海洋污染物对菲律宾蛤仔的免疫毒性

环境污染能够影响养殖贝类的免疫能力,是导致贝类大规模死亡的重要原因之一.探讨了大连周边4个海区污染物对采集的菲律宾蛤仔(Ruditapes philippinarum)免疫毒性影响.结果发现:污染物浓度和种类对蛤仔的免疫和生理指标具有重要影响,在重金属和石油污染物浓度较低的皮口海区,血细胞总数、亮氨酸氨基肽酶活性和血淋巴溶菌酶活性均显著高于其它3个海区(P＜0.05),而蛤仔的脂质氧化水平则较低;在重金属和石油污染物浓度较高的黑石礁海区,蛤仔血淋巴谷胱甘肽含量显著高于其它3个海区(P＜0.05);在重金属浓度较高的庄河海区,蛤仔表现出较高的超氧化物歧化酶活性(P＜0.05).     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters

Quahog Parasite Unknown (QPX) is a protistan parasite affecting hard clams Mercenaria mercenaria along the Northeastern coast of the United States. The geographic distribution and occurrence of disease epizootics suggests a primary role of temperature in disease development. This study was designed to investigate the effect of temperature on constitutive and QPX-induced defense factors in M. mercenaria. Control and QPX-challenged (both experimentally and naturally) clams were maintained at 13, 21 and 27 °C for 4 months. Control and experimentally-infected clams originated from a southern broodstock (Florida, no prior reports of disease outbreak) while naturally-infected clams originated from a northern broodstock (Massachusetts, enzootic area). Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. Measured parameters included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated a strong influence of temperature on constitutive clam defense factors with significant modulation of cellular and humoral parameters of control clams maintained at 13 °C compared to 21 and 27 °C. Similarly, clam response to QPX challenge was also affected by temperature. Challenged clams exhibited no difference from controls at 27 °C whereas different responses were observed at 21 °C and 13 °C compared to controls. Despite differences in infection mode (experimentally or naturally infected) and clam origin (northern and southern broodstocks), similarities were observed at 13 °C and 21 °C between QPX infected clams from Florida and Massachusetts. Clam response to temperature and to QPX exhibited interesting relationship with QPX disease development highlighting major influence of temperature on disease development.     

Behavioral plasticity of the soft-shell clam, Mya arenaria (L.), in the presence of predators increases survival in the field

Soft-shell clams, Mya arenaria, are sessile, suspension-feeding bivalves that are preyed upon by the exotic green crab, Carcinus maenas. Clams evade crab consumers by burrowing deeper into the sediment after perceiving a threat from a nearby predator. The purpose of this study was to determine the types of signals that M. arenaria use to detect predators and the types of behaviors clams use to avoid being eaten. In a field study, clams increased their burial depth in the presence of green crab predators consuming conspecifics that were caged nearby, and also increased burial depth after artificial tactile stimulation in the laboratory assay. These results indicate that clams can use chemical and mechanical cues to detect potential predatory threats. We performed a field study to examine the difference in survivability of clams that had burrowed deeper into the sediment in response to predators vs. control clams that were burrowed less deeply. Significantly higher survival rates were observed in clams that had initially burrowed more deeply, suggesting that increasing burial depth is a valid predator avoidance strategy. Some bivalves also alter their pumping rates in the presence of predators, making them less apparent and providing more structural defense by covering soft tissue, and we measured pumping time of soft-shell clams in the presence and absence of predators, when burrowing was not an option for escape. Soft-shell clams did not alter their pumping time in the presence of green crab predators, possibly because they employ a burrowing method called “hydraulic” or “jet-propelled” burrowing, where it is necessary for the clam to pump in order to burrow. Chemical signals and tactile cues instigated behavioral changes in M. arenaria, and this change in behavior (increasing burial depth) increased clam survival in the field.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r² = 0.68, p < 0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.     

Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: I. Dynamics of QPX disease

Quahog Parasite Unknown (QPX) causes disease and mortality in hard clams, Mercenaria mercenaria. Seasonality of QPX disease prevalence in the field and changes in QPX growth and survival in vitro suggest a role of temperature in the hard clam-QPX interaction and disease development. This study specifically examined the effect of temperature on QPX disease development and dynamics. Naturally and experimentally infected clams were separately maintained in the laboratory at 13 °C, 21 °C, or 27 °C for 4 months. Following this initial treatment, temperature was adjusted to 21 °C for 5 additional months to simulate seasonal changes of temperature in the field and to investigate the effect of temperature variations on QPX disease dynamics. Mortality was continuously monitored during the experiment and clams were sampled at 2, 4 and 9 months for the assessment of QPX disease prevalence and intensity using our standard histological and quantitative PCR techniques. Results demonstrated significantly higher QPX disease prevalence and intensity, as well as higher mortality, in naturally-infected clams maintained at 13 °C as compared to those held at 21 °C or 27 °C. Similarly, disease development was significantly higher in experimentally infected clams maintained at the colder temperature (70% prevalence after 4 months) as compared to those maintained under warmer conditions (<10%). Additionally, our results demonstrated an improvement in the condition of clams initially maintained at 13 °C for 4 months after transfer to 21 °C for 5 additional months, with a significant reduction of QPX prevalence (down to 19%). Interestingly, disease development or healing in clams maintained at different temperatures exhibited a strong relationship with clam defense status (jointly submitted paper) and highlighted the impact of temperature on clam activity and QPX disease dynamics. These findings should be taken into account for the timing of activities involving the monitoring, movement (e.g. relays, transplants) or grow out (e.g. commercial culture, municipal enhancement) of hard clams in enzootic areas.     

Pathology survey of the short-neck clam Ruditapes philippinarum occurring on sandy tidal flats along the coast of Ariake Bay, Kyushu, Japan

The pathological condition of the short-neck clam Ruditapes philippinarum was surveyed along the coast of Kumamoto, Japan, in June 2004. DNA sequences of the non-transcribed spacer region and internal transcribed spacer region flanking 5.8S rRNA identified Perkinsus olseni among the clams. Ray’s fluid thioglycollate medium assay indicated that 96.7% of the clams surveyed from the Kiguchi River tidal flat (native clams, Stn KR-N) and 96.7% of the clams surveyed from the Midori River tidal flat (Stn MR) were infected with P. olseni with an infection intensity of 464,278 and 199,937 Perkinsus cells/gram tissue wet weight (gWW), respectively. In contrast, 66.7% of the clams imported from China and stored along the Kiguchi River tidal flat (Stn KR-I) and 20.2% of clams from the Arao tidal flat (Stn AT) were infected with P. olseni with an infection intensity of 37,547 and 3382 Perkinsus cells/gWW, respectively. Brown ring disease was detected in the clam population from Stn KR-I at a prevalence of 90.0%. Polymerase chain reaction and the 16S rRNA sequence suggested that the agents of brown ring disease observed at Stn KR-I were Vibrio tapetis-like bacteria. Sporocysts and metacercariae of unidentified trematodes were also observed in the gonads and mantle of the clams from Stn KR-I, Stn MR, and Stn AT, at prevalences of 7.1-42.9%. Metacestodes (larval tapeworms) were found in the foot and digestive gland at a prevalence of 52.5%, 30.0%, and 14.3% in clams from Stns MR, AT, and KR-N, respectively. Histology also showed massive hemocyte infiltration and inflammation among clams heavily infected with P. olseni. Castration of the follicle was typical among clams infected with the trematode. The data indicate that most of the clams along the coast of Kumamoto are infected with various pathogens at various rates of infection, and these pathogens could have negative effects on the clam population in the long term.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

Movement and aggregation in the fluted giant clam (Tridacna squamosa L.)

Aquaculture has been the traditional focus of tridacnid giant clam research whereas their ecology and behaviour have received much less attention. This study was based on the observation that juvenile fluted giant clams (Tridacna squamosa), when evenly distributed in a tank, will move and aggregate over time. We observed movement in clams ranging from 10 to 313 mm in shell length and ‘climbing’ up the sides of tanks was noted for clams with lengths between 10 and 22 mm. Locomotion also occurred after byssal attachment to the substrate; there was a highly significant association between type of movement (i.e. translation, rotation and no movement) and presence of attachment. Tests for phototaxis were negative. Aggregation was examined by placing clams in regular patterns on grids. After three days in the aquarium and 24 h in the field, their positions were analysed to obtain a statistical parameter for ‘clumpiness’. This was found to be greater in the live clam runs than both random walk and random distribution simulations, suggesting that clams were attracted to conspecifics. The latter was tested by recording clam movement with respect to five types of fixed ‘targets’ (i.e. live clam, fouled clam shell, foul-free clam shell, random inanimate object and none). The test clams moved, non-significantly, towards live clam targets and displayed higher mobility compared to tests with other target objects; a negative correlation between mobility and clam length was also observed. A choice experiment using bidirectional water inflow with clam effluent as one source resulted in clams moving toward the effluent, offering the first direct support for positive chemotaxis among conspecifics in Bivalvia. Together, our results indicate the presence of chemical signalling among clams, leading to movement toward one another and clumping. Aggregation could serve several ecological functions, such as defence against predation, physical stabilisation and facilitation of reproduction. With worldwide decline in natural giant clam densities, the opportunity for conspecific clumping is reduced, and local stocks could be facing increased vulnerability to Allee affects.     

