Influences of dietary vitamin D3 on growth, antioxidant capacity, immunity and molting of Chinese mitten crab (Eriocheir sinensis) larvae.

This study investigates the effects of vitamin D3 (VD3) on growth performance, antioxidant capacity, immunity and molting of larval Chinese mitten crab Eriocheir sinensis. A total of 6,000 larvae (7.52 ± 0.10 mg) were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary VD3 (0, 3000, 6000, 9000, 12000 and 36000 IU/kg) respectively for 23 days. The highest survival and molting frequency were found in crabs fed 6000 IU/kg VD3. Weight gain, specific growth rate, and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg VD3 compared to the control. Broken-line analysis of molting frequency, weight gain and specific growth rate against dietary VD3 levels indicates that the optimal VD3 requirement for larval crabs is 4825-5918 IU/kg. The highest whole-body VD3 content occurred in the 12000 IU/kg VD3 group, and the 25-dihydroxy VD3 content decreased with the increase of dietary VD3. The malonaldehyde content was lower than the control. Moreover, the superoxide dismutase activity, glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg VD3 were significantly higher than in control. Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress, and the relative mRNA expressions indicate vitamin D receptor (VDR) is the important regulatory element in molting and innate immunity. The molting-related gene expressions showed that the response of crab to salinity was self-protective. This study would contribute to a new understanding of the molecular basis underlying molting and innate immunity regulation by vitamin D3 in E. sinensis.

Dietary soybean oil aggravates the adverse effects of low salinity on intestinal health in juvenile mud crab Scylla paramamosain.

Salinity is one of the important factors affecting the physiological state of crustaceans in marine environments. Lipid plays major roles in energy supply and is main sources of essential fatty acids for membrane integrity, which is critical in adaptations to changes in salinity. Here we evaluated the effects of salinity (medium, 23 ppt and low, 4 ppt) and dietary lipid source (fish oil, FO and soybean oil, SO) on intestinal health of the marine crustacean mud crab Scylla paramamosain. The results indicated that low salinity and dietary SO (LSO group) significantly affected intestinal histomorphology, with a significant decrease of intestinal fold height and width as well as down-regulation of intestinal mRNA levels of tight junction genes compared to crab reared at medium salinity and fed FO diets (MFO group). Crabs reared at low salinity and fed SO showed an increased inflammatory response in intestine, which stimulated a physiological detoxification response together with apoptosis compared to crab in the MFO group. Low salinity and SO diets also could be responsible for multiply the pathogenic bacteria of Photobacterium and inhibit the beneficial bacteria of Firmicutes and Rhodobacteraceae in intestine, and act on a crucial impact on the development of intestinal microbial barrier disorders. The results of microbial function predictive analysis also support these inferences. The findings of the present study demonstrated that soybean oil as the main dietary lipid source could exacerbate the adverse effects of low salinity on intestinal health of mud crab, and provided evidence suggesting that dietary lipid source and fatty acid composition may play vital roles in intestinal health and the process of adaptation to environmental salinity in marine crustaceans.

Evidences for Red Pigment Concentrating Hormone (RPCH) and Beta-Pigment Dispersing Hormone (ß-PDH) Inducing Oocyte Meiotic Maturation in the Chinese Mitten Crab, Eriocheir sinensis.

