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Transcriptome mining is a powerful method for crustacean peptide discovery, especially when large sequence datasets are available and an appropriate reference is extant. Recently, a 206,041-sequence transcriptome for the copepod Calanus finmarchicus was mined for peptide-encoding transcripts, with ones for 17 families/subfamilies identified. Here, the deduced Calanus pre/preprohormones were used as templates for peptide discovery in the copepods Tigriopus californicus and Lepeophtheirus salmonis; large transcriptome shotgun assembly datasets are publicly accessible for both species. Sixty-five Tigriopus and 17 Lepeophtheirus transcripts, encompassing 22 and 13 distinct peptide families/subfamilies, respectively, were identified, with the structures of 161 and 70 unique mature peptides predicted from the deduced precursors. The identified peptides included members of the allatostatin A, allatostatin C, bursicon α, bursicon β, CAPA/periviscerokinin/pyrokinin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, FLRFamide, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, and tachykinin-related peptide families, most of which possess novel structures, though isoforms from other copepods are known. Of particular note was the discovery of novel isoforms of adipokinetic hormone-corazonin-like peptide, allatotropin, corazonin, eclosion hormone and intocin, peptide families previously unidentified in copepods. In addition, Tigriopus precursors for two previously unknown peptide groups were discovered, one encoding GSEFLamides and the other DXXRLamides; precursors for the novel FXGGXamide family were identified from both Tigriopus and Lepeophtheirus. These data not only greatly expand the catalog of known copepod peptides, but also provide strong foundations for future functional studies of peptidergic signaling in members of this ecologically important crustacean subclass., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Remipedia is a small, recently described crustacean class that inhabits submerged marine/anchialine cave systems. Phylogenetic and morphological investigations support a sister group relationship between these animals and the hexapods. The recent deposition of numerous (>100,000) transcriptome shotgun assembly sequences for Speleonectes cf. tulumensis provides a unique resource to identify proteins of interest from a member of the Remipedia. Here, this dataset was mined for sequences encoding putative neuropeptide pre/preprohormones, with the mature peptides predicted from the deduced precursors using an established workflow. The structures of 40 mature peptides were obtained via this strategy, including members of 11 well-known arthropod peptide families (adipokinetic hormone/corazonin-like peptide [ACP], allatostatin A, allatostatin C, diuretic hormone 31, eclosion hormone, ion transport peptide/crustacean hyperglycemic hormone, neuropeptide F, proctolin, SIFamide, sulfakinin and tachykinin-related peptide); these are the only peptides thus far described from any member of the Remipedia. Comparison of the Speleonectes isoforms with those from other crustaceans and hexapods revealed the peptidome of this species to have characteristics of both subphyla (e.g. it possesses the stereotypical decapod crustacean SIFamide and tachykinin-related peptide isoforms, while simultaneously being the only crustacean with an insect AKC). Moreover, BLAST searches in which the deduced Speleonectes precursors were compared to the pancrustacean protein database most frequently returned insect homologs as the closest matches. The peptidomic analyses presented here are consistent with the hypothesized phylogenetic position of the Remipedia within the Pancrustacea, and serve as a foundation from which to launch future investigations of peptidergic signaling in remipedes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The concentration of 12 organochlorine pesticides (OCPs) were measured in water, sediment, aquatic plant, and animal (shrimp and fish) of Nansi Lake by gas chromatography equipped with an electron capture detector. The total OCPs concentrations were 65.31-100.31 ng L(-1) in water, 2.9-6.91 ng g(-1) dry weight (dw) in sediments, 1.29-6.42 ng g(-1) dw in aquatic plants and 7.57-17.22 ng g(-1) dw in animals. The OCPs composition profiles showed that heptachlor compounds was also the predominant OCPs contaminants in addition to hexachlorocyclohexanes (HCHs) and dichlorodiphenyltrichloroethanes (DDTs) in Nansi Lake. According to the source of HCHs and DDTs in sediment samples, there was no new input and the HCHs pollution mainly came from the use of Lindane in Nansi Lake. Bioaccumulation of OCPs in aquatic biota indicated that DDTs and heptachlor compounds had a strong accumulation, followed by HCHs and drins. The accumulation abilities of fish for OCPs were higher than those of plants and shrimps. The OCPs biota-sediment accumulation factor values of Channa argus was the highest in fish samples, followed by Carassius auratus, and Cyprinus caspio. Risk assessment of sediment showed that heptachlor epoxide had a higher occurrence possibility of adverse ecological effects to benthic species. Based on the calculation of acceptable daily intake and hazard ratio, HCHs in fish and shrimps from Nansi Lake had a lifetime cancer risk of greater than one per million. The risk assessment of water, sediment, and fish indicated the water environment of Nansi Lake is at a safe level at present.

