Sex identification of Carcinus maenas by analysis of carapace geometrical morphometry

The European green crab Carcinus maenas is a widely spread invasive species. Studying the most recently introduced population of the species in the Atlantic coast off Patagonia, it became apparent to us that carapaces (the dorsal cover of the visceral-cavity detaching from the rest of the exoskeleton after ecdysis) accumulated on the intertidal may be used as a valuable complementary material to analyze the size structure of crabs. However, since growth rate (and consequently size at instar) of crabs differs between males and females, finding a method to distinguish crab sex by observation of carapace morphology was necessary to allow the construction of independent size frequency distributions for each sex. In this work, we examined the shape of the carapaces from both sexes of C. maenas, and using Elliptical Fourier Analysis successfully identified sexual dimorphism. Thus, a reliable method to identify sexes by visual observation of the carapace was developed. Based on our results, we discuss the evolutionary significance of carapace form differentiation of both sexes.     

Toxicokinetics of heavy metals from a mining spill using Carcinus maenas

The knowledge of the reproduction and growth background related to the shore crab Carcinus maenas promotes the use of this crab as a model crustacean to asses the potential for endocrine disruption in crustaceans. In addition, an enzyme linked inmunosorbent assay (ELISA), sensitive to the shore crab vitellogenin in serial hemolymph samples allows determination of the extent of disruption of the process of vitellogenesis in female crabs and its likely impact on reproductive output. Intermoult females Carcinus maenas were exposed to concentrations of Cd: 3 microgl(-1), Cu: 15 microgl(-1) and Zn: 700 microgl(-1) determined at the Guadalquivir estuary after the Aznalcóllar mining spill, during 21 days. Crab hemolymph samples, were taken every seven days, and analyzed through an ELISA for Carcinus maenas vitellogenin. Vitellogenin concentration along the time was fitted to a first order kinetic approach. Results showed a good correlation among experimental values and estimated ones. Metal exposure resulted in an increase in vitellogenin concentration in hemolymph, especially for cadmium.     

Cypermethrin induces glutathione S-transferase activity in the shore crab,Carcinus maenas

Cypermethrin is a synthetic pyrethroid that is particularly toxic to crustacea. It is therefore applied as a chemotherapeutant in the salmonid aquaculture industry for the treatment of sea lice infestations. After use, cypermethrin is released directly into the marine environment, to be diluted by fresh seawater. The shore crab, Carcinus maenas is found in the vicinity of fish farms, and may come into contact with released cypermethrin. The detoxification enzyme, glutathione S-transferase (GST) has been implicated in cypermethrin metabolism in terrestrial arthropods, but this has not yet been demonstrated in crustacea. In this paper we investigate the response of GST activity in Carcinus to cypermethrin exposure, and also the time course of the induction process. GST activity is significantly increased in Carcinus exposed to nominal concentrations of 50 and 500 ng/l of water-borne cypermethrin. Carcinus demonstrate a significant elevation in GST activity following intra-cephalothoracic injection with 10 ng of cypermethrin. GST activity returns to basal levels after 36 h. The potential application of GST activity in Carcinus as a biomarker of cypermethrin exposure is discussed.     

Reassessing the invasion of South African waters by the European shore-crab Carcinus maenas

The European shore-crab Carcinus maenas has been present in South Africa since 1983. Despite this species’ international reputation as a biological invader, its distribution in this region has only been considered by three outdated ‘snapshot surveys.’ The present study is the most comprehensive to date, providing an update on the species’ range and the first temporal assessment of its abundance and demographics. Along South Africa's Cape Peninsula and surrounding areas, C. maenas was absent from 12 intertidal sites surveyed, except for Sea Point, and no crabs were found during subtidal surveys along the open coastline. Subtidal harbour populations were recorded in the Cape Town harbours of Table Bay and Hout Bay (previously estimated as comprising approximately 164 200 and 6 500 individuals, respectively). Table Bay was surveyed monthly for one year, using baited traps, crab condos and postlarvae settlement collectors, to assess size distributions and reproductive seasonality of the crab. Reproductive females were recorded throughout most of the year. These results suggest that the harbour populations could be targeted by control programmes, but provide no strong evidence to support the initiation of management action during a particular season. The lack of detection of postlarval settlement, even among well-established populations, suggests this will not be a useful monitoring tool for detecting incursions.     