Red pigment concentrating hormone (RPCH) and pigment dispersing hormone (PDH) are crustacean neuropeptides involved in broad physiological processes including body color changes, circadian rhythm, and ovarian growth. In this study, the full-length cDNA of RPCH and PDH were identified from the brain of the Chinese mitten crab Eriocheir sinensis. The deduced RPCH and PDH mature peptides shared identical sequence to the adipokinetic hormone/RPCH peptides family and the ß-PDH isoforms and were designated as Es-RPCH and Es-ß-PDH, respectively. Es-RPCH and Es-ß-PDH transcripts were distributed in the brain and eyestalks. The positive signals of Es-RPCH and Es-ß-PDH were localized in the neuronal clusters 6, 8, 9, 10, and 17 of the brain as revealed by in situ hybridization. The expression level of Es-RPCH and Es-ß-PDH mRNA in nervous tissues were all significantly increased at vitellogenic stage, and then decreased at the final meiotic maturation stage. The administrated with synthesized Es-RPCH peptide results in germinal vesicles shift toward the plasma membrane in vitellogenic oocyte, and significant decrease of the gonad-somatic index (GSI) and mean oocyte diameter as well as the expression of vitellogenin mRNA at 30 days post injection in vivo. Similar results were also found when injection of the Es-ß-PDH peptide. In vitro culture demonstrated that Es-RPCH and Es-ß-PDH induced germinal vesicle breakdown of the late vitellogenic oocytes. Comparative ovarian transcriptome analysis indicated that some reproduction/meiosis-related genes such as cdc2 kinase, cyclin B, 5-HT-R and retinoid-X receptor were significantly upregulated in response to Es-RPCH and Es-ß-PDH treatments. Taken together, these results provided the evidence for the inductive effect of Es-RPCH and Es-ß-PDH on the oocyte meiotic maturation in E. sinensis.

Toxicological mechanism of excessive copper supplementation: Effects on coloration, copper bioaccumulation and oxidation resistance in mud crab Scylla paramamosain.

Copper is a widespread pollutant in marine environments, and marine animals can ingest large amounts of copper through the food chain. Here, an 8-week feeding trial was designed to investigate the effects of different dietary copper levels on coloration, copper bioaccumulation, stress response and oxidation resistance of juvenile mud crab Scylla paramamosain. The results indicated that crabs fed the diet with 162 mg/kg copper exhibited a dark-blue carapace and hemolymph. The accumulation of copper in tissues was positively correlated with the level of copper in feed. High/excess dietary copper (162 mg/kg) up-regulated the expression of stress response related genes, and reduced the expression/activities of anti-oxidation genes/enzymes. The activity of phenoloxidase decreased significantly when dietary copper level was 86-162 mg/kg, and the expression of hemocyanin was up-regulated in crab fed the diets with 28-162 mg/kg copper. Overall, the results of the present study indicated that high dietary copper led to parachrea in carapace and hemolymph of mud crab, and caused copper deposition abnormality in carapace and hepatopancreas. The data suggested that the toxic effects of dietary copper were concentration-dependent such that, excess dietary copper (162 mg/kg) had adverse impacts on oxidation resistance.

Dietary glutathione supplementation enhances antioxidant activity and protects against lipopolysaccharide-induced acute hepatopancreatic injury and cell apoptosis in Chinese mitten crab, Eriocheir sinensis.

Eriocheir sinensis (E. sinensis) is an important aquaculture species in China. However, deteriorating water environments lead to oxidative stress in these crabs, which subsequently reduces their quality and yield. Glutathione (GSH) is an endogenous antioxidant that is used to mitigate oxidative stress. However, whether dietary GSH can enhance the resistance of E. sinensis to oxidative stress remains unclear. Herein, crabs were fed dietary GSH (the basal diet was supplemented with 0, 300, 600, 900, and 1200 mg/kg diet weight of GSH) for up to 3 weeks and, then, challenged with lipopolysaccharide (LPS; 400 µg/kg body weight). After 6 h, their hepatopancreas were sampled. Diet supplementation with 600 and 900 mg/kg diet weight GSH not only increased the content of GSH in the hepatopancreas, but also enhanced the activities and mRNA expressions of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) (P < 0.05), compared to that in control crabs challenged with LPS alone. Diet supplementation with 600 or 900 mg/kg GSH also significantly increased the enzyme activities of GSH reductase and γ-glutamyl cysteine synthetase (γ-GCS) in LPS-treated crabs. Haematoxylin-eosin (HE) staining, electron microscopy, and flow cytometry were used to examine the structure and subcellular structure of and apoptosis in the hepatopancreas. The histopathology and sub-microstructure analysis results also showed that diet supplementation with 600 or 900 mg/kg GSH significantly alleviated damage in crabs challenged with LPS and decreased reactive oxygen species (ROS) levels and cell apoptosis ratios in the hepatopancreas, compared to the LPS-treated crabs. To further understand the effect of dietary GSH on LPS-induced apoptosis, the activities and gene or protein expressions of apoptosis-related factors were evaluated. As a result, diet supplementation with 600 or 900 mg/kg GSH significantly decreased the activities of caspases-3, -8, and -9 and inhibited the relative expression of caspase-3 and -8 but increased the expression of B-cell lymphoma-2 (bcl-2) and B-cell lymphoma-2-associated X inhibitor (bax inhibitor) in crabs challenged with LPS. This treatment further significantly downregulated the relative protein levels of caspase-3, -8, -9 and Bax and upregulated those of Bcl-2 in crabs challenged with LPS. However, treatment with 1200 mg/kg GSH caused the opposite effects. Overall, our results reveal that appropriate diets supplemented with 600 or 900 mg/kg GSH could enhance the antioxidant capacity and anti-apoptotic mechanisms in crabs after LPS injection, thereby providing a theoretical basis for the application of dietary GSH in E. sinensis.