The first set of competitive inhibitors of molt inhibiting hormone (MIH) has been developed using the effective approaches such as Hip-Hop, virtual screening and manual alterations. Moreover, the conserved residues at 71 and 72 positions in the molt inhibiting hormone is known to be significant for selective inhibition of ecdysteroidogenesis; thus, the information from mutation and solution structure were used to generate common pharmacophore features. The geometry of the final six-feature pharmacophore was also found to be consistent with the homology-modeled MIH structures from various other decapod crustaceans. The Hypo-1, comprising six features hypothesis was carefully selected as a best pharmacophore model for virtual screening created on the basis of rank score and cluster processes. The hypothesis was validated and the database was virtually screened using this 3D query and the compounds were then manually altered to enhance the fit value. The hits obtained were further filtered for drug-likeness, which is expressed as physicochemical properties that contribute to favorable ADME/Tox profiles to eliminate the molecules exhibit toxicity and poor pharmacokinetics. In conclusion, the higher fit values of CI-1 (4.6), CI-4 (4.9) and CI-7 (4.2) in conjunction with better pharmacokinetic profile made these molecules practically helpful tool to increase production by accelerating molt in crustaceans. The use of feeding sub-therapeutic dosages of these growth enhancers can be very effectively implemented and certainly turn out to be a vital part of emerging nutritional strategies for economically important crustacean livestock.

Glacial-relict species of the genus Mysis (opossum shrimps) inhabiting both fresh-water lakes and brackish sea waters in northern Europe show a consistent lake/sea dichotomy in eye spectral sensitivity. The absorbance peak (λmax) recorded by microspectrophotometry in isolated rhabdoms is invariably 20-30 nm red-shifted in "lake" compared with "sea" populations. The dichotomy holds across species, major opsin lineages and light environments. Chromophore exchange from A1 to A2 (retinal → 3,4-didehydroretinal) is a well-known mechanism for red-shifting visual pigments depending on environmental conditions or stages of life history, present not only in fishes and amphibians, but in some crustaceans as well. We tested the hypothesis that the lake/sea dichotomy in Mysis is due to the use of different chromophores, focussing on two populations of M. relicta from, respectively, a Finnish lake and the Baltic Sea. They are genetically very similar, having been separated for less than 10 kyr, and their rhabdoms show a typical lake/sea difference in λmax (554 nm vs. 529 nm). Gene sequencing has revealed no differences translating into amino acid substitutions in the transmembrane parts of their opsins. We determined the chromophore identity (A1 or A2) in the eyes of these two populations by HPLC, using as standards pure chromophores A1 and A2 as well as extracts from bovine (A1) and goldfish (A2) retinas. We found that the visual-pigment chromophore in both populations is A1 exclusively. Thus the spectral difference between these two populations of M. relicta is not due to the use of different chromophores. We argue that this conclusion is likely to hold for all populations of M. relicta as well as its European sibling species.

The tumor suppressor gene p53 plays a critical role in safeguarding the integrity of genome in mammalian cells. It acts as a sequence-specific transcription factor. Once activated by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 as well as its downstream target genes in invertebrates. A full length cDNA that encodes a 453-amino-acid p53 protein (Lvp53) was characterized in the Pacific white shrimp (Litopenaeus vannamei) to explore the potential relationships between p53 and two antioxidant enzyme genes: Mn-superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) in eliminating cell stresses in L. vannamei. Sequence analysis revealed a close phylogenetic relationship between Lvp53 and that of Marsupenaeus japonicus, and a high degree of conservation in critical amino acids residues is involved in DNA and zinc binding among species. Quantitative real-time PCR showed that Lvp53 was expressed with varied levels in all the 11 tissues under investigation. In response to acute pH challenge, the relative expression of Lvp53 was induced in a pH- and time-dependent manner, with the peak observed at pH 6.1 and after 24 h of treatment, in which condition, both the relative mRNA expressions and the enzymatic activities of LvMnSOD and LvGPx were increased correspondingly. In response to acute cadmium (Cd) exposure, the relative expression of Lvp53 was upregulated in a time- and concentration-dependent manner, with the maximum detected at Cd 6.6 μM and after 48 h of exposure, in which case, both the transcripts and the enzymatic activities of LvMnSOD and LvGPx were also induced. After Lvp53 transcripts were declined by double-strand RNA injection, the relative mRNA expressions of LvMnSOD and LvGPx were decreased correspondingly. Meanwhile, pH 6.1 or 6.6 μM Cd could not induce the transcripts or the enzymatic activities of LvMnSOD or LvGPx any more in Lvp53-silenced shrimp, but increased shrimp mortalities. These results indicated the involvement of Lvp53, LvMnSOD and LvGPx in mediating cell stress caused by suboptimal pH and elevated levels of Cd in L. vannamei, and that the expressions of LvMnSOD and LvGPx were positively regulated by Lvp53, which is a potential mechanism for shrimp to survive the oxidative stress that occurs during short-term exposure to Cd or challenge with acidic pH. This finding will contribute to better understanding of p53 signaling pathways and redox regulation in invertebrate organisms., (© 2013.)

