Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure

Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.     

Carbonyl reductase mRNA abundance and enzymatic activity as potential biomarkers of oxidative stress in marine fish

Carbonyl reductase (CBR) is an enzyme involved in protection from oxidative stress. In rainbow trout (Oncorhynchus mykiss), the hepatic mRNA abundance of the two isoforms (A and B) is increased after exposure to treated sewage effluents, as well as after exposure with β-naphthoflavone (β-NF) and the pro-oxidant paraquat. In this study, we show that the same chemicals similarly increase the single known hepatic CBR mRNA level and CBR catalytic activity in the coastal living eelpout (Zoarces viviparus). Hepatic CBR mRNA abundance and catalytic activity were also compared between eelpout collected at contaminated and reference sites on the Swedish west coast, but no differences were observed. In conclusion, CBR is a potential biomarker candidate for monitoring the exposure and effects of AhR agonists and/or pro-oxidants in the marine environment, but more research is needed to investigate temporal regulation as well as dose dependency for different chemicals. The mRNA and enzymatic assays presented in this study provide two additional tools for researchers interested in expanding their biomarker battery.     

Relationship between metallothionein, copper and zinc in perch (Perca fluviatilis) environmentally exposed to heavy metals

Hepatic levels of Cu, Zn and metallothionein (MT) in perch, caught in a Cu/Zn gradient from a brassworks, reflected the water concentration of Cu (1·0–9·4 ppb) and Zn (0·56–59 ppb). Significant correlations were found between hepatic Cu and MT levels (r = 0·72), and between Zn and MT levels (r = 0·69). There was an increase of the amount of Cu and Zn in the cytosolic fraction of the liver with increased hepatic levels of the metals. When liver samples. from perch caught at the most contaminated location, were run on a gel filtration column (Sephadex G-75) 78% of the cytosolic Cu and 24% of the Zn in the cytosol eluted together with MT.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium

Although the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5 +/- 0.5 cm and large: 5.2 +/- 0.7 cm) were exposed to Cd (100 microg l(-1)) at different water temperatures (5, 18 and 25 degrees C) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 degrees C) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 degrees C, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 degrees C there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E(2)) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E(2) and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E(2) and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Character and distribution of exposed glaciodeltaic deposits off outer Cape Cod, Massachusetts, and their effects on hydrogeology and benthic habitats

Seabed outcrops of glaciodeltaic sediments were identified in four places east of Cape Cod, Massachusetts, during seismic-reflection, multibeam bathymetric and backscatter, bottom photographic, and sediment sampling surveys. These strata record coarser-grained ice-proximal glaciofluvial topset to finer-grained distal glaciolacustrine bottomset deposition within deltaic systems that prograded southwestward into glacial lakes from the South Channel lobe about 18 ka b.p. These beds are important because they (1) influence the outer Cape’s hydrogeologic framework, and (2) constitute relatively stable, locally rough habitats within an area of seafloor dominated by mobile sand and gravelly sediment, and benefit the benthic fauna by providing shelter and a substrate amenable to burrow construction.     

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goks?yr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E₂) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E₂ and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E₂ and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

厦门湾红树林生境适宜性评估及修复潜力分析

为定量评估多元环境因子对红树林分布的影响,探索区域红树林生境适宜性和修复潜力的空间分布格局,本研究应用最大熵模型评估厦门湾红树林生境适宜性,叠加土地利用/覆被数据分析红树林修复潜力。结果表明,在生物气候、地形、水质、沉积物、水文等5组环境变量中,影响厦门湾研究区内红树林分布的主要环境变量类型为温度、地形和水质。在单项环境因子中,离岸线距离、营养盐浓度、盐度等对厦门湾红树林生境适宜性的贡献度最大。适宜性和修复潜力较高的区域主要分布于九龙江河口、翔安下潭尾、海沧东屿等区域。与现存红树林的分布相对照,理论上还有约406.4 hm^2的高修复潜力区和1001.2 hm^2的中修复潜力区可考虑用于未来红树林修复。现有的保护区和修复工程已基本覆盖了上述大部分区域,未来厦门湾内的红树林修复选址可考虑泉州市安海湾沿岸、龙海市九龙江河口、漳州港沿岸和附近岛屿。本研究尝试采用新兴模型方法判断红树林的综合适生条件,并实现空间化、定量化的评估,其结果可为红树林修复选址提供重要的科学依据,并为区域红树林保护与修复管理提供决策支持。     