Dietary nano cerium oxide promotes growth, relieves ammonia nitrogen stress, and improves immunity in crab (Eriocheir sinensis).

Oxidative stress plays a crucial role in ammonia nitrogen toxicity. In this study, the beneficial effects of dietary nano cerium oxide (nano CeO2) as a potent antioxidant were examined in the Chinese mitten crab (Eriocheir sinensis). Crabs were fed a diet supplemented with 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, or 12.8 mg/kg nano CeO2 for 60 d. The optimum supplementation level of nano CeO2 that significantly increased weight gain rate and decreased feed coefficient was 0.8 mg/kg. This level also offered immune protection when crabs were kept under ammonia nitrogen stress and/or exposed to pathogen infection (Aeromonas hydrophila). Supplementation with 0.8 mg/kg of CeO2 (i) relieved pathological damage to the hepatopancreas; (ii) increased hemocyte counts, including total number of hemocytes, granulocytes, and hyalinocytes; (iii) decreased malondialdehyde content and increased antioxidant enzyme activities of superoxide dismutase and catalase in the hemolymph; (iv) increased the activities of lysozyme, acid phosphatase, and alkaline phosphatase in the hemolymph; and (v) increased gene and protein expression of cathepsin L in the hepatopancreas. Mortality increased when crabs were injected with bacteria under ammonia nitrogen stress, but dietary supplementation with 0.8 mg/kg nano CeO2 decreased the mortality rate. Thus, the results of this study suggested that dietary supplementation with nano CeO2 in crabs promoted growth and up-regulated immunity to bacterial infection under ammonia nitrogen stress.

Evaluation of Formulated Diets Enriched by Spinach Extracts for the Broodstock Females, Portunus pelagicus (Linnaeus, 1758).

BACKGROUND AND OBJECTIVE: Spinach extract (Amaranthacea tricolor) is a very prospective feed raw materials to stimulate the molting and growth of crab female broodstock. The purpose of this study was to evaluate the quality of broodstock females, Portunus pelagicus (Linnaeus, 1758) at different levels of spinach extract based on physical, organoleptic and chemical tests. MATERIALS AND METHODS: Four different treatments of spinach extract (0, 250, 500 and 750 ng g-1 crab, respectively) were used in this study. The female crab samples were collected from coastal region of Padang, West Sumatera and placed randomly in four concrete tanks (200×100×100 cm). Each concrete tank consist of five units of plastic box (45.5×32.5×16.5 cm) with the maximum density was one crab per box. RESULTS: The organoleptic test showed that formulated diets enriched with spinach extract (0, 250 and 500 ng g-1 crab) had a smooth texture, pungent aroma and brown. Whereas from the physics test results obtained good water stability (rupture velocity ranged from 89.20-105.40 min and solids dispersion ranged from 4.97-7.17%), hardness (92.66-98.07%) and sinking velocity (3.88-5.88 cm sec-1) (p<0.05). The results also showed that formulated diet enriched with spinach extract doses of 250-750 ng g-1 crab gave a value of delicacy of feed (0.195-0.386 g crab-1 weight/day) which was significantly different (p<0.05) with 0 ng g-1 crab (0.445 g crab-1 weight/day). The chemical test shows the moisture content were 11.60%, ash 9.31%, protein 44.38%, fat 7.64% and carbohydrate 14.46%. CONCLUSION: It was concluded that there was a linear relationship between dose of spinach extracts in formulated diet and rupture velocity and a quadratic relationship between dose of spinach extracts in formulated diet and solid disperse, hardness and sinking velocity and delicacy of feed.