A transcriptomic assembly originated from hypodermis and Y organ of the crustacean Pontastacus leptodactylus is used here for in silico characterization of oxi-reductase enzymes potentially involved in the metabolism of ecdysteroid molting hormones. RNA samples were extracted from male Y organ and its neighboring hypodermis in all stages of the molt cycle. An equimolar RNA mix from all stages was sequenced using next generation sequencing technologies and de novo assembled, resulting with 74,877 unique contigs. These transcript sequences were annotated by examining their resemblance to all GenBank translated transcripts, determining their Gene Ontology terms and their characterizing domains. Based on the present knowledge of arthropod ecdysteroid metabolism and more generally on steroid metabolism in other taxa, transcripts potentially related to ecdysteroid metabolism were identified and their longest possible conceptual protein sequences were constructed in two stages, correct reading frame was deduced from BLASTX resemblances, followed by elongation of the protein sequence by identifying the correct translation frame of the original transcript. The analyzed genes belonged to several oxi-reductase superfamilies including the Rieske non heme iron oxygenases, cytochrome P450s, short-chained hydroxysteroid oxi-reductases, aldo/keto oxireductases, lamin B receptor/sterol reductases and glucose-methanol-cholin oxi-reductatses. A total of 68 proteins were characterized and the most probable participants in the ecdysteroid metabolism where indicated. The study provides transcript and protein structural information, a starting point for further functional studies, using a variety of gene-specific methods to demonstrate or disprove the roles of these proteins in relation to ecdysteroid metabolism in P. leptodactylus., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Extracellular traps (ETs) are formed from the DNA, histones and cytoplasmic antimicrobial proteins that are released from a range of vertebrate immune-cells in response to pathogenic stimulation. This novel defense mechanism has not been demonstrated in invertebrates. In this study, we investigated the formation of ETs in the crustacean Litopenaeus vannamei. We found that stimulation of shrimp hemocytes with phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and live Escherichia coli all led to the formation of the characteristic ET fibers made from host cell DNA. After E. coli stimulation, we found that histone proteins were co-localized with these extracellular DNA fibers. The results further showed that E. coli were trapped by these ET-like fibers and that some of the trapped bacteria were permeabilized. All of these results are characteristic of the ETs that are seen in vertebrates and we therefore conclude that shrimp are also capable of forming extracellular traps., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The first step of host fighting against pathogens is that pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. In the present study, we investigated how invertebrate pattern recognition receptor (PRR) C-type lectins might be involved in the antimicrobial response in crustacean. Based on our previously obtained completed coding regions of EsLecA and EsLecG in Eriocheir sinensis, the recombinant EsLectin proteins were produced via prokaryotic expression system and affinity chromatography. Subsequently, both rEsLecA and rEsLecG were discovered to have wide spectrum binding activities towards microorganisms, and their microbial-binding was calcium-independent. Moreover, the binding activities of both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. Both microorganism growth inhibitory activities assays and antibacterial activities assays revealed their capabilities of suppressing microorganisms growth and directly killing microorganisms respectively. Furthermore, the encapsulation assays signified that both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. Collectively, data presented here demonstrated the successful expression and purification of two C-type lectins proteins in the Chinese mitten crab, and their critical role in the innate immune system of an invertebrate., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Whole-organism concentration ratios (CRwo-media) for plutonium (Pu) in wildlife were calculated using data from the broad range of organism types and environmental settings of the US nuclear research program. Original sources included site-specific reports and scientific journal articles typically from 1960s to 80s research. Most of the calculated CRwo-media values are new to existing data sets, and, for some wildlife categories, serve to fill gaps or add to sparse data including those for terrestrial reptile; freshwater bird, crustacean and zooplankton; and marine crustacean and zooplankton. Ratios of Pu concentration in the whole-organism to that in specific tissues and organs are provided here for a range of freshwater and marine fish. The CRwo-media values in fish living in liquid discharge ponds were two orders of magnitude higher than those for similar species living in lakes receiving Pu from atmospheric fallout, suggesting the physico-chemical form of the source Pu can dominate over other factors related to transfer, such as organism size and feeding behavior. Small rodent data indicated one to two order of magnitude increases when carcass, pelt, and gastrointestinal tract were included together in the whole-organism calculation compared to that for carcass alone. Only 4% of Pu resided in the carcass of small rodents compared to 75% in the gastrointestinal tract and 21% in the pelt., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Environmental assessments to evaluate potentials risks to humans and wildlife often involve modelling to predict contaminant exposure through key pathways. Such models require input of parameter values, including concentration ratios, to estimate contaminant concentrations in biota based on measurements or estimates of concentrations in environmental media, such as water. Due to the diversity of species and the range in physicochemical conditions in natural ecosystems, concentration ratios can vary by orders of magnitude, even within similar species. Therefore, to improve model input parameter values for application in aquatic systems, freshwater concentration ratios were collated or calculated from national grey literature, Russian language publications, and refereed papers. Collated data were then input into an international database that is being established by the International Atomic Energy Agency. The freshwater database enables entry of information for all radionuclides listed in ICRP (1983), in addition to the corresponding stable elements, and comprises a total of more than 16,500 concentration ratio (CRwo-water) values. Although data were available for all broad wildlife groups (with the exception of birds), data were sparse for many organism types. For example, zooplankton, crustaceans, insects and insect larvae, amphibians, and mammals, for which there were CRwo-water values for less than eight elements. Coverage was most comprehensive for fish, vascular plants, and molluscs. To our knowledge, the freshwater database that has now been established represents the most comprehensive set of CRwo-water values for freshwater species currently available for use in radiological environmental assessments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.

Shrimp is one of the most popular seafoods worldwide, and its lipids have been studied for biological activity in both, muscle and exoskeleton. Free fatty acids, triglycerides, carotenoids, and other lipids integrate this fraction, and some of these compounds have been reported with cancer chemopreventive activities. Carotenoids and polyunsaturated fatty acids have been extensively studied for chemopreventive properties, in both in vivo and in vitro studies. Their mechanisms of action depend on the lipid chemical structure and include antioxidant, anti-proliferative, anti-mutagenic, and anti-inflammatory activities, among others. The purpose of this review is to lay groundwork for future research about the properties of the lipid fraction of shrimp.