Physiological responses of juvenile and adult shore crabs Carcinus maenas (Crustacea: Decapoda) to pyrene exposure

This study measured aspects of the physiology of juvenile [(<35mm carapace width (CW)] and adult (>60mm CW) Carcinus maenas to test the hypothesis that these different life-history stages exhibit different sensitivities to environmental contamination. Newly-collected juveniles had significantly (P<0.05) lower immunocompetence (phagocytosis and cellular integrity), lower metabolic energy (haemolymph glucose) and increased scope for growth compared with adults. Seven day exposure to a sub-lethal concentration (200microgL(-1)) of pyrene significantly (P<0.01) reduced immunocompetence, elevated basal heart rate and decreased respiration (at rest) for juveniles but had no overall impact on adult crab physiology. Results confirm that physiological differences exist between juvenile and adult shore crabs, and cause juveniles to be more susceptible to the effects of pyrene exposure than adults. Such differences in sensitivity to contamination between life-cycle stages of the same species have to be taken into account during the risk assessment process.     

Acute response versus reproductive performance in five strains of brine shrimp exposed to copper sulphate

Five geographically and genetically diverse strains of the brine shrimp, Artemia salina, were tested for response to copper sulphate. Mean survival times (MST) were determined for each strain following continuous exposure to concentrations of 10^?1, 1, 10¹, 10², 10³, 10⁴ and 10⁵ ppm CuSO₄. In addition, lifetime reproductive performance parameters were evaluated for 15 females from each strain after 24 h exposure to 0·067 × 10^?9 CuSO₄.Two-factorial ANOVA revealed that treated females produced fewer offspring per brood and fewer broods per female, resulting in an overall decline in the mean number of offspring per day per female over the reproductive span. When the standard 24 h MST results are compared with the 24 h dose level of CuSO₄ found to impair reproductive performance, the latter was from 24,000 to 156,000 times more sensitive, depending upon strain.     

Sex differences in body morphology and multitrophic interactions involving the foraging behaviour of the crab Carcinus maenas

Many animals are sexually dimorphic, but the underlying evolutionary causes and ecological consequences of sexual dimorphism are not fully understood. One predicted consequence for sexual dimorphism is that different sexes show niche differentiation. If sexual dimorphism is in feeding appendages, then differences may be manifested by different diets and thus contrasting behavioural responses to potential prey. Sexual dimorphisms in feeding appendages may also result in different handling times, which may then be correlated with differences in exposure, and, hence predation risk to the predator while feeding. In addition, the prey of the sexually dimorphic predator may respond differently to cues from each sex according to the predation risk each presents to the prey. We tested these predictions using a crab (Carcinus maenas) with sexual dimorphism in chelae dimensions, its predator the cuttlefish Sepia officinalis and prey with known differences in handling times; the gastropod molluscs Gibbula umbilicalis and Littorina littorea. We demonstrated that male C. maenas orientated more frequently to cues from L. littorea whereas females orientated more towards G. umbilicalis in contradiction of patterns predicted by handling times. Male crabs had a faster heart rate than females but this was not influenced by food‐based cues. We also showed no difference in foraging times with respect to changing levels of predator risk and also no differences in gastropod responses to odours from male or female crabs. Our results showed that predictions of handling time and sexual dimorphism are not associated. The experiments indicated the male and female crabs are probably ecological equivalents and thus niche differentiation is less likely.     