Effects of glycinin and ß-conglycinin on growth performance and intestinal health in juvenile Chinese mitten crabs (Eriocheir sinensis).

This study investigates the effects of two soybean antigens (glycinin and ß-conglycinin) as an antinutritional substance in the diet on the growth, digestive ability, intestinal health and microbiota of juvenile Chinese mitten crabs (Eriocheir sinensis). The isonitrogenous and isolipidic diets contained two soybean antigens at two levels each (70 and 140 g/kg ß-conglycinin, 80 and 160 g/kg glycinin) and a control diet without ß-conglycinin or glycinin supplementation, and were used respectively to feed juvenile E. sinensis for seven weeks. Dietary inclusion of either glycinin or ß-conglycinin significantly reduced crab survival and weight gain. The crabs fed diets containing soybean antigens had higher malondialdehyde concentrations and lower catalase activities in the intestine than those in the control. The activities of trypsin and amylase in the intestine were suppressed by dietary ß-conglycinin and glycinin. Dietary glycinin or ß-conglycinin impaired the immunity and morphological structure of intestine, especially the peritrophic membrane. The mRNA expression of constitutive and inducible immune responsive genes (lipopolysaccharide-induced TNF-α factor and interleukin-2 enhancer-binding factor 2) increased while the mRNA expression of the main genes related to the structural integrity peritrophic membrane (peritrophin-like gene and peritrophic 2) significantly decreased in the groups with soybean antigen addition. Soybean antigen could also change the intestinal microbial community. The abundance of pathogenic bacteria (Ochrobactrum, Burkholderia and Pseudomonas) increased significantly in both soybean antigen groups. Although pathogenic bacteria Vibrio were up-regulated in the glycinin group, the abundance of Dysgonomonas that degraded lignocellulose and ameliorated the gut environment decreased in the glycinin group. This study indicates that existence of soybean antigens (glycinin or ß-conglycinin) could induce gut inflammation, reshape the community of gut microbiota, and cause digestive dysfunction, ultimately leading to impaired growth in crabs.

Effects of dietary yeast extract supplementation on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis).

This study was conducted to evaluate the effects of dietary supplementation with yeast extract on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis). A total of 432 crabs (initial average weight, 4.62 ±â€¯0.11 g) were randomly distributed into four treatment groups with six replicates of 18 crabs. The crabs were fed a basal diet or the same diet supplemented with 2.5, 5, and 10 g/kg yeast extract for 8 weeks. The results showed that dietary yeast extract inclusion enhanced the edible viscera index (linear, P < 0.001), edible viscera crude protein (CP) content (linear, P = 0.025) and serum phenoloxidase (ProPO) activity (quadratic, P = 0.023) at 56 day, increased the total superoxide dismutase (T-SOD) activity at 28 day (quadratic, P = 0.037) and catelase (CAT) activity at 56 day (quadratic, P = 0.034) of edible viscera, and muscular T-SOD activity (quadratic, P = 0.020) at 56 day in Chinese mitten crab. Compared with the control group, the inclusion of 5 g/kg yeast extract in the diet increased the edible viscera index, enhanced the CAT activity of edible viscera at 56 day in Chinese mitten crab (P < 0.05). Dietary 10 g/kg yeast extract inclusion enhanced the edible viscera index at 56 day in Chinese mitten crab than that of the control group (P < 0.05). These results implied that dietary yeast extract inclusion improved the edible viscera index and crude protein content of edible viscera, enhanced serum immunity, and increased the antioxidant status of edible viscera and muscle in Chinese mitten crab, especially when it is supplemented at 5 g/kg yeast extract in the diet.