Background: After cellulose and starch, chitin is the third-most abundant biopolymer on earth. Chitin or its deacetylated derivative chitosan is a valuable product with a number of applications. It is one of the main components of shrimp shells, a waste product of the fish industry. To obtain chitin from Penaeus monodon, wet and dried shrimp shells were deproteinated with two specifically enriched proteolytic cultures M1 and M2 and decalcified by in-situ lactic acid forming microorganisms. The viscosity of biologically processed chitin was compared with chemically processed chitin. The former was further investigated for purity, structure and elemental composition by several microscopic techniques and (13)C solid state NMR spectroscopy., Results: About 95% of the protein of wet shrimp shells was removed by proteolytic enrichment culture M2 in 68 h. Subsequent decalcification by lactic acid bacteria (LAB) took 48 h. Deproteination of the same amount of dried shrimps that contained a 3 × higher solid content by the same culture was a little bit faster and was finished after 140 h. The viscosity of chitin was in the order of chemically processed chitin > bioprocessed chitin > commercially available chitin. Results revealed changes in fine structure and chemical composition of the epi-, exo- and endocuticle of chitin from shrimp shells during microbial deproteination and demineralization. From transmission electron microscopy (TEM) overlays and electron energy loss spectroscopy (EELS) analysis, it was found that most protein was present in the exocuticle, whereas most chitin was present in the endocuticle. The calcium content was higher in the endocuticle than in the exocuticle.13C solid state NMR spectra of different chitin confirmed < 3% impurities in the final product., Conclusions: Bioprocessing of shrimp shell waste resulted in a chitin with high purity. Its viscosity was higher than that of commercially available chitin but lower than that of chemically prepared chitin in our lab. Nevertheless, the biologically processed chitin is a promising alternative for less viscous commercially available chitin. Highly viscous chitin could be generated by our chemical method. Comprehensive structural analyses revealed the distribution of the protein and Ca matrix within the shrimp shell cuticle which might be helpful in developing shrimp waste processing techniques.

Concentrations of several persistent organic pollutants (POPs: PCBs, PBDEs, OCPs) in aquatic species from the Scheldt estuary were related with factors (body size, lipids, trophic position) possibly influencing their bioaccumulation. Stable nitrogen isotope ratios (δ(15)N) were used as a measure for trophic position. A decreasing trend in POP levels toward the sea was observed. For POP concentrations in sediments, this trend could be attributed to a dilution effect from mixing with seawater. However, concentrations in biota more downstream were higher than expected after taking into account the dilution effect, possibly due to differences in bioavailability. Tissue concentrations were correlated with the lipid content in biota, but not with body size. Biomagnification was only significant for some PCB congeners and p,p'-DDE at the most marine sampling location (Terneuzen, L1) and for p,p'-DDD and BDE 100 at the second sampling location (Bath, L2). A significant decreasing relationship was found for γ-HCH concentrations with increasing δ(15)N at Terneuzen. For Antwerpen (L3), no significant relationships were detected. TMFs ranged from 0.64 for γ-HCH up to 1.60 for PCB 194. These results suggest that biomagnification was more important in the marine part of the estuary, although the presence of multiple carbon sources at the freshwater side might have led to an underestimation of the influence of trophic position.

Exposure and depuration experiments for Gammarus pulex and Daphnia magna were conducted to quantitatively analyze biotransformation products (BTPs) of organic micropollutants (tramadol, irgarol, and terbutryn). Quantification for BTPs without available standards was performed using an estimation method based on physicochemical properties. Time-series of internal concentrations of micropollutants and BTPs were used to estimate the toxicokinetic rates describing uptake, elimination, and biotransformation processes. Bioaccumulation factors (BAF) for the parents and retention potential factors (RPF), representing the ratio of the internal amount of BTPs to the parent at steady state, were calculated. Nonlinear correlation of excretion rates with hydrophobicity indicates that BTPs with lower hydrophobicity are not always excreted faster than the parent compound. For irgarol, G.pulex showed comparable elimination, but greater uptake and BAF/RPF values than D.magna. Further, G. pulex had a whole set of secondary transformations that D. magna lacked. Tramadol was transformed more and faster than irgarol and there were large differences in toxicokinetic rates for the structurally similar compounds irgarol and terbutryn. Thus, predictability of toxicokinetics across species and compounds needs to consider biotransformation and may be more challenging than previously thought because we found large differences in closely related species and similar chemical structures.

Melatonin (N-acetyl-5-methoxy-tryptamine) was first discovered from the bovine pineal gland extract in 1958. Since then, its synthesis, metabolism, physiological, and patho-physiological functions are well studied in vertebrates; there is an increasing recognition of melatonin in invertebrates and especially in crustaceans. The presence of melatonin in crustaceans is now well documented and some functional aspects in the framework of crustacean biology have been demonstrated. This review aims at giving a comprehensive overview of the various physiological events regulated by this pleiotropic hormone. Topics include: glucose homeostasis, regulation of reproduction, molting, limb regeneration, and antioxidant properties. Finally, perspectives on current and possible research are offered., (Copyright © 2013 Wiley Periodicals, Inc.)