Selective crab predation on native and introduced bivalves in British Columbia

Experiments were conducted to determine whether locally abundant crab species prefer co-occurring littleneck clams, Protothaca staminea (Conrad, 1837) and Tapes philippinarum (A. Adams and Reeve, 1850), relative to a recently introduced species, the varnish clam, Nuttallia obscurata, (Reeve, 1857). Prey preference, handling time, pick-up success, profitability and consumption rates were investigated for two crab species, Dungeness crab, Cancer magister (Dana, 1852) and red rock crab, Cancer productus (Randall, 1839) crabs. Both crab species preferred varnish clams over the native species. This may be attributable to the lower handling time, higher pick-up success and increased profitability of consuming varnish clams. Handling time appeared to be a factor not only in species preference, but also in the degree of preference, with shorter handling times corresponding to stronger preference values. Both native and introduced bivalves burrow into the substratum, with the varnish clam burrowing deepest. When feeding on clams in limited substratum both crab species preferred the varnish clam. In the unlimited substratum trials Dungeness crabs preferred varnish clams (although to a lesser degree) while red rock crabs preferred littleneck clams. This was likely due to the significantly deeper burial of the varnish clam, making it less accessible. Although the morphology (i.e. thin shell, compressed shape) of the invader increases its vulnerability to predation, burial depth provides a predation refuge. These results demonstrate how interactions between native predators and the physical characteristics and behaviour of the invader can be instrumental in influencing the success of an invasive species.     

Identification and characterization of the pathogenic effect of a Vibrio parahaemolyticus-related bacterium isolated from clam Meretrix meretrix with mass mortality

A mass mortality of clam, Meretrix meretrix, occurred in Jiangsu Province of China in the late September of 2007. Of the isolates obtained from the diseased clams, MM21 had the strongest virulence to the clam in the virulence test, with a LD50 value of ∼6 × 10⁶ CFU ml⁻¹. MM21 was identified as Vibrio parahaemolyticus by the VITEK 2 Compact system and 16S rDNA sequencing. Detection of virulence-associated genes by PCR indicated that MM21 was positive for toxR and tlh, and negative for tdh. Compared with control group, histiocytes from MM21-infected clams displayed a variety of cytopathological changes by transmission electron microscopy examination, which included increased lipid droplets in hepatocytes, deposition of granules in the mantle, excessive secretion in the gill. The results of our study suggested that MM21 may have been an etiological element in the mass mortalities of hard clam (M. meretrix) in Jiangsu Province of China in 2007.     

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.     

Contents of Cd,Fe, Mn and Zn in the tissue ofKatelysia hiantina collected from Tolo Harbour,an almost land-locked sea

Summary Samples ofK.hiantina and sediments were collected from iron ore tailings and other nearby sites within Tolo Harbour for heavy metal analysis. It was found that sediments as well as the clams at the tailings contained higher concentrations of Cd, Fe, Mn and Zn. Positive correlations between the metals in the sediments and clam tissues were found. The clams collected from the tailings showed a bustantial loss of Fe, Mn and Zn when reared in clean filtered seawater for a period of 8 days.     

Air exposure and functionality of Chamelea gallina haemocytes: effects on haematocrit, adhesion, phagocytosis and enzyme contents

The Venus clam Chamelea gallina is fairly common along the western coasts of the Adriatic and is subjected to intense fishing. Since over the last 20 years extensive hypoxic and anoxic conditions have repeatedly damaged this natural resource, we decided to study the effects of anoxic stress on the functionality of clam haemocytes and the consequences on immune responses. Clams, exposed to air, close their valves and tissues become anoxic and metabolism processes switch to anaerobiosis. In these conditions, a significant decrease in the haematocrit value and in the percentage of acid phosphatase-positive haemocytes was observed, while the number of cells with beta-glucuronidase significantly increased after day 1. The above indices generally returned to control values when clams were re-immersed in seawater after 1 day of treatment. Clams exposed to air for 2 days and then re-immersed, attempted to recover in the subsequent 3 days. Animals had fully recovered on day 4. Three-day-exposed clams did not recover. Phagocytic and adhesion indices decreased significantly after the first day of air exposure. The change in frequency of three types of circulating cells (spreading, round, apoptotic) was also monitored.     

