有机锡污染与海产腹足类性畸变

综述了海产腹足类性畸变发生的原因和形态变化,并对利用海产腹足类性畸变进行有机锡污染的生物监测,海产腹足类性畸变的生态毒理学等方面的研究进展及我国的研究现状作了详细的介绍。     

Laboratory studies of cryopreservation of sperm and trochophore larvae of the eastern oyster

The eastern oyster, Crassostrea virginica, is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States. Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlled-rate freezer at a rate of 2.5 degrees C per min until reaching a final temperature of -30 degrees C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70 degrees C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol. Thawed sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration of larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries.     

Survival of eastern oysters Crassostrea virginica from three lines following experimental challenge with bacterial pathogens

Shellfish production is often affected by bacterial pathogens that cause high losses in hatcheries and nurseries. We evaluated the relative survival of larvae and juveniles of 3 Crassostrea virginica oyster lines: (1) GHP, a Rhode Island line; (2) NEHY, a line resistant to dermo and multinucleated sphere X diseases; and (3) FLOWERS, a line resistant to Roseovarius oyster disease, experimental challenge with Vibrio spp. isolates RE22 and RE101, causative agents of bacillary necrosis in Pacific oyster larvae, and the type strain of Roseovarius crassostreae, causative agent of Roseovarius oyster disease. All of the isolates were able to induce significant mortalities in oyster larvae and juveniles. Susceptibility to bacterial challenge in larvae was significantly higher at 25 degrees C than at 20 degrees C. Susceptibility decreased with oyster age; mean survival time ranged from 24 h in oyster larvae to more than 6 wk in juveniles. Significant differences in susceptibility to bacterial challenge were observed between oyster lines; NEHY was the most resistant line overall. Extracellular products (ECPs) from Vibrio sp. RE22 and R. crassostreae, as well as viable bacteria, were toxic to hemocytes from the 3 oyster lines, suggesting that ECPs are involved in pathogenesis and that external and mucosal barriers to infection are major contributors to resistance to bacterial challenge. These protocols will be useful in the elucidation of mechanisms of bacterial pathogenesis and resistance to infection in oysters.     

象山港熊本牡蛎幼体分布、附着及生长的空间格局

牡蛎幼体迁移与附着影响着牡蛎的空间分布、资源补充、种群维持及生态服务功能。本研究通过野外调查,监测了1个完整潮汐周期(9个潮时采样)内象山港2个试验点(采苗场和产卵场)表层、底层水体中熊本牡蛎幼体的时空动态,并开展野外附着试验,检验了熊本牡蛎在2个试验点和3个潮区(上层T、中层M和底层B)附着及生长的空间格局。结果表明: 在1个完整潮汐周期内,2个试验点表层水体中牡蛎幼体丰度均有显著的时间变化,采苗场表层水体中熊本牡蛎幼体丰度最大值(20.8±5.6 ind·L^-1)出现于高平潮时,显著高于其他8个潮时;而产卵场相反,高平潮时牡蛎幼体丰度最低(0.1±0.1 ind·L^-1);2个试验点底层水体中牡蛎幼体丰度在不同潮时之间均无显著差异。采苗场附着牡蛎总丰度、成活率和壳高在不同潮区间差异显著,牡蛎总丰度大小顺序为B>T=M,成活率和壳高的大小顺序为T>M>B,活体牡蛎丰度在潮区之间无显著差异。产卵场附着牡蛎总丰度、成活率、活体牡蛎丰度和壳高在不同潮区之间均无显著差异。表明高平潮时是熊本牡蛎附着的主要时段,产卵场和采苗场具有相似的牡蛎稚贝附着密度。     

环境因子对两种海洋双壳类幼体趋性行为中的调节作用

本文研究了温度、盐度和重金属等几种因子对缢蛏和长牡蛎幼体趋地性和趋光性行为的影响。结果表明:1)降低温度能增强缢蛏幼体的正趋地性,而长牡蛎幼体却由正趋地性转变为负趋地性,同时两种幼体的正趋光性受到抑制作用;升高温度对缢蛏幼体趋光性的影响不明显,但能加强牡蛎幼体的正趋光性。2)盐度提高时缢蛏幼体的正趋光性趋于增强,正趋地性趋于减弱。3)重金属Cu和Zn在各自的亚致死浓度下具有提高缢蛏幼体正趋地性和降低正趋光性的作用,而牡蛎幼体的正趋地性却趋于减弱,正趋光性趋于增强。本文据上述幼体的行为变化,还讨论了这几种因子对幼体垂直分布的调节作用。     