The sublethal stress of the organophosphate insecticide chlorpyrifos was investigated in different tissues of the freshwater crab (Barytelphusa guerini). Crabs were exposed to 1/3 of LC50 concentrations for 7, 14, 21, and 28 days. After 28 days, they were released into fresh water and kept for 18 days for recovery. The study was conducted by estimating total proteins, amino acids, ammonia, urea, and glutamine levels, and protease, transaminases, and phosphatases activities. Total proteins level was decreased whereas amino acids and ammonia were increased. The urea content was decreased in all tissues and glutamine exhibited a mixed response. Protease activities and those of alanine and aspartate aminotransferase, respectively, were elevated. Acid phosphatase activity was reduced in hepatopancreas and brain and induced in gills and muscle. Alkaline phosphatase activity was enhanced in gills and hepatopancreas and reduced in muscle and brain. The crabs recovered from the biochemical stress caused by chlorpyrifos after their release into fresh water.

As the use of single-walled carbon nanotubes (SWNTs) increases over time, so does the potential for environmental release. This research aimed to determine the toxicity, bioavailability, and bioaccumulation of SWNTs in marine benthic organisms at the base of the food chain. The toxicity of SWNTs was tested in a whole sediment exposure with the amphipod Ampelisca abdita and the mysid Americamysis bahia. In addition, SWNTs were amended to sediment and/or food matrices to determine their bioavailability and bioaccumulation through these routes in A. abdita, A. bahia, and the estuarine amphipod Leptocheirus plumulosus. No significant mortality to any species via sediment or food matrices was observed at concentrations up to 100 ppm. A novel near-infrared fluorescence spectroscopic method was utilized to measure and characterize the body burdens of pristine SWNTs in nondepurated and depurated organisms. We did not detect SWNTs in depurated organisms but quantified them in nondepurated A. abdita fed SWNT-amended algae. After a 28-d exposure to [(14) C]SWNT-amended sediment (100 µg/g) and algae (100 µg/g), [(14) C]SWNT was detected in depurated and nondepurated L. plumulosus amphipods at 0.50 µg/g and 5.38 µg/g, respectively. The results indicate that SWNTs are bioaccessible to marine benthic organisms but do not appear to accumulate or cause toxicity., (Copyright © 2013 SETAC.)

The principal aim of this work was to explore the responses of the groundwater crustacean Niphargus rhenorhodanensis to oxidative stress caused by short- and long-term drastic variations in oxygen level. To this end, we investigated thiobarbituric acid reactive substances (TBARS) levels and anti-oxidative enzyme (SOD and GPx) activities during 24 h anoxia and post-anoxia recovery, and during 10 days of severe hypoxia and post-hypoxia recovery. We observed a decrease in TBARS amounts during recovery from severe hypoxia. Parallel to these results, we observed an overactivation of SOD activity after a 24 h anoxic stress. GPx activity measured at the end of anoxia or severe hypoxia and in the early hours of post-stress recovery also showed an overactivation compared to the control group. We can hypothesize that this overproduction of GPx corresponded to an anticipatory mechanism coping with the overproduction of reactive oxygen species (ROS) during the recovery phase in subterranean animals. This response could be considered as a major asset for life in alternately normoxic and hypoxic conditions, and therefore in extreme biotopes such as groundwaters., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The effect of different seasons on sterol content of seafoods was investigated. There were four sterols (cholesterol, sitosterol, desmosterol and stigmasterol) identified, with cholesterol being the predominant sterol. Stigmasterol was a minor component in fish muscle, whilst sitosterol was one of the main phytosterols found in fish muscle. Cholesterol content of fish consisted of 38-100% of total sterols in fish and 54-80% of total sterols in shellfish. The highest cholesterol content of fish muscle was found in summer and the lowest in autumn, whereas season did not have any effect on cholesterol level of green tiger prawn and speckled shrimp. Total sterol content of fish muscle ranged from 49 to 110 mg/100 g, although the range of total sterols in shrimp muscle was between 62 and 91 mg/100 g. The result of the study showed that total sterols in fish were generally found at lower levels in winter compared with other seasons.

Sediment amendment with activated carbon (AC) is a promising technique for in situ sediment remediation. To date it is not clear whether this technique sufficiently reduces sediment-to-water fluxes of sediment-bound hydrophobic organic chemicals (HOCs) in the presence of bioturbators. Here, we report polychlorobiphenyl (PCB) pore water concentrations, fluxes, mass transfer coefficients, and survival data of two benthic species, for four treatments: no AC addition (control), powdered AC addition, granular AC addition and addition and subsequent removal of GAC (sediment stripping). AC addition decreased mass fluxes but increased apparent mass transfer coefficients because of dissolved organic carbon (DOC) facilitated transport across the benthic boundary layer (BBL). In turn, DOC concentrations depended on bioturbator activity which was high for the PAC tolerant species Asellus aquaticus and low for AC sensitive species Lumbriculus variegatus. A dual BBL resistance model combining AC effects on gradients, DOC facilitated transport and biodiffusion was evaluated against the data and showed how the type of resistance differs with treatment and chemical hydrophobicity. Data and simulations illustrate the complex interplay between AC and contaminant toxicity to benthic organisms and how differences in species tolerance affect mass fluxes from sediment to the water column.