Evaluation of iron loading in four types of hepatopancreatic cells of the mangrove crab Ucides cordatus using ferrocene derivatives and iron supplements.

The mangrove crab Ucides cordatus is a bioindicator of aquatic contamination. In this work, the iron availability and redox activity of saccharide-coated mineral iron supplements (for both human and veterinary use) and ferrocene derivatives in Saline Ucides Buffer (SUB) medium were assessed. The transport of these metallodrugs by four different hepatopancreatic cell types (embryonic (E), resorptive (R), fibrillar (F), and blister (B)) of U. cordatus were measured. Organic coated iron minerals (iron supplements) were stable against strong chelators (calcein and transferrin). Ascorbic acid efficiently mediated the release of iron only from ferrocene compounds, leading to redox-active species. Ferrous iron and iron supplements were efficient in loading iron to all hepatopancreatic cell types. In contrast, ferrocene derivatives were loaded only in F and B cell types. Acute exposition to the iron compounds resulted in cell viability of 70-95%, and to intracellular iron levels as high as 0.40 µmol L-1 depending upon the compound and the cell line. The easiness that iron from iron metallodrugs was loaded/transported into U. cordatus hepatopancreatic cells reinforces a cautionary approach to the widespread disposal and use of highly bioavailable iron species as far as the long-term environmental welfare is concerned.

A Multifaceted Mass Spectrometric Method to Probe Feeding Related Neuropeptide Changes in Callinectes sapidus and Carcinus maenas.

Food intake is regulated by various neuromodulators, including numerous neuropeptides. However, it remains elusive at the molecular and cellular level as to how these important chemicals regulate internal processes and which regions of the neuronal organs are responsible for regulating the behavior. Here we report a comparative neuropeptidomic analysis of the brain and pericardial organ (PO) in response to feeding in two well-studied crustacean physiology model organisms, Callinectes sapidus and Carcinus maenas, using mass spectrometry (MS) techniques. A multifaceted MS-based approach has been developed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and PO. The method employs stable isotope labeling of brain and PO extracts for relative MS quantitation, capillary electrophoresis (CE)-MS for fractionation and high-specificity analysis, and mass spectrometric imaging (MSI) for in-situ molecular mapping of peptides. A number of neuropeptides, including RFamides, B-type allatostatins (AST-B), RYamides, and orcokinins exhibit significant changes in abundance after feeding in this investigation. Peptides from the AST-B family found in PO tissue were shown to have both altered expression and localization changes after feeding, indicating that they may be a class of vital neuropeptide regulators involved in feeding behavior. Graphical Abstract ᅟ.

Influence of Macrofaunal Burrows on Extracellular Enzyme Activity and Microbial Abundance in Subtropical Mangrove Sediment.

Bioturbation and bioirrigation induced by burrowing macrofauna are recognized as important processes in aquatic sediment since macrofaunal activities lead to the alteration of sediment characteristics. However, there is a lack of information on how macrofauna influence microbial abundance and extracellular enzyme activity in mangrove sediment. In this study, the environmental parameters, extracellular enzyme activities, and microbial abundance were determined and their relationships were explored. Sediment samples were taken from the surface (S) and lower layer (L) without burrow, as well as crab burrow wall (W) and bottom of crab burrow (B) located at the Mai Po Nature Reserve, Hong Kong. The results showed that the burrowing crabs could enhance the activities of oxidase and hydrolases. The highest activities of phenol oxidase and acid phosphatase were generally observed in B sediment, while the highest activity of N-acetyl-glucosaminidase was found in W sediment. The enzymatic stoichiometry indicated that the crab-affected sediment had similar microbial nitrogen (N) and phosphorous (P) availability relative to carbon (C), lower than S but higher than L sediment. Furthermore, it was found that the highest abundance of both bacteria and fungi was shown in S sediment, and B sediment presented the lowest abundance. Moreover, the concentrations of phosphorus and soluble phenolics in crab-affected sediment were almost higher than the non-affected sediment. The alterations of phenolics, C/P and N/P ratios as well as undetermined environmental factors by the activities of crabs might be the main reasons for the changes of enzyme activity and microbial abundance. Finally, due to the important role of phenol oxidase and hydrolases in sediment organic matter (SOM) decomposition, it is necessary to take macrofaunal activities into consideration when estimating the C budget in mangrove ecosystem in the future.

