Cadmium-binding proteins in the blue crab, callinectes sapidus: laboratory-field comparison

The blue crab, Callinectes sapidus, is distributed along the east coast of the United States from Cape Cod, Mass., through the Gulf of Mexico, including both relatively unpolluted coastal areas and estuaries contaminated with trace metals. Cadmium is of particular concern because it is concentrated in the digestive glands of blue crabs and can be passed on to consumer organisms. Tissue concentrations and partitioning of trace metals from crabs exposed in the laboratory to 10 ppb dissolved cadmium for 40 days were compared with blue crabs collected from two locations on the Hudson River, NY, Foundry Cove and Haverstraw Bay, Foundry Cove and Haverstraw Bay, both of which have elevated trace metal levels relative to estuarine areas near Beaufort, NC. Crab digestive glands, gills and muscle were removed and analyzed for total cadmium, copper, zinc and nickel concentrations using acid digestion and atomic absorption spectrophotometry, and metal-binding (metallothionein-like) proteins were determined by gel-filtration chromatography. In crabs exposed to cadmium in the laboratory, the cytosolic partitioning was similar to previous investigations at our laboratory where higher levels of cadmium (100 ppb) and shorter exposure times (14 days) were used. The similarity in cadmium partitioning from these two separate experiments indicates dose independence. In crabs from polluted environments the digestive glands contained the highest concentrations of trace metals. Chromatograms of the cytosol from the digestive glands and gills from both field and laboratory exposed crabs showed similar distributions of cadmium, copper and zinc. The gills of both field and laboratory-exposed crabs had metal-binding proteins that contained mostly cadmium, and the digestive glands had metallothionein-like proteins that contained cadmium, copper and zinc. Estimated molecular weights for these proteins were similar to the metallothioneins found in other crustaceans and mammals.     

Effects of toxicants on developing oocytes and embryos of the blue crab, Callinectes sapidus

Oocytes (90 μm in diameter) were isolated from ovaries of 14-day post-molt adult female crabs and maintained in culture media. The addition of cadmium (20 μg/ liter) or tributyltin (2 μg/liter) to developing oocytes resulted in decreases in the growth of oocytes as measured by protein and lipovitellin accumulation relative to controls. Crab embryos isolated from yellow ‘sponge’ of female blue crabs were maintained in seawater until hatching (6–8 days). Toxicants tested included cadmium, copper, tributyltin and endosulfan. Some of the processes or events which were followed included water uptake, lipovitellin utilization rate, formation of appendages, formation of a heart, formation of eyespots and hatching to zoea stage. The primary effects from addition of toxicants were deformed eyespots and reduced hatching success. The hatching EC₅₀ (concentration at which 50% of embryos failed to hatch) for copper, cadmium, tributyltin and endosulfan were 3.1, 0.25, 0.047 and 450 μg/liter, respectively. Crab embryos appear to be suitable for testing of the effects of a variety of toxicants with advantages including low cost, reproducibility, low variability and sensitivity.     

Detection of the water-soluble fraction of crude oil by the blue crab, Callinectes sapidus

The ability of the blue crab, Callinectes sapidus, to detect petroleum hydrocarbons was measured with behavioural techniques. When presented with a water-soluble fraction of Prudhoe Bay crude oil, blue crabs abruptly changed antennular orientation, began rhythmic beating of the maxillipedal flagellae, and increased antennular flicking rate. The threshold concentration at which 50% of the crabs detected the water-soluble fraction was 2 × 10^?6 mg/litre. The blue crab apparently can readily detect petroleum hydrocarbons at concentrations found in chronically polluted areas as well as oil spill situations.     

Effect of reproductive toxicants on lipovitellin in female blue crabs, Callinectes sapidus

Changes in the concentration of lipovitellin, a lipoprotein that is the major protein in mature oocytes of crustaceans, were determined during ovarian development in female blue crabs, Callinectes sapidus. To test the effects of reproductive toxins on vitellogenesis, crabs were fed food containing cadmium or injected with the juvenile hormone antagonist, precocene II. Monoclonal antibodies prepared against one of the lipovitellin peptides (peptide A—107 kDa) were used in an enzyme linked immunosorbent assay to determine lipovitellin concentrations. Recently molted female crabs fed food containing cadmium (0.02 mg/g) for 18 days had oocytes with the same lipovitellin concentration as controls, but after 24 days the oocytes in cadmium treated crabs had significantly lower lipoprotein contents than controls (30 ± 4 ng/oocyte—cadmium treated; 45 ± 4 ng/oocyte—control). Precocene II-treated crabs had significantiy lower lipovitellin concentrations after 18 and 24 days. Diameters of oocytes were the same in both treated and control crabs. The results suggest that some pollutants may affect reproduction by acting during secondary vitellogenesis i.e., period when there is a large increase in lipovitellin synthesis. Since lipovitellins serve many of the nutritional needs of recently emerged crab embryos, poor larval survival may occur as a result of lower lipovitellin content.     