With a model invertebrate animal, we have assessed the fate of magnetic nanoparticles in biologically relevant media, i.e., digestive juices. The toxic potential and the internalization of such nanoparticles by nontarget cells were also examined. The aim of this study was to provide experimental evidence on the formation of Co(2+), Fe(2+), and Fe(3+) ions from CoFe₂O₄ nanoparticles in the digestive juices of a model organism. Standard toxicological parameters were assessed. Cell membrane stability was tested with a modified method for measurement of its quality. Proton-induced X-ray emission and low energy synchrotron radiation X-ray fluorescence were used to study internalization and distribution of Co and Fe. Co(2+) ions were found to be more toxic than nanoparticles. We confirmed that Co(2+) ions accumulate in the hepatopancreas, but Fe(n+) ions or CoFe₂O₄ nanoparticles are not retained in vivo. A model biological system with a terrestrial isopod is suited to studies of the potential dissolution of ions and other products from metal-containing nanoparticles in biologically complex media.

Metal concentrations were evaluated in water, bottom sediments, and biota in four field campaigns from 2002 to 2004 in the Potiguar Basin, northeastern Brazil, where offshore oil exploration occurs. Analyses were performed by inductively coupled plasma mass spectrometry and inductively coupled plasma optical emission spectrometry. Total metal concentrations in water (dissolved + particulate) and sediments were in the range expected for coastal and oceanic areas. Abnormally high concentrations in waters were only found for Ba (80 μg l(-1)) and Mn (12 μg l(-1)) at the releasing point of one of the outfalls, and for the other metals, concentrations in water were found in stations closer to shore, suggesting continental inputs. In bottom sediments, only Fe and Mn showed abnormal concentrations closer to the effluent releasing point. Metal spatial distribution in shelf sediments showed the influence of the silt-clay fraction distribution, with deeper stations at the edge of the continental shelf, which are much richer in silt-clay fraction showing higher concentrations than shallower sediments typically dominated by carbonates. Metal concentrations in estuarine (mollusks and crustaceans) and marine (fish) organisms showed highest concentrations in oysters (Crassostrea rhizophorae). Fish tissues metal concentrations were similar between the continental shelf influenced by the oil exploration area and a control site. The results were within the range of concentrations reported for pristine environments without metals contamination. The global results suggest small, if any, alteration in metal concentrations due to the oil exploration activity in the Potiguar Basin. For monitoring purposes, the continental inputs and the distribution of the clay-silt fraction need to be taken into consideration for interpreting environmental monitoring results.

Background: Gammarus minus, a freshwater amphipod living in the cave and surface streams in the eastern USA, is a premier candidate for studying the evolution of troglomorphic traits such as pigmentation loss, elongated appendages, and reduced eyes. In G. minus, multiple pairs of genetically related, physically proximate cave and surface populations exist which exhibit a high degree of intraspecific morphological divergence. The morphology, ecology, and genetic structure of these sister populations are well characterized, yet the genetic basis of their morphological divergence remains unknown., Results: We used degenerate PCR primers designed to amplify opsin genes within the subphylum Crustacea and discovered two distinct opsin paralogs (average inter-paralog protein divergence ≈ 20%) in the genome of three independently derived pairs of G. minus cave and surface populations. Both opsin paralogs were found to be related to other crustacean middle wavelength sensitive opsins. Low levels of nucleotide sequence variation (< 1% within populations) were detected in both opsin genes, regardless of habitat, and dN/dS ratios did not indicate a relaxation of functional constraint in the cave populations with reduced or absent eyes. Maximum likelihood analyses using codon-based models also did not detect a relaxation of functional constraint in the cave lineages. We quantified expression level of both opsin genes and found that the expression of both paralogs was significantly reduced in all three cave populations relative to their sister surface populations., Conclusions: The concordantly lowered expression level of both opsin genes in cave populations of G. minus compared to sister surface populations, combined with evidence for persistent purifying selection in the cave populations, is consistent with an unspecified pleiotropic function of opsin proteins. Our results indicate that phototransduction proteins such as opsins may have retained their function in cave-adapted organisms because they may play a pleiotropic role in other important processes that are unrelated to vision.

Background: Environmental signals to maternal organisms can result in developmental alterations in progeny. One such example is environmental sex determination in Branchiopod crustaceans. We previously demonstrated that the hormone methyl farnesoate could orchestrate environmental sex determination in the early embryo to the male phenotype. Presently, we identify a transcription factor that is activated by methyl farnesoate and explore the extent and significance of this transgenerational signaling pathway., Methodology/principal Findings: Several candidate transcription factors were cloned from the water flea Daphnia pulex and evaluated for activation by methyl farnesoate. One of the factors evaluated, the complex of two bHLH-PAS proteins, dappuMet and SRC, activated a reporter gene in response to methyl farnesoate. Several juvenoid compounds were definitively evaluated for their ability to activate this receptor complex (methyl farnesoate receptor, MfR) in vitro and stimulate male sex determination in vivo. Potency to activate the MfR correlated to potency to stimulate male sex determination of offspring (pyriproxyfen>methyl farnesoate>methoprene, kinoprene). Daphnids were exposed to concentrations of pyriproxyfen and physiologic responses determined over multiple generations. Survivial, growth, and sex of maternal organisms were not affected by pyriproxyfen exposure. Sex ratio among offspring (generation 2) were increasingly skewed in favor of males with increasing pyriproxyfen concentration; while, the number of offspring per brood was progressively reduced. Female generation 2 daphnids were reared to reproductive maturity in the absence of pyriproxyfen. Sex ratios of offspring (generation 3) were not affected in this pyriproxyfen lineage, however, the number of offspring per brood, again, was significantly reduced., Conclusions: Results reveal likely components to a hormone/receptor signaling pathway in a crustacean that orchestrates transgenerational modifications to important population metrics (sex ratios, fecundity of females). A model is provided that describes how these signaling processes can facilitate population sustainability under normal conditions or threaten sustainability when perturbed by environmental chemicals.