重金属Cd胁迫对红树蚬的抗氧化酶、消化酶活性和MDA含量的影响

摘要：本文在实验室条件下,观测不同Cd胁迫处理（时间和水平）对红树蚬（Geloina coaxans (Gmelin)）的胃组织中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、丙二醛(MDA)含量,及淀粉酶、脂肪酶和蛋白酶等3种消化酶活性的影响效应。结果表明,高浓度组（4.00 mg?L-1和8.00 mg?L-1）在较短的暴露时间即出现SOD 活性显著诱导,而低浓度组则需要更长的暴露时间。所有处理组的CAT活性均先受诱导而后抑制,但去除胁迫后低浓度组活性上升,高浓度组则持续下降。低浓度处理组在胁迫初期MDA含量上升,但随后下降至较低水平;高浓度处理组MDA含量上升稍为滞后,但随后上升至较高水平。在消化酶方面,Cd对红树蚬胃组织淀粉酶的影响基本上表现为抑制;在胁迫初期脂肪酶活性受到显著诱导,随着胁迫时间延长酶活性则下降,同时胁迫解除后不同程度恢复;对蛋白酶的影响效应规律性不明显。显著的效应-剂量间相关关系存在于特定时间的SOD活性（1d）,CAT、淀粉酶活性和MDA含量（7d和恢复6d）。本文还探讨了这些指标作为生物标记物应用于监测海洋重金属污染的可能性。 关键词：红树林;红树蚬;Cd胁迫;消化酶;抗氧化酶;脂质过氧化     

The course and mortality of a hematopoietic neoplasm in the soft-shell clam,Mya arenaria

Results from two experiments containing approximately 280 Mya arenaria indicated that significantly higher (P < 0.05) mortality occurred within the neoplastic clam population than in the nonneoplastic clam population. Using an in vivo blood cytological technique, five levels of severity were established. The levels were based on the number of neoplastic cells in the circulation with level 1 as the lowest severity and level 5 as the highest severity. Neoplastic clams that were diagnosed as the lowest level had higher survival rates (60%) than those clams diagnosed as the highest level (0%). The hematopoietic neoplasm in M. arenaria followed one of three courses: (1) in approximately 50% of the neoplastic clams the disease progressed to a higher severity and resulted in death; (2) in approximately 40% of the neoplastic clams the disease was chronic, i.e., remained at a stable level; and (3) in approximately 10% of the neoplastic clams the disease diminished in severity or disappeared entirely. In addition, 10% of the clams diagnosed as nonneoplastic at the beginning of the experiments were neoplastic by the termination of the experiment. The contraction of the disease may have been de novo or by stimulation of latent infections.The prevalence of the neoplasm in clams collected from an epizootic area followed a biphasic seasonal pattern. The highest prevalences occurred in October, November, and in May. The hematopoietic neoplasm in M. arenaria was also age and species specific.     

Survivorship of juvenile surf clams Donax serra (Bivalvia, Donacidae) exposed to severe hypoxia and hydrogen sulphide

Toxic “sulphide eruptions” sporadically occur in the highly productive inshore regions of the central Namibian Benguela upwelling system. The surf clam Donax serra (Röding, 1798) dominates the intertidal and upper subtidal of large exposed sandy beaches of southern Africa and its recruitment seems to be affected by sulphide events. The reaction of juvenile surf clams to low oxygen concentrations and sulphide occurrence (0.1 mmol l⁻¹) was examined by in vitro exposure experiments in a gas-tight continuous flow system. After 2 h of hypoxic- and hypoxic/sulphidic conditions, clams moved to the sediment surface, aiding their passive transport to areas with more favourable conditions. The clams showed a high sulphide detoxification capacity by oxidising the penetrating hydrogen sulphide to non-toxic thiosulphate. Moreover, juvenile D. serra switched to anaerobic energy production, indicated by the significant accumulation of succinate and, to some extent, alanine. Test animals were not able to reduce their energy requirements enough to withstand long periods of exposure, leading to a median survival time (LT₅₀) of 80 h under hypoxic sulphide incubation. In conclusion, natural “sulphide eruptions”, especially those with a large spatial and temporal extension, have to be considered as an important factor for D. serra recruitment failures. Hydrogen sulphide is assumed to be a potential community structuring factor.