Impact of disease on the survival of three commercially fished species.

Recent increases in emergent infectious diseases have raised concerns about the sustainability of some marine species. The complexity and expense of studying diseases in marine systems often dictate that conservation and management decisions are made without quantitative data on population-level impacts of disease. Mark-recapture is a powerful, underutilized, tool for calculating impacts of disease on population size and structure, even in the absence of etiological information. We applied logistic regression models to mark-recapture data to obtain estimates of disease-associated mortality rates in three commercially important marine species: snow crab (Chionoecetes opilio) in Newfoundland, Canada, that experience sporadic epizootics of bitter crab disease; striped bass (Morone saxatilis) in the Chesapeake Bay, USA, that experience chronic dermal and visceral mycobacteriosis; and American lobster (Homarus americanus) in the Southern New England stock, that experience chronic epizootic shell disease. All three diseases decreased survival of diseased hosts. Survival of diseased adult male crabs was 1% (0.003-0.022, 95% CI) that of uninfected crabs indicating nearly complete mortality of infected crabs in this life stage. Survival of moderately and severely diseased striped bass (which comprised 15% and 11% of the population, respectively) was 84% (70-100%, 95% CI), and 54% (42-68%, 95% CI) that of healthy striped bass. The disease-adjusted yearly natural mortality rate for striped bass was 0.29, nearly double the previously accepted value, which did not include disease. Survival of moderately and severely diseased lobsters was 30% (15-60%, 95% CI) that of healthy lobsters and survival of mildly diseased lobsters was 45% (27-75%, 95% CI) that of healthy lobsters. High disease mortality in ovigerous females may explain the poor recruitment and rapid declines observed in this population. Stock assessments should account for disease-related mortality when resource management options are evaluated.

Sublethal Toxicity of Crude Oil Exposure in The Blue Crab, Callinectes sapidus, at Two Life History Stages.

The 2010 Deepwater Horizon oil spill occurred during peak spawning season for many Gulf of Mexico fish and invertebrates. Early life stages of important fishery species were at risk to encounter crude oil. In this study, we investigated the effect of crude oil exposure on two life stages of the blue crab (Callinectes sapidus). We tested the effects of oil exposure on the survival and growth of larval (pelagic) and juvenile (estuarine) blue crabs as well as the effects of oil on the molt frequency of juveniles. Larval crabs exposed to crude oil showed no discernable growth or mortality differences when compared to non-exposed controls. Juvenile crabs exposed to oil also showed no differences in size but exhibited increased intermolt duration (time between molts). Our study suggests that different life-stages may respond differently when exposed to crude oil and that oil exposure negatively affects growth rate of juvenile blue crabs.

Assessing Fish and Motile Fauna around Offshore Windfarms Using Stereo Baited Video.