墨西哥湾海洋环境对蓝蟹(Callinectes sapidus)幼体分布和扩散的影响

本文以蓝蟹为例,研究海洋环境对甲壳动物幼体迁移规律和机制的影响。利用不规则三角形网格和有限体积模型(finite-volume coastal ocean model, FVCOM)耦合kinesis模型的方法,分析研究了墨西哥湾物理环境对蓝蟹(Callinectessapidus)幼体的分布和扩散途径的影响。蓝蟹在每年的四、五月份海水落潮期间产卵,通过跟踪算法从产卵区域沿着墨西哥湾海域进行模拟,获得了80天内蓝蟹幼体的粒子移动轨迹,记录并分析了幼体经过海域的盐度值。研究结果证明了该方法可有效模拟蓝蟹幼体在特定海域的迁移规律和扩散机制,进一步研究可为了解海洋物理环境对蓝蟹和其他渔业资源的影响提供借鉴。     

Movement patterns and trajectories of ovigerous blue crabs Callinectes sapidus during the spawning migration

Female blue crabs (Callinectes sapidus Rathbun) migrate from low salinity estuarine regions to high salinity regions near the ocean to release larvae. During this migration, ovigerous females use ebb-tide transport, a vertical migratory behavior in which they ascend into the water column during ebb tides, to move seaward to larval release areas. In order to determine the relationship of ebb-tide vertical migrations to local currents and the influence of these vertical migrations on the horizontal transport of blue crabs in the estuary, ovigerous females with mature embryos (1–3 days from hatching) were tracked near Beaufort Inlet, North Carolina (USA), in July and August 2001 and 2002. Crabs were tagged and tracked using ultrasonic telemetry, and currents near the crabs were measured simultaneously with a shipboard acoustic Doppler current profiler.During the two seasons, eight crabs were successfully tracked for periods ranging from 3.9–37.0 h and for distances ranging from 1.9–10.6 km. All crabs migrated seaward during the tracking periods. Crabs moved episodically during all tidal phases with periods of movement on the order of minutes to an hour. They moved with local currents in terms of both speed and direction during ebb tides, consistent with ebb-tide transport, and moved down-estuary (seaward) in opposition to local currents during flood tides. The percentage of time that crabs were active was higher during night ebb tides than during day ebb tides or flood tides and increased with increasing ebb-tide current speed. Mean migratory speeds were 0.11, 0.04, 0.08 and 0.02 m s⁻¹ during night ebb, night flood, day ebb and day flood tides, respectively, and net migratory speeds were on the order of 5 km day⁻¹. Due to the episodic nature of the crabs' movements, the total distances that crabs traveled during ebb tides ranged from 10–40% of the distances that passive particles could have traveled under the same conditions.     

三疣梭子蟹精子保存研究

于2004年5月到2004年7月对三疣梭子蟹精子进行了常温、低温和超低温保存研究。利用离子载体A23187人工诱导顶体反应以判断精子活力。常温和低温保存实验的结果显示,4℃下无钙人工海水(calcium-free artificial sea water,Ca2 FASW)保存236h的精子有25%以上的成活率。在4℃,25℃两种温度与Ca2 FASW、天然海水(natural sea water,NSW)两种基础液构成的4种组合中,最优组为4℃、Ca2 FASW,最差组为25℃、NSW。将精子浓度与Ca2 FASW对照组及诱导组比较,结果显示,25℃和4℃下,精子浓度与对照组顶体反应率相关性不显著(P=0.068>0.05,P=0.265>0.05),与诱导组顶体反应率极显著相关(P<0.01)。以二甲亚砜(Dimethyl sulfoxide,DMSO)和甘油(Glvcerol,G)作为抗冻剂进行超低温冷冻实验,72h后解冻,包括对照组在内的所有组均有98%以上的精子不经诱导就发生顶体反应。     

Properties of structure and function of cadmium-binding proteins/peptides from Euglena gracilis

Euglena gracilis makes two small Cd-binding proteins/peptides (Cd-BP I and Cd-BP II) in response to exposure to Cd²⁺. These proteins migrate slower than mammalian Cd-metallothionein (Cd-Mt) through Sephadex G-75, but like Cd-Mt also contain Zn and are separated into two species when chromatographed over a DEAE column. Both BPs are much less stable than Cd-Mt under acidic conditions. One source of this instability is that at least Cd-BP II contains acid-labile sulfide co-ordinated to the metals. It has been quantitated in Cd-BP II and shown to have a value of 1·25 ± 0·10 S²⁻/Cd. The reactivity of the Cd-binding sites in ligand substitution and thioldisulfide exchange reactions has been assessed. The reactions of Cd-BP I and II with pyridylazoresorcinol (PAR) were slow and displayed complex kinetic behavior. Similarly, both Cd-BPs react slowly and with complicated kinetics with 5,5′-dithiobis-(2-nitrobenzoate).     