The influence of cooling rate,developmental stage,and the addition of sugar on the cryopreservation of larvae of the pearl oyster Pinctada fucata martensii

This paper examines the effects of cooling rate, developmental stage, and the addition of sugar on the cryopreservation of the larvae of the pearl oyster, Pinctada fucata martensii. The survival rates of frozen-thawed trochophores was 43.1% at a cooling rate of 1.0 degrees C/min. The survival rate of frozen-thawed larvae increased with developmental stage, except for umbo stage larvae, and the late D-shaped larvae showed a survival rate as high as 91%. The addition of sugar (0.2M glucose or sucrose) improved the survival rate of larvae. These results indicate that the preferred cooling rate, developmental stage, and sugar for the cryopreservation of pearl oyster larvae are 1 degrees C/min, late D-shaped larvae and 0.2M glucose or sucrose.     

Organotin in the Tagus estuary

The tri(n-butyl)tin (TBT) moiety has been shown to have complex ecotoxicological effects on estuarine populations. In the Tagus the massive die-off of the Portuguese oyster (Crassostrea angulata Lmk), has been attributed to the introduction and use of this contaminant by shipyards. This paper reviews previous work by others and reassesses our results obtained in water and sediments of the Tagus estuary, as a contribution to the understanding of tin geochemistry and organotin speciation in this estuary. Contrary to earlier results, the latest surveys show that in the open waters of the Tagus measurable concentrations of all the three butyltin species occur, exhibiting peak concentrations at two locations that indicate two previously undetected sources of TBT. TBT also presents a peak concentration in sediments near the Lisnave shipyards, as previously suggested. The latest surveys still detected methylated forms of tin, particularly monomethyltin (Me Sn³⁺), in Tagus sediments, with maximum concentrations in the vicinity of urban effluent discharges. The concentrations for TBT and its degradation products found in the Tagus are potentially harmful to the populations of gastropods and endemic bivalves.     

Establishing Restoration Strategy of Eastern Oyster via a Coupled Biophysical Transport Model

For marine fish and invertebrates, larval dispersal plays a critical role in determining connections among source and sink habitats, and the lack of a predictive understanding of larval dispersal is a fundamental obstacle to the development of spatially explicit restoration plans for marine populations. We investigated larval dispersal patterns of eastern oyster in an estuary along the Northern Gulf of Mexico under different simulation scenarios of tidal amplitude and phase, river discharge, wind direction, and larval vertical migration, using a coupled biophysical transport model. We focused on the dispersal of larvae released from the commercially exploited (Cedar Point, CP) and non‐exploited (Bon Secour Bay, BSB) oyster populations. We found that high flushing rates through the dominant inlet prevented larval exchange between the commercially exploited and non‐exploited populations, resulting in negligible connectivity between them. Variations in tidal amplitude, river discharge and wind direction played a more important role in the amount of larvae retained in Mobile Bay when they are released from CP than from BSB. Under most of the scenarios, larvae from BSB were retained around the spawning area, while larvae from CP showed a predominant westward flow. Net sinking behavior of late‐stage larvae increased larval retention in the bay, but physical transport showed a higher impact in the amount of larvae retained. These findings have enhanced our understanding of larval dispersal of eastern oyster in a wide, shallow estuarine system, and been used to establish spatially explicit strategies for oyster restoration in the Mobile Bay system, Alabama.     