Survival, growth, metallothionein and glycogen levels of Nucella lapillus (L.) exposed to subchronic cadmium stress: the influence of nutritional state and prey type

Dogwhelks Nucella lapillus feed mainly on mussels and barnacles, and may experience periods of starvation. We report effects of nutritional state and prey type on the survival, growth, cadmium (Cd) accumulation, metallothionein (MT) induction and glycogen stores in N. lapillus exposed to Cd in water. Adult dogwhelks, with similar shell length (30.0+/-1.5 mm), were either starved or fed to satiation with barnacles Semibalanus balanoides, mussels Mytilus edulis or Cd-dosed M. edulis, and kept in filtered natural seawater (< 0.01 microg Cd 1(-1)) or Cd-contaminated (400 microg Cd 1(-1)) seawater for 80 days. Mortality and individual growth rate were determined. Cd, MT and glycogen were measured in different tissues. Prolonged starvation and exposure to Cd significantly reduced the survivorship of N. lapillus, but feeding could help dogwhelks to combat Cd toxicity and minimise mortality. Extended starvation also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of dogwhelks and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves than eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. The use of growth, condition index, MT and glycogen as biomarkers of environmental pollution are discussed. These results indicate a need to incorporate biological data including growth (or at least condition index) and prey type into biomonitoring programmes to allow sound interpretation.     

伶仃洋海域潜在性富营养化评价与研究

根据2006年7月至2007年12月的调查资料,一年四季分析珠江口伶仃洋海域营养盐的空间分布特征及季节变化,该海域DIP表层年平均值为0.054mg/L,底层年平均值为0.53mg/L,DIN表层平均值为1.20mg/L,底层平均值为1.03mg/L;采用近海海域水体潜在性富营养化等级的划分标准和评价方法进行评价,评价结果表明,伶仃洋海域营养化水平基本上为磷限制潜在性富营养化至III级富营养水平。     

Rapid narcosis and delayed mortality in larvae of king crabs and kelp shrimp exposed to the water-soluble fraction of crude oil

Sensitivity of larval Paralithodes camtschatica and Eualus suckleyi exposed 20 min–96 h to the water-soluble fraction (WSF) of crude oil was determined. Swimming cessation in half the animals occurred within 20 minutes' exposure to 2 ppm WSF for shrimp larvae and only 0·5 ppm for crab larvae. Half the larvae of both species died after 6 hours' exposure to 8 ppm or 24 hours' exposure to 2–4 ppm, but deaths did not occur until several days after exposure ended. In the field, most non-swimming larvae would probably die as well.     

Biodiversity and seasonal variation of benthic macrofauna in Minicoy Island, Lakshadweep, India

From the Indian coast only limited data are available on the benthic fauna of the seagrass communities. In this study, seasonal variation in the distribution of macrobenthos and influence of environmental parameters was explored at four seagrass beds and two mangrove stations along the Minicoy Island, Lakshadweep, India, from September 1999 to August 2001. A total of 160 macrobenthic species from eight major groups represented the macrofauna of the Minicoy Island. Of the identified taxa, molluscs 70（gastropods 41.46%, bivalves 7.5%）, polychaetes 27（16.88%）, crustaceans 30（18.75%）, echinoderms 11（6.88%） and remaining others. Average seasonal abundance of benthic macrofauna ranged from 219 to 711 ind./m2, species diversity varied from 1.45 to 3.64 bits per individual, species richness index ranged from 4.01 to 26.17, evenness 0.69 to 1.66. In general, the higher abundance and species diversity was noticed in southern seagrass stations and northern seagrass stations, but in the mangrove stations comparatively low species diversity was observed. Three-way analysis of variance indicated that all communities resulted as being significantly different between seagrass and mangrove station, mainly when the seasonal interaction was considered. Multivariate analyses were employed to help define benthic characteristic and the relationship between environmental parameters at the six monitoring stations. Results of cluster analyses and multidimensional scale plot suggest that for mangrove region, different physiographic provinces, lower salinity, dissolved oxygen and sediment biotic structure have a higher influence on the species composition and diversity than other oceanographic conditions.     