Heavy metal stress induced hyperglycemia in blue swimmer crab, Portunus pelagicus

The study was to find out the effect of cadmium and mercury on levels of hemolymph glucose, crustacean hyperglycemic hormone (CHH) and hepatopancreas glycogen in the blue swimmer crab Portunus pelagicus. The experiments were performed in both intact and eyestalk ablated crabs. Quantification of CHH was done by the indirect ELISA with the aid of primary anti-Carcinus maenas-CHH antibody. Higher glucose concentration was observed on exposure to 8×10^-6 of cadmium ((825.6±5.42) μg/mL) and 6×10^-6 of mercury ((90.5±6.25) μg/mL) after 48 h and 24 h respectively. Higher level of hemolymph glucose was observed in eyestalk intact crabs on exposure to cadmium and mercury than eyestalk ablated crabs. Decrease in the levels of CHH was observed in both eyestalk intact and ablated crabs on heavy metal exposure. Decline of the hepatopancreas glycogen level was also witnessed with the exposure to heavy metal, which validated its utilization in the production of glucose. Thus this study brings to light, the variations in hemolymph glucose, CHH and hepatopancreas glycogen on heavy metal stress. These carbohydrate metabolites can be used as biomarkers in assessing heavy metal contamination in water bodies.     

日本蟳酚氧化酶的分离纯化及其部分生物化学性质研究

利用离子交换层析和凝胶过滤层析等方法,从日本(Charybdis japonica)血淋巴中分离纯化出了酚氧化酶,并以L-二羟苯丙氨酸(L-DOPA)作为特异性底物对其生化性质和酶性质进行了研究。结果表明,酚氧化酶和酚氧化酶原的分子质量分别为64.5 ku和69.5 ku。以L-DOPA为底物对酚氧化酶纯品进行研究发现,其最适pH值为6.0、最适温度为40℃。对底物L-DOPA和儿茶酚的米氏常数Km值分别为3.41和7.97 mmol/L。该酶对亚硫酸钠、苯硫脲极为敏感,对硫脲、苯甲酸非常敏感,表明该酶很可能是一种儿茶酚酶型的酶。此外,EDTA,DETC,Zn2 ,Mg2 和Cu2 均能显著抑制该酶活性,且10 mmol/L Cu2 能有效地回复该酶被DETC所抑制的酶活性,表明该酶确为一种Cu-金属酶。     

日本蟳捕食栉孔扇贝机制的初步研究

在现场和室内条件下研究了日本蟳(Charybdis japonica)对栉孔扇贝(Chlamys farreri)的捕食机制。现场实验表明,成年日本蟳可以捕食壳高小于5.0 cm的栉孔扇贝,其捕食强度随着水温的升高而增大,而壳高大于5.9 cm的栉孔扇贝则可以免遭日本蟳的捕食;相对于栉孔扇贝,日本蟳更倾向于捕食贻贝;室内实验表明水温低于10℃时,日本蟳对大规格扇贝的捕食作用不明显。相同温度条件下,室内实验日本蟳的捕食强度要低于现场,但其温度系数(Q10)差别不大。在上述实验基础上,本文提出了提高底播栉孔扇贝成活率的方法,即选择大规格的扇贝在水温较低的秋、冬季进行底播,同时添加一部分贻贝作为牺牲诱饵以减少日本蟳对扇贝的捕食。     

Cadmium acclimation and limb regeneration in the fiddler crab, Uca pugilator: Sex differences

After multiple autotomy in the fiddler crab, Uca pugilator, cadmium retards limb regeneration and ecdysis. To study the effects of pre-exposure to lower levels of cadmium, groups were pre-exposed to 0·1, 0·5 or 1·0 mg liter^?1 for a week prior to autotomy. Crabs were then placed in 1·0 mg liter^?1 for regeneration. Pre-exposed groups were compared with groups of crabs which had not been pre-exposed to Cd. It was found that males pre-exposed to 0·5 mg liter^?1 regenerated more rapidly in 1·0 mg liter^?1 than those not pre-exposed. This phenomenon was not observed in females, in which none of the three pre-exposure concentrations were able to accelerate regeneration in 1·0 mg liter^?1 relative to those not pre-exposed. In the pre-exposed males in which regeneration was accelerated and was comparable to control levels, no accompanying decrease of time until molting was observed. Therefore, the protective effect of pre-exposure would seem to be on the regeneration processes themselves and is not dependent on the neuroendocrine system which controls the molt cycle.     