Effects of habitat complexity on cannibalism rates in European green crabs (Carcinus maenas Linnaeus, 1758)

The habitat in which predator–prey interactions take place may have a profound influence on the outcome of those interactions. Cannibalism is an intriguing form of predation whereby foraging by predators may contribute to the regulation of their own populations.This is particularly interesting in the case of invasive species, like the widely distributed European green crab (Carcinus maenas). This study explores how habitat complexity influences cannibalism rates in green crab populations of Prince Edward Island, Atlantic Canada. Both laboratory and field experiments were conducted to measure feeding rates by individual adult green crabs on a standard number of smaller conspecifics. In the laboratory, experimental treatments mimicked unstructured to increasingly structured habitats: water, sandy bottom, oyster shells, mussel shells, oyster shells with sandy bottom and mussel shells with sandy bottom. In those trials, adult green crabs consumed several times more juveniles on unstructured habitats than on the most structured ones, with a gradual decrease in predation rates across increasingly complex habitats. Field inclusion experiments used the same approach and were conducted in sandy bottoms, sandy bottoms with a layer of oyster shells and sandy bottoms with a layer of mussel shells. These trials showed similar patterns of decreasing feeding rates across increasingly complex habitats, but differences among treatments were not significant. These results support the idea that complex habitats have the potential to mediate predator–prey interactions, including adult–juvenile cannibalism in green crabs.     

Partitioning of copper among copper-binding proteins in the mussel Mytilus edulis exposed to soluble copper

Partitioning of copper among copper-binding proteins was evaluated in digestive glands of Mytilus edulis exposed to soluble copper. Groups of mussels were held in flow-through bioassay systems and exposed to 25 μg Cu liter^?1 for up to 21 weeks. At three-week intervals, groups of 25 mussels were removed and the digestive glands were analyzed for copper-binding proteins by gel-permeation chromatography and atomic absorption spectrometry.Chronic exposure to copper resulted in increased amounts of copper in the low molecular weight (LMW) protein fraction, which contains metallothioneins, and in the high molecular weight (HMW) protein fraction, which contains metalloenzymes. Concentrations of copper in the LMW protein fraction increased and then appeared to plateau with long exposure times, whereas those in the HMW protein fraction continued to increase with exposure time.     

Seasonal, Geographical and Size Differences in Oxygen Consumption, Digestive Gland and Gills in Carcinus maenas (L), a Study of Condition for Ecologists

Abstract. The excited rate of oxygen consumption, and the wet weights of the digestive gland and the gills were studied in relation to overall size. One experiment was made in Roscoff, France, one in Portaferry, N. Ireland, and three in Wicklow, Ireland. Higher levels of oxygen consumption were found in Roscoff together with greater gill weight. Seasonal changes in the level of oxygen consumption in Wicklow may be influenced by the dry wt/wet wt ratio of the gills - probably an index of condition for the crabs as a whole. The power functions for allometric growth were virtually constant for each factor in all five experiments. All levels of factors were reduced in males weighing more than 60 g. Disproportions in the weights of the chelae marked the populations in Roscoff and Portaferry, compared with Wicklow. These suggest a genetic basis for the geographical differences in oxygen consumption and gill weight. The importance of local experiments for local conditions and the possibilities for predicting physiological rates from morphology are discussed.     

Metabolic responses in gills of Manila clam Ruditapes philippinarum exposed to copper using NMR-based metabolomics

Copper is an important heavy metal contaminant with high ecological risk in the Bohai Sea. In this study, the metabolic responses in the bioindicator, Manila clam (Ruditapes philippinarum), to the environmentally relevant copper exposures were characterized using NMR-based metabolomics. The significant metabolic changes corresponding to copper exposures were related to osmolytes, intermediates of the Krebs cycle and amino acids, such as the increase in homarine, branched chain amino acids and decrease in succinate, alanine and dimethylamine in the copper-exposed clam gills during 96 h exposure period. Overall, Cu may lead to the disturbances in osmotic regulation and energy metabolism in clams during 96 h experimental period. These results demonstrate that NMR-based metabolomics is applicable for the discovery of metabolic biomarkers which could be used to elucidate the toxicological mechanisms of marine heavy metal contaminants.     