In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 μg/L, and no effects against the molting process and nymphal development were scored. In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100 μg/L) in vivo., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Absorption of elevated atmospheric CO2 is causing surface ocean pH to decline, a process known as ocean acidification (OA). To date, few studies have assessed the physiological impacts of OA on early life-history stages of intertidal organisms, which transition from habitats with fluctuating pH (intertidal zone) to relatively stable (pelagic zone) pH environments. We used the intertidal crab Petrolisthes cinctipes to determine whether metabolic responses to year 2300 predictions for OA vary among early developmental stages and to examine whether the effects were more pronounced in larval stages developing in the open ocean. Oxygen consumption rate, total protein, dry mass, total lipids and C/N were determined in late-stage embryos, zoea I larvae and newly settled juveniles reared in ambient pH (7.93 ± 0.06) or low pH (7.58 ± 0.06). After short-term exposure to low pH, embryos displayed 11% and 6% lower metabolism and dry mass, respectively, which may have an associated bioenergetic cost of delayed development to hatching. However, metabolic responses appeared to vary among broods, suggesting significant parental effects among the offspring of six females, possibly a consequence of maternal state during egg deposition and genetic differences among broods. Larval and juvenile metabolism were not affected by acute exposure to elevated CO2. Larvae contained 7% less nitrogen and C/N was 6% higher in individuals reared at pH 7.58 for 6 days, representing a possible switch from lipid to protein metabolism under low pH; the metabolic switch appears to fully cover the energetic cost of responding to elevated CO2. Juvenile dry mass was unaffected after 33 days exposure to low pH seawater. Increased tolerance to low pH in zoea I larvae and juvenile stages may be a consequence of enhanced acid-base regulatory mechanisms, allowing greater compensation of extracellular pH changes and thus preventing decreases in metabolism after exposure to elevated PCO2. The observed variation in responses of P. cinctipes to decreased pH in the present study suggests the potential for this species to adapt to future declines in near-shore pH.

So far, there is limited information on biotransformation mechanisms and products of polar contaminants in freshwater crustaceans. In the present study, metabolites of biocides and pharmaceuticals formed in Gammarus pulex and Daphnia magna were identified using liquid chromatography-high resolution mass spectrometry. Different confidence levels were assigned to the identification of metabolites without reference standards using a framework based on the background evidence used for structure elucidation. Twenty-five metabolites were tentatively identified for irgarol, terbutryn, tramadol, and venlafaxine in G. pulex (21 via oxidation and 4 via conjugation reactions) and 11 metabolites in D. magna (7 via oxidation and 4 via conjugation reactions), while no evidence of metabolites for clarithromycin and valsartan was found. Of the 360 metabolites predicted for the four parent compounds using pathway prediction systems and expert knowledge, 23 products were true positives, while 2 identified metabolites were unexpected products. Observed oxidative reactions included N- and O-demethylation, hydroxylation, and N-oxidation. Glutathione conjugation of selected biocides followed by subsequent reactions forming cysteine conjugates was described for the first time in freshwater invertebrates.

Juvenile Pacific white shrimp (Litopenaeus vannamei) were exposed to trifluralin at 0.1 and 0.01 mg L(-1) for 72 h under controlled conditions. Samples of shrimp and tank water were collected at intervals up to 48 days after exposure. Analysis of the shrimp tissues by gas chromatography-mass spectrometry (GC-MS) and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-qToF-MS) in combination with profiling and metabolite identification software (Agilent MET-ID and Mass Profiler Professional) detected the presence of parent trifluralin together with two main transformation products (TPs), 2-ethyl-7-nitro-5-(trifluoromethyl)benzimidazole (TP1) and 2-amino-6-nitro-4-(trifluoromethyl)phenyl)propylamine (TP2). The highest concentration of trifluralin, determined by GC-MS, was 120 μg kg(-1) at 0 day withdrawal. Residues of trifluralin (CCα = 0.25 μg kg(-1), CCβ = 0.42 μg kg(-1)) were detectable for up to 7 days after exposure. Similarly, the highest concentrations of TP1 and TP 2, determined by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), were 14 and 18 μg kg(-1), respectively. Residues of TP1 (CCα = 0.05 μg kg(-1), CCβ = 0.09 μg kg(-1)) and TP2 (CCα = 0.1 μg kg(-1), CCβ = 0.17 μg kg(-1)) were detectable for up to 4 and 24 withdrawal days, respectively.

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops., Competing Interests: The authors have declared that no competing interests exist.