杂色鲍血蓝蛋白多克隆抗体的制备与血蓝蛋白35kDa片段鉴定

以杂色鲍(Haliotis diversicolor Reeve)为研究对象,通过密度梯度离心方法纯化得到高纯度血蓝蛋白,以其作为抗原皮下注射免疫新西兰大白兔,从而获得高效价的兔源多克隆抗血清。进一步通过Protein A抗原亲和纯化的方法对该抗血清纯化,最终获得效价更高、检测特异性更好的血蓝蛋白多克隆抗体。应用该抗体进行Western检测发现,鲍血淋巴中存在着多样的血蓝蛋白衍生产物;进一步结合质谱技术对其中35 kDa条带进行鉴定发现,其来源于血蓝蛋白I型亚基的H结构域。     

红海湾远海梭子蟹Portunus pelagicus的食物组成及营养位置分析

利用稳定同位素技术;分析了2015年5月在红海湾海域采集的远海梭子蟹Portunus pelagicus及其潜在食物的碳（δ13C）和氮（δ15N）稳定同位素比值;通过稳定同位素混合模型（SIAR）;估算不同食物对远海梭子蟹食性的相对贡献。结果表明:远海梭子蟹的δ13C值在-18.1‰~-14.1‰之间;δ15N值的范围为9.8‰~13.8‰;平均值分别为-16.9‰±1.3‰和12.1‰±2.3‰。δ13C和δ15N值的变化范围均较大;表明远海梭子蟹食物来源广泛、多样。远海梭子蟹的食物主要由双壳贝类、螺类、鱼类、头足类、虾类、蟹类和藻类组成。SIAR模型计算结果显示:藻类为远海梭子蟹的主要食物;其平均贡献为32.5%;双壳贝类的相对食性贡献为21.9%;虾类、螺类、鱼类和蟹类的平均贡献差异较小;分别为9.7%、9.7%、9.6%和9.3%;头足类的贡献最低;仅为7.3%。此外;根据δ15N值及营养位置的计算公式得出:远海梭子蟹在红海湾海域位于2.91±0.68营养级。远海梭子蟹的7类潜在食物中;藻类的营养位置最低;为1.21±0.58;其次为双壳贝类;为2.00±0.25;蟹类、螺类、虾类和鱼类的营养位置分别为2.85±0.33、2.87±0.26、3.01±0.16和3.08±0.18;头足类的最高;为3.41±0.17。     

Cadmium accumulation,distribution and elimination in the bivalve Macoma balthica: Neither metallothionein nor metallothionein-like proteins are involved

Accumulation and elimination of cadmium was investigated in the tellinid clam, Macoma balthica (L.). Accumulation of Cd was linear in soft tissues (0·35 μg g⁻¹ day⁻¹ dry weight) throughout a 29-day exposure period at 100 μg Cd litre⁻¹. Loss of Cd, measured after transfer of experimentally-contaminated clams to clean seawater, was exponential and relatively slow (biological half-time, 70 days). In contrast, Cd uptake onto the shell of Macoma was characteristic of saturation kinetics, indicative of physical adsorption processes. Amounts of Cd accumulated by the shell were low, and the elimination rate rapid (half-time, 7 days) compared with soft tissues.Gel chromatographic profiles of cytosol extracts from control and experimental groups of M. balthica revealed that most of the Cd was bound to high molecular weight ligands, an unusual feature in bivalve molluscs. Although the proportion of cytosolic Cd associated with very low molecular weight ligands is small (< 15%) this pool may be important in regulating Cd fluxes in Macoma during uptake and elimination phases. No evidence was found of metallothionein (or similar metal-binding protein) involvement in Cd accumulation and storage in either field or laboratory exposed clams.The metabolism of Cd in M. balthica is different from that reported for other bivalve groups in several respects. In particular, the relatively slow rate of Cd accumulation in Macoma may be an important adaptive advantage for survival in contaminated areas and may compensate for the absence of a recognised detoxifying (MT) system in this species.     