Differential response to ocean acidification in physiological traits of Concholepas concholepas populations

Phenotypic adaptation to environmental fluctuations frequently occurs by preexisting plasticity and its role as a major component of variation in physiological diversity is being widely recognized. Few studies have considered the change in phenotypic flexibility among geographic populations in marine calcifiers to ocean acidification projections, despite the fact that this type of study provides understanding about how the organism may respond to this chemical change in the ocean. We examined the geographic variation in CO₂ seawater concentrations in the phenotype and in the reaction norm of physiological traits using a laboratory mesocosm approach with short-term acclimation in two contrasting populations (Antofagasta and Calfuco) of the intertidal snail Concholepas concholepas. Our results show that elevated pCO₂ conditions increase standard metabolic rates in both populations of the snail juveniles, likely due to the higher energy cost of homeostasis. Juveniles of C. concholepas in the Calfuco (southern) population showed a lower increment of metabolic rate in high-pCO₂ environments concordant with a lesser gene expression of a heat shock protein with respect to the Antofagasta (northern) population. Combined these results indicate a negative effect of ocean acidification on whole-organism functioning of C. concholepas. Finally, the significant Population × pCO₂ level interaction in both studied traits indicates that there is variation between populations in response to high-pCO₂ conditions.     

Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations.

Protein expression and lysosomal stability were observed in Mytilus edulis exposed to 0, 20, 40, 60, and 80 ppb copper sulfate for 24 h in a static exposure system. Haemolymph was removed from each mussel and analyzed for lysosomal damage using the Neutral Red retention assay expressed as the ratio of lysosomal size to cell area, the lysosomal destabilization ratio. Gill tissue from the same individuals was dissected, homogenized and the proteins extracted and then separated using two-dimensional gel electrophoresis. Two-dimensional protein maps from each concentration were analyzed using MELANIE II 2D gel analysis software (Biorad). Lysosomal damage increased with each dosage, with LDRs ranging from 0.18 +/- 0.01 at 0 ppb copper sulfate to 0.32 +/- 0.11 at 80 ppb copper sulfate. Orthogonal comparisons showed that the control, 20 and 40 ppb classes together differed significantly (P < 0.05) from the 60 and 80 ppb classes together. Specific protein expression signatures were identified at each dosage.     

Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress.

Applied to environmental toxicology, proteome analysis may be used to isolate chemical-specific protein expression signatures (PES). In this project specific PES were isolated in mussels, Mytilus edulis, from the Baltic Sea subjected in the laboratory to treatment with copper (70 ppb), Aroclor 1248 (1 ppb), and to lowered salinity. Four mussels in each treatment group were acclimated in the laboratory for 24 h before beginning the 7-day exposure. Whole body tissue was homogenized and separated using two-dimensional gel electrophoresis. The protein gels were scanned to TIFF files and compared using MELANIE II 2D gel analysis software (BioRad). Protein expression signatures including proteins induced and repressed by exposure were isolated for each treatment group. The specificity of PES due to environmental changes shows promise in bioindication, toxicity testing and in helping identify possible toxicity mechanisms.     

Elucidating cellular and behavioural effects of contaminant impact (polycyclic aromatic hydrocarbons,PAHs) in both laboratory-exposed and field-collected shore crabs,Carcinus maenas (Crustacea: Decapoda)

Monitoring effects of contaminants at lower levels of biological organisation (e.g. biochemical and cellular) allows for mechanistic evaluation of effects of contaminant exposure through laboratory exposures. However, higher level organism effects (e.g. physiological and behavioural) are deemed more ecologically-relevant. In the present study, cellular (cell viability and immune function), physiological (cardiac activity) and behavioural (foraging behaviour) responses were evaluated in field-collected shore crabs Carcinus maenas from three estuaries [a ‘relatively’ (polycyclic aromatic hydrocarbon) PAH-contaminated site (Plym Estuary) and two ‘comparatively clean’ field sites (Avon and Yealm Estuaries)] and compared with responses of crabs exposed in the laboratory to a model organic contaminant (PAH) pyrene (200 μg l⁻¹ for 28 days). The hypothesis that behavioural indicators may be more sensitive than other more traditional methods was tested. No significant impacts were observed at the cellular or physiological level in Plym-collected crabs (the ‘relatively’ contaminated site), but foraging behaviour was significantly altered (increased prey handling time) compared to individuals collected from the ‘relatively’ uncontaminated sites (Avon and Yealm). When given a cockle as a prey item, both Plym-collected and laboratory-exposed crabs took longer to handle and break into cockle shells. Therefore, ecologically-relevant behavioural observations may serve as valuableindicators of environmental quality.     