Top predators like the Neotropical otter, Lontra longicaudis annectens, are usually considered good bioindicators of habitat quality. In this study, we evaluated heavy metal contamination (Hg(tot), Pb, Cd) in the riverine habitat, prey (crustaceans and fish), and otter feces in two Ramsar wetlands with contrasting upstream contamination discharges: Río Blanco and Río Caño Grande in Veracruz, Mexico, during the dry, the wet, and the nortes seasons. Most comparisons revealed no differences between sites while seasonal differences were repeatedly detected for all of the compartments. Higher concentrations of Pb during the dry season and of Cd during the wet season in otter feces mirrored differences detected in the most seasonally consumed prey. Compared with fecal methylmercury values reported for the European otter (0.25-0.75 mg kg(-1)) in unprotected areas, the Hg(tot) levels that we measured were lower (0.02-0.17 mg kg(-1)). However, Pb (117.87 mg kg(-1)) and Cd (9.14 mg kg(-1)) concentrations were higher (Pb, 38.15 mg kg(-1) and Cd, 4.72 mg kg(-1)) in the two Ramsar wetlands. Protected areas may shelter species, but those with water-linked diets may suffer the effect of chemicals used upstream.

Fluoranthene (FLU) is a priority polycyclic aromatic hydrocarbon (PAH) commonly detected in estuarine sediments, water and biota. Despite this, information on FLU detection, accumulation and effects on marine crustaceans is scarce. This work investigated the accumulation of FLU in Carcinus maenas and the responses of several early-warning biomarkers after a 7-day laboratory exposure to five FLU concentrations (2.56 to 100 μg L(-1)). After exposure to FLU, sub-samples of the crabs' digestive gland and muscle were collected for biomarker determinations. The remaining digestive gland and muscle, together with the rest of the whole-body soft tissues, were analysed for FLU residues by gas chromatography-mass spectrometry (GC-MS). The biomarkers assessed were: i) the quantification of FLU-type compounds by fixed wavelength fluorescence (FF); ii) the activities of glutathione S-transferases (GST) and glutathione reductase (GR), and the levels of total glutathione (GT) and lipid peroxidation (LPO) for oxidative stress; iii) the activity of acetylcholinesterase (AChE) for neurotoxicity; iv) the activities of isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH) enzymes, and total protein, glycogen and lipids as indicators of changes in energy metabolism and storage; and v) the lysosomal membrane stability (LMS) as a measure of cell damage. The results showed strong (R(2)>0.95) concentration-dependent accumulation of FLU residues (as measured by GC-MS) in the remaining whole-body soft tissues and of FLU-type compounds (as measured by FF) in the digestive gland and muscle. A strong positive linear relationship (R(2)=0.91) between FLU residues and FLU-type compounds was also found. Comparing to controls, activities of GST and GR were significantly higher in crabs exposed to ≥16 and ≥40 μg L(-1) FLU, respectively. TG levels and IDH activity showed a significant trend to increase with FLU concentrations whereas AChE activity exhibited the opposite trend. FF measurements in the digestive gland and muscle proved to be an expeditious cost-effective method to assess the uptake and availability of FLU and its metabolites in C. maenas. The results suggest that under continuous environmental exposure, FLU may enhance detoxification and anti-oxidant defences, and cause alterations in the aerobic energy pathway, as well as neuromuscular toxic effects that may increase C. maenas risk of predation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Skeletal muscle fibre size is highly variable, and while diffusion appears to limit maximal fibre size, there is no paradigm for the control of minimal size. The optimal fibre size hypothesis posits that the reduced surface area to volume in larger fibres reduces the metabolic cost of maintaining the membrane potential, and so fibres attain an optimal size that minimizes metabolic cost while avoiding diffusion limitation. Here we examine changes during hypertrophic fibre growth in metabolic cost and activity of the Na⁺-K⁺-ATPase in white skeletal muscle from crustaceans and fishes. We provide evidence for a major tenet of the optimal fibre size hypothesis by demonstrating that larger fibres are metabolically cheaper to maintain, and the cost of maintaining the membrane potential is proportional to fibre surface area to volume. The influence of surface area to volume on metabolic cost is apparent during growth in 16 species spanning a 20-fold range in fibre size, suggesting that this principle may apply widely.

Oxygen is recognized as a structuring factor of metazoan communities in marine sediments. The importance of oxygen as a controlling factor on meiofauna (32 µm-1 mm in size) respiration rates is however less clear. Typically, respiration rates are measured under oxic conditions, after which these rates are used in food web studies to quantify the role of meiofauna in sediment carbon turnover. Sediment oxygen concentration ([O(2)]) is generally far from saturated, implying that (1) current estimates of the role of meiofauna in carbon cycling may be biased and (2) meiofaunal organisms need strategies to survive in oxygen-stressed environments. Two main survival strategies are often hypothesized: 1) frequent migration to oxic layers and 2) morphological adaptation. To evaluate these hypotheses, we (1) used a model of oxygen turnover in the meiofauna body as a function of ambient [O(2)], and (2) performed respiration measurements at a range of [O(2)] conditions. The oxygen turnover model predicts a tight coupling between ambient [O(2)] and meiofauna body [O(2)] with oxygen within the body being consumed in seconds. This fast turnover favors long and slender organisms in sediments with low ambient [O(2)] but even then frequent migration between suboxic and oxic layers is for most organisms not a viable strategy to alleviate oxygen limitation. Respiration rates of all measured meiofauna organisms slowed down in response to decreasing ambient [O(2)], with Nematoda displaying the highest metabolic sensitivity for declining [O(2)] followed by Foraminifera and juvenile Gastropoda. Ostracoda showed a behavioral stress response when ambient [O(2)] reached a critical level. Reduced respiration at low ambient [O(2)] implies that meiofauna in natural, i.e. suboxic, sediments must have a lower metabolism than inferred from earlier respiration rates conducted under oxic conditions. The implