MT)-like proteins. Cd concentration in the lipids of the hepatopancreas of control and experimental scallops was equal.