There remains limited knowledge of how offshore windfarm developments influence fish assemblages, particularly at a local scale around the turbine structures. Considering the existing levels of anthropogenic pressures on coastal fish populations it is becoming increasingly important for developers and environmental regulators to gain a more comprehensive understanding of the factors influencing fish assemblages. Improving our ability to assess such fish populations in close proximity to structures will assist in increasing this knowledge. In the present study we provide the first trial use of Baited Remote Underwater Stereo-Video systems (stereo BRUVs) for the quantification of motile fauna in close proximity to offshore wind turbines. The study was conducted in the Irish Sea and finds the technique to be a viable means of assessing the motile fauna of such environments. The present study found a mixture of species including bottom dwellers, motile crustaceans and large predatory fish. The majority of taxa observed were found to be immature individuals with few adult individuals recorded. The most abundant species were the angular crab (Goneplax rhomboides) and the small-spotted catshark (Scyliorhinus canicula). Of note in this study was the generally low abundance and diversity of taxa recorded across all samples, we hypothesise that this reflects the generally poor state of the local fauna of the Irish Sea. The faunal assemblages sampled in close proximity to turbines were observed to alter with increasing distance from the structure, species more characteristic of hard bottom environments were in abundance at the turbines (e.g. Homarus gammarus, Cancer pagarus, Scyliorhinus spp.) and those further away more characteristic of soft bottoms (e.g. Norwegian Lobster). This study highlights the need for the environmental impacts of offshore renewables on motile fauna to be assessed using targeted and appropriate tools. Stereo BRUVs provide one of those tools, but like the majority of methods for sampling marine biota, they have limitations. We conclude our paper by providing a discussion of the benefits and limitations of using this BRUV technique for assessing fauna within areas close to offshore windfarms.

Effects of dietary peppermint (Mentha piperita) essential oil on survival, growth, cannibalism and hepatopancreatic histopathology of Portunus pelagicus juveniles.

A two-part experiment was performed to determine whether dietary peppermint oil could improve the growth and/or decrease aggression among blue swimmer crab, Portunus pelagicus early juveniles. A total of five isonitrogenous diets were made that contained increasing peppermint oil levels of 0.00, 0.05, 0.10, 0.50 or 1.00%.? These diets were fed to 45 replicate crabs in each treatment (total of 225 crabs) for 12 days, the final sizes and weights were measured, and then placed in 3 replicate containers (30 in total/treatment) to allow the opportunity for cannibalism over 10 days.? After 10 days, the remaining crabs were examined for any histopathological changes in gills or hepatopancreas.? Results showed dietary peppermint oil, at the tested levels, had no effect on the growth or cannibalism, in either experiments (p > 0.05).? However, there were substantial changes in the hepatopancreatic histopathology that included thinner tubules and significantly less B- and R-cells from 0.10% dietary peppermint oil and above.? The unaffected growth or cannibalism indicate that the levels of dietary peppermint oil used were insufficient and further investigations are required, particularly on the implications to the hepatopancreatic changes. ?

What can an ecophysiological approach tell us about the physiological responses of marine invertebrates to hypoxia?

Hypoxia (low O2) is a common and natural feature of many marine environments. However, human-induced hypoxia has been on the rise over the past half century and is now recognised as a major problem in the world's seas and oceans. Whilst we have information on how marine invertebrates respond physiologically to hypoxia in the laboratory, we still lack understanding of how they respond to such stress in the wild (now and in the future). Consequently, here the question 'what can an ecophysiological approach tell us about physiological responses of marine invertebrates to hypoxia' is addressed. How marine invertebrates work in the wild when challenged with hypoxia is explored using four case studies centred on different hypoxic environments. The recent integration of the various -omics into ecophysiology is discussed, and a number of advantages of, and challenges to, successful integration are suggested. The case studies and -omic/physiology integration data are used to inform the concluding part of the review, where it is suggested that physiological responses to hypoxia in the wild are not always the same as those predicted from laboratory experiments. This is due to behaviour in the wild modifying responses, and therefore more than one type of 'experimental' approach is essential to reliably determine the actual response. It is also suggested that assuming it is known what a measured response is 'for' can be misleading and that taking parodies of ecophysiology seriously may impede research progress. This review finishes with the suggestion that an -omics approach is, and is becoming, a powerful method of understanding the response of marine invertebrates to environmental hypoxia and may be an ideal way of studying hypoxic responses in the wild. Despite centring on physiological responses to hypoxia, the review hopefully serves as a contribution to the discussion of what (animal) ecophysiology looks like (or should look like) in the 21st century.