Influence of cadmium exposure on plasma calcium, vitellogenin and calcitonin in vitellogenic rainbow trout

Effects of cadmium exposure on plasma levels of calcitonin and free and protein-bound calcium were studied in vitellogenic female rainbow trout kept in brackish water (7%(.)). Fish were exposed to 100 μg cadmium litre⁻¹ for four weeks. Exposure of female rainbow trout in the stage of vitellogenesis, with increased total plasma levels of calcium, resulted in a complex hypocalcemic response. Thus, hypocalcemia was found to be due to three different processes: (1) a decrease in the free plasma calcium, and a reduction in protein-bound calcium; due both to (2) decreased plasma levels of vitellogenin; and (3) a reduced binding of calcium to vitellogenin. These findings support the concept of an interference of cadmium with ionregulating tissues as a mechanism for hypocalcemia in rainbow trout. A direct effect on the vitellogenin-binding of calcium was also observed and reproductive function in the females was affected by decreased plasma levels of vitellogenin. In spite of the marked changes of plasma calcium in exposed fish, no significant effects on plasma calcitonin were observed, indicating a lack of a direct relationship between plasma calcium and calcitonin levels in rainbow trout.     

Distribution of larvae ofPinnixa rathbuni Sakai (Decapoda: pinnotheridae) in Ise Bay and its neighbouring coastal waters,central Japan part 3. Aggregation of the benthic adult population with reference to the spatial distribution of the planktonic larvae

According to studies made to date, the planktonic larvae ofPinnixa rathbuni, especially the early zoeal stages, have been found mainly in the eastern part of Ise Bay (Pacific coast of central Japan), while the benthic adult population is restricted to the southwestern part of the bay. The present study was made to obtain a better understanding of the origin of the dense population of early zoea found along the coast of the Chita Peninsula in the eastern part of the bay. Field surveys made with a Smith-McIntyre grab from May to November 1981 (every two months) ruled out the possibility that the benthic adult crabs in the southwestern part of the bay migrated to the eastern part of the bay to release their larvae, or that a substantial population of the crabs occurred in the eastern part of the bay. Thus, the larvae may be derived wholly from the benthic adult populations localized in the southwestern part of the bay. The spatial distribution of the early larvae can be explained by assuming that the larvae, released by the benthic adult population in the southwestern part of the bay, are rapidly transported toward the Chita Peninsula and form a dense larval population along the coast in the eastern part of the bay.     

Mercury pollution in sediments,benthic organisms and inshore fishes of Haifa Bay,Israel

Total mercury concentrations were determined in surficial sediments, eleven species of benthic organisms and six species of fish from Haifa Bay, Israel. The results show that essentially all of the shallow water zone of the Bay receives anthropogenic mercury. A mercury-cell chlor-alkali plant was identified as the source of pollution. Surficial sediments in the vicinity of the plant, containing up to 0·99 μg Hg/g dry weight, were up to 157 times enriched in mercury relative to an unpolluted area. Mercury levels in the benthic organisms reflected the levels in the sediments. Maximal concentrations reaching 38·7 and 18·2 μg Hg/g dry weight were found in the carnivorous gastropod molluscs Arcularia circumcinta and Arcularia gibbosula, respectively. In all fish species, specimens caught in Haifa Bay had higher mercury concentrations in the muscle tissue than specimens caught south of the Bay. A maximal value of 1·66 μg Hg/g wet weight was recorded in Diplodus sargus.