Preliminary results of providing various combinations of live foods to grouper (Epinephelus coioides) larvae

Fertilized oyster eggs, S-rotifer (Brachionusrotundiformis) and SS-rotifer (Brachionus sp.),were tested either solely or in combination fortheir suitability as feed for early stage grouper (Epinephelus coioides) larvae. Sizes ofS-rotifers ranged between 143-224 µm lorica lengthwith mean 182±21µm, and SS-rotifersbetween 122-176µmlorica length with mean 154±13µm. Theresults indicated that both fertilized oyster eggs andSS-rotifers were suitable as feed at F1 (first feedingday). However, poor growth was recorded when providing oyster eggs solely for the periodsF1-F3. Although growth of larvae at F6 had nodifference between the sole SS-rotifers and the oystereggs additionally provided for F1-F3, better survivalof larvae at F15 was obtained when providingcombinations of SS-rotifers with oyster eggs for F1-F3.Besides, better growth and survival of larvae at F15was found when providing S-rotifers enriched withKirin yeast for F7-F15. The highest survival andfastest growth of larvae at F15 was found whenproviding oyster eggs for F1-F3, SS-rotifers forF1-F6, S-rotifers for F7-F15, both rotifers enrichedwith Kirin yeast, and Isochrysis for F1-F15.Total fatty acid (TFA), EPA, DHA content, and DHA/EPA(D/E) ratio of larvae changed with their sizes andcorresponded to that of their feeds. The F15 larvaehaving a higher TFA grew faster, having higher DHA orEPA survived better.     

The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for pacific oyster (Crassostrea gigas) larvae

Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.     

Comparative susceptibility of veliger larvae of four bivalve mollusks to a Vibrio alginolyticus strain

The susceptibility of 7 d old veliger larvae of the scallops Argopecten ventricosus and Nodipecten subnodosus, the penshell Atrina maura, and the Pacific oyster Crassostrea gigas to a pathogenic strain of Vibrio alginolyticus was investigated by challenging the larvae with different bacterial concentrations in a semi-static assay. The results indicate that the larvae of the 2 scallop species are more susceptible to the V. alginolyticus strain than those of the oyster and the penshell. Signs of the disease were similar to bacillary necrosis described in previous work. Interspecies differences in susceptibility to pathogens are discussed.     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

Estrogens counteract the masculinizing effect of tributyltin in zebrafish

Recently, it has been demonstrated that the biocide tributyltin (TBT) can interfere with fish sex differentiation, leading to a bias of sex toward males. On the contrary, it is well known that estrogenic compounds can induce fish feminization. Yet, the combined effects of mixtures of androgenic and estrogenic compounds on fish sex differentiation have never been investigated before, even though in the environment animals are frequently exposed to both groups of xenobiotics. Therefore, in order to investigate whether exposure to estrogenic compounds can block the masculinizing effect of TBT, 5 days post-fertilization zebrafish (Danio rerio) larvae were exposed for a four month period to TBT and to the synthetic estrogen-ethinylestradiol (EE2). The fish were fed a diet containing TBT at nominal concentrations of 25 and 100 ng TBT/g, and two groups of animals were also dosed with TBT plus EE2 at nominal water concentration of 3.5 ng/L, using a flow-through design. As expected, fish exposed to TBT showed a bias of sex toward males (62.5% males in control tanks and 86% and 82% in TBT 25 and TBT 100 ng TBT/g, respectively). Co-exposure to EE2 completely blocked the masculinizing effect of TBT, with 7% males in the TBT 25 ng/g + EE2 treatment and 0% in the EE2 alone and in the TBT 100 ng/ + EE2 exposed groups. These results clearly indicate that EE2, at environmentally relevant concentrations, can block the TBT masculinizing effects in zebrafish, which suggests that in the aquatic environment the presence of estrogens may neutralize the fish masculinizing effect of TBT. Our findings highlight the need of testing the combined effects of contaminants, as single exposure studies may not be sufficient to predict the effects of mixtures of xenobiotics with antagonistic properties.     

Fate of Bacillus sphaericus 2362 spores following ingestion by nontarget invertebrates.

Elimination of Bacillus sphaericus spores ingested by midge larvae, snails, and oysters was most rapid among midge larvae. Spores remained in oysters up to 21 days and in snails up to 49 days. Viable spores were recovered in snail and oyster feces for these same periods. There was no indication of actively growing B. sphaericus in the animals. Passage through oyster gut detoxified the B. sphaericus mosquito larval toxin, but there was a 33% retention of toxicity following snail gut passage. Midge larvae reared to adults in spore-containing water carried spores in/on the adult body. This suggests that these animals could carry the bacteria to sites beyond the application area.     

Detection of ostreid herpesvirus-1 (OsHV-1) by PCR using a rapid and simple method of DNA extraction from oyster larvae

A DNA extraction procedure was developed for the detection of ostreid herpesvirus-1 (OsHV-1) using the polymerase chain reaction (PCR) in oyster larvae. The DNA extraction procedure developed was tested on 8 larval samples. Abnormal nuclei with characteristic features associated with OsHV-1 infections were only observed in samples in which the viral DNA was detected by PCR. A previously described competitive PCR method was applied to detect inhibition during PCR reactions. The results show that the method can be used on small amounts of oyster larvae (3 mg) for the detection of OsHV-1 DNA by PCR.     

Influence of environmental variables on the growth and reproductive cycle of Crassostrea (Mollusca,Bivalvia) in Guaratuba Bay,Brazil

The production of oysters in Guaratuba Bay, in the state of Paraná, Brazil, is still a mixed activity of mariculture and extractivism. The sustainable development of this production requires the monitoring of environmental, genetic, reproductive, and zootechnical variables. This study evaluated the importance of these variables on Crassostrea oyster production. Data were obtained between September 2009 and February 2011 from six evaluations: water quality, oyster larvae in plankton, capture of oyster spat by artificial collectors, molecular identification of collected spat and larvae, continuous evaluation of the reproductive maturity stage of adult oysters, and zootechnical performance achieved by experimental production. Temperature, dissolved oxygen, and water transparency were the environmental factors that had the most influence on the reproductive cycle (e.g. gonadal development and spat capture) and on the growth of the oyster. The highest rates for spat uptake were recorded in the summer, coinciding with the peak of sexual maturity of adults. The growth of the oysters, in terms of the weight of the meat, was related to the gonadal stage.     

The effects of some selected bacteria on embryos and larvae of the American oyster,Crassostrea virginica

An investigation was conducted to determine the effects of selected bacteria on healthy embryos and larvae of the American oyster, Crassostrea virginica. A total of 156 bacterial types were isolated from moribund larvae and particulate matter collected with a 36 μm Nitex screen. Most of the isolates were nonpigmented motile, gram-negative rods sensitive to chloramphenicol and neomycin, and were classified as Pseudomonas and Vibrio.Three oyster developmental stages were exposed to selected bacteria: embryos in the first 48 hr of development, 2-day-old larvae, and presetting, 10-day-old larvae. Twenty of the bacterial strains tested caused gross morphological abnormalities, decreased growth, and/or increased mortality for one or more of the three developmental stages. Neither culture filtrate nor heat-killed bacteria produced these effects.     

Oyster Larvae Settle in Response to Habitat-Associated Underwater Sounds

Following a planktonic dispersal period of days to months, the larvae of benthic marine organisms must locate suitable seafloor habitat in which to settle and metamorphose. For animals that are sessile or sedentary as adults, settlement onto substrates that are adequate for survival and reproduction is particularly critical, yet represents a challenge since patchily distributed settlement sites may be difficult to find along a coast or within an estuary. Recent studies have demonstrated that the underwater soundscape, the distinct sounds that emanate from habitats and contain information about their biological and physical characteristics, may serve as broad-scale environmental cue for marine larvae to find satisfactory settlement sites. Here, we contrast the acoustic characteristics of oyster reef and off-reef soft bottoms, and investigate the effect of habitat-associated estuarine sound on the settlement patterns of an economically and ecologically important reef-building bivalve, the Eastern oyster (Crassostrea virginica). Subtidal oyster reefs in coastal North Carolina, USA show distinct acoustic signatures compared to adjacent off-reef soft bottom habitats, characterized by consistently higher levels of sound in the 1.5–20 kHz range. Manipulative laboratory playback experiments found increased settlement in larval oyster cultures exposed to oyster reef sound compared to unstructured soft bottom sound or no sound treatments. In field experiments, ambient reef sound produced higher levels of oyster settlement in larval cultures than did off-reef sound treatments. The results suggest that oyster larvae have the ability to respond to sounds indicative of optimal settlement sites, and this is the first evidence that habitat-related differences in estuarine sounds influence the settlement of a mollusk. Habitat-specific sound characteristics may represent an important settlement and habitat selection cue for estuarine invertebrates and could play a role in driving settlement and recruitment patterns in marine communities.     

The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non‐native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density‐dependent effects between the native and non‐native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed some acceptable limit, but also how patch formation modifies this.