Preliminary results of the in vivo and in vitro characterization of a tentacle venom fraction from the jellyfish Aurelia aurita.

The neurotoxic effects produced by a tentacle venom extract and a fraction were analyzed and correlated by in vivo and in vitro approaches. The tentacle venom extract exhibited a wide range of protein components (from 24 to >225 kDa) and produced tetanic reactions, flaccid paralysis, and death when injected into crabs. Two chromatography fractions also produced uncontrolled appendix movements and leg stretching. Further electrophysiological characterization demonstrated that one of these fractions potently inhibited ACh-elicited currents mediated by both vertebrate fetal and adult muscle nicotinic acetylcholine receptors (nAChR) subtypes. Receptor inhibition was concentration-dependent and completely reversible. The calculated IC(50) values were 1.77 µg/µL for fetal and 2.28 µg/µL for adult muscle nAChRs. The bioactive fraction was composed of a major protein component at ~90 kDa and lacked phospholipase A activity. This work represents the first insight into the interaction of jellyfish venom components and muscle nicotinic receptors.

Molecular cloning and sequence of retinoid X receptor in the green crab Carcinus maenas: a possible role in female reproduction.

Retinoid X receptor (RXR) belongs to an ancient superfamily of nuclear hormone receptors, and plays an important role in reproduction of vertebrates. However, the reproductive role of RXR has not been clarified in crustaceans. In this investigation, we first report the cloning of two alternative splice variants of RXR cDNA from green crab ovarian RNA. RXR mRNA levels were quantified in different vitellogenic stages of the crab hepatopancreas (HP) and ovary. The expression of RXR mRNA relative to the arginine kinase mRNA was significantly increased in the HP of vitellogenic crabs in a stage-dependent manner. The relative levels of RXR mRNA in the ovary were significantly lower in vitellogenic stage III crabs than in crabs in the other three stages. These data indicate that the HP and ovary of the crab are capable of expressing RXR, which may regulate, in part, vitellogenesis in the crab. We also examined the effects of methyl farnesoate (MF) and RXR-dsRNA treatments on vitellogenin and RXR gene expression. Vitellogenin and RXR mRNA levels in HP and ovarian fragments incubated in MF were significantly (P<0.001) higher than in control tissue fragments prepared from the same animal. Treatment of crabs with RXR-dsRNA significantly (P<0.001) reduced mRNA levels for RXR and for vitellogenin as well as MF levels in hemolymph. These results indicate that, MF and RXR form a complex (MF-RXR) directly and together stimulate ovarian development in these green crabs. This interaction of RXR, MF, and ovary development axis is a novel finding and is the first report to the best of our knowledge.

Dietary copper effects in the estuarine crab, Neohelice (Chasmagnathus) granulata, maintained at two different salinities.

We analyzed the dietary copper effects in the estuarine crab Neohelice (Chasmagnathus) granulata and its interaction with water salinity. Crabs were maintained at 2 per thousand and 30 per thousand salinity for 5 weeks and they were fed with commercial food supplemented with the green alga Scenedesmus vacuolatus previously exposed to copper. No mortalities were observed, but crabs maintained at 2 per thousand salinity accumulated on average 40% more copper compared to animals maintained at 30 per thousand salinity. At 2 per thousand salinity, superoxide dismutase (SOD) activity and reduced glutathione (GSH) levels were increased at the first and second weeks, respectively, while lipid peroxidation and protein oxidation were evident after 4 weeks of copper exposure. At 30 per thousand salinity, all measured variables increased progressively but were significantly higher only at the end of the assay (5th week), except for protein oxidation that remained unchanged throughout the experiment. The hepatosomatic index (HSI) was significantly decreased in response to copper exposure, but only in crabs acclimated to 2 per thousand. These findings have suggested that dietary copper exposure induces greater metal accumulation and larger oxidative stress responses in crabs maintained at 2 per thousand salinity.