A comparison between the physiological effects in fish exposed to lethal and sublethal concentrations of a dispersant and dispersed oil

The acute and sublethal effects of a dispersant and crude oil on anisosmotic and isosmotic regulation in flounder have been tested. Flounder were exposed to different concentrations of Corexit 9527 and crude oil by use of a biotest system. Fourteen days' exposure to 20 ppm of Corexit 9527, alone or in a 1:1 mixture with crude oil, had no effect on the blood parameters. On the other hand, 96 hours' exposure to 80 ppm of the same compounds led to 50 % mortality and significant effects on the blood parameters in surviving fish. The comparability between effects obtained in fish exposed to lethal and sublethal concentrations of toxicants, respectively, is discussed.     

Differences in physiological response to increased seawater temperature in nearshore and offshore corals in northern Vietnam

Effects of elevated seawater temperature show high spatial heterogeneity and variation within and among coral species. The objective of this study was to investigate how two coral species, Porites lutea and Galaxea fascicularis, from two high latitude reefs differently exposed to chronic disturbance, respond to elevated seawater temperatures. Corals were collected from reefs nearshore (i.e. subjected to high sediment load, higher chlorophyll α concentrations, turbidity etc.) and offshore (i.e. less exposed). The corals were exposed in the lab to gradually increasing temperatures (25.5-33.5?°C) for 72?h after which they were allowed to recover to ambient temperature (25.5?°C) for 24?h. Production and respiration were measured after 24, 48, 72 and 96?h. The results show that P. lutea from nearshore reefs suffered an initial decrease in gross primary production/respiration (GP/R) ratio after 24?h, after only a moderate temperature increase (+2?°C, from 25.5 to 27.5?°C), while there was no difference in GP/R ratio between heat-exposed and controls the other days, indicating that the chronic disturbance in the nearshore reef had no effect on their thermotolerance. Furthermore, P. lutea from the offshore reef showed a decrease in GP/R ratio both after 24?h and 72?h (33.5?°C) of exposure. In comparison, G. fascicularis showed a decrease in GP/R ratio after 48?h, 72?h and 96?h of exposure for the nearshore corals. Also, after 72?h these corals had withdrawn their polyps. There were no differences between heat-treated and controls for the offshore G. fascicularis. This implies that the chronically disturbed G. fascicularis had lower thermotolerance when exposed to a temperature increase. This study, hence, shows that the response of corals to elevated seawater temperature varies with species and environmental background history.     

Different cardiac response to copper in limpets from metal polluted and clean shores of Hong Kong

The gastropod limpet, Cellana grata, is common on moderately exposed to exposed Hong Kong rocky shores. On the basis of the existing literature on trace metal contamination in Hong Kong, four sites were identified, representative of highly polluted (Aberdeen and Lei Yue Mun) and clean (Cape d'Aguilar and Clear Water Bay) conditions. Limpets from the two polluted sites showed similar, but significantly higher body concentrations of copper than limpets from the two clean sites, which were also similar. Non-invasive measurements of cardiac activity of limpets from the four sites showed between site differences in baseline heart rates under standard seawater, irrespective of pollution level. When acutely exposed to water borne copper (2 h, 0.5+/-0.06 SD ppm), however, limpets from clean sites showed a significantly higher increase in inter-beating time (bradycardia) than those from polluted sites. These results highlight the potential use of cardiac activity to assess the exposure of natural populations of limpets to trace metal pollution.     

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.