Cadmium uptake and accumulation by the decapod crustacean Penaeus indicus

Juveniles of the dendrobranchiate decapod Penaeus indicus take up radiolabelled cadmium from solution over the exposure concentration range of 1.8-31.5 microg L(-1), with an uptake rate constant of 0.090 L g(-1)d(-1) at 15 salinity and 25 degrees C. New cadmium taken up is added to the existing cadmium content of the prawn with no significant excretion, and the rate of accumulation of radiolabelled cadmium is a measure of the absolute cadmium uptake rate from solution. Moulting had no significant effect on the accumulation of cadmium. Newly accumulated cadmium is distributed to all organs with the highest proportions of body content being found in the hepatopancreas, exoskeleton, gills and remaining soft tissues, the hepatopancreas and gills containing the highest labelled cadmium concentrations. Like other crustaceans, penaeid prawns inhabiting anthropogenically contaminated coastal waters with raised cadmium bioavailabilities can be expected to contain raised body concentrations of cadmium. Cadmium concentrations of most field-collected adult penaeids are relatively low, as a probable consequence of the growth dilution of their cadmium contents as a result of the rapid growth rates of penaeid prawns.     

Gill fouling and parasitism in the rock crab, Cancer irroratus say

Rock crabs, Cancer irroratus, were examined grossly and microscopically to estimate the influence of molting activity, geographical source and seabottom sediments on gill color, carapace color and tissue pathology. Collections were made from deepwater stations (20–55 m) in the Hudson Shelf Valley near dredge- and sewage-sludge disposal sites in the New York Bight apex, and from nearshore waters near Boothbay Harbor, Maine. Extensive gill blackening was noted in up to 30 % of the crabs from the New York stations when all specimens were in the intermolt condition. Blackening was not noted in intermolt crabs collected in Maine. Carapace blackening was present in up to 79 % of the New York crabs but absent in those from Maine. Severe blackening was attributed to direct contact of Cancer irroratus with blackened, highly organic sediments in the Hudson Shelf Valley. Fouling microorganisms on gill epicuticle included bacteria, diatoms, stalked ciliates, amoebae, flagellates and copepods. Bacteria, diatoms, amoebae and flagellates were common on crab gills from both locations. Ciliates and copepods counted in histological sections were considerably fewer in crabs from the New York stations than in those from Maine. Melanized nodules (hemocyte clumps) were present in gill tissue in up to 58 % of the New York crabs and in up to 68 % of Maine crabs. Unidentified microsporidans were found in up to 55 % of the New York specimens and in less than 2 % of those from Maine. Histological data accumulated since 1975 are summarized to provide a list of endoparasites, polychaete worms and barnacles observed in Cancer irroratus from the northeastern United States. Gill blackening, but not parasitism or microbial fouling, proved to be the best indicator or monitoring the effects of ocean dumping on the health of a benthic crustacean species.     

锯缘青蟹大颚器的显微和超微结构

采用组织切片和电镜技术,对锯缘青蟹大颚器的形态结构进行了观察.大颚器位于大颚肌几丁质腱的后侧基部.腺细胞胞质丰盈,质膜大量内陷和作不同程度的卷绕,内含大量具管状脊的线粒体和滑面内质网.卵巢成熟时大颚器腺细胞内线粒体增多,少数线粒体形态异样,高尔基体易见,散布的小管状滑面内质网(tSER)少,高度汇合的tSER增多,大小形状不同的空泡数量明显增加,且近质膜分布,质膜内陷和卷绕程度加剧.表明此时锯缘青蟹大颚器的合成与分泌能力增强,其生理功能与卵巢发育密切相关.     

Relationship between heavy metal accumulation and genetic variability decrease in the intertidal crab Pachygrapsus marmoratus (Decapoda; Grapsidae)

The “genetic erosion” hypothesis posits that heavy metal stress is related to a loss of genetic diversity at the population level. The genetic diversity of natural populations can, however, be affected by natural processes as well as by human impact. We studied the relationship between heavy metal bioaccumulation and genetic variability in the intertidal crab Pachygrapsus marmoratus. Tissue samples were collected from 40 individuals inhabiting four polluted and four unpolluted sites along the Tuscan coast (Mediterranean basin), and were examined for four heavy metals (arsenic, As, cadmium, Cd, lead, Pb, and copper, Cu). We also assessed the genetic variability of 235 crabs from the same localities using six microsatellite loci.Our results show that the bioaccumulation levels of these individuals accurately reflect the levels of pollution in their immediate environment, and that heavy metals accumulate more in the hepatopancreas than in the gills. Moreover, populations from polluted sites have significantly less genetic variability, measured as mean standardized d², and a significantly lower percentage of unrelated individuals, than populations from unpolluted sites. This evidence supports the “genetic erosion” hypothesis for metal heavy exposure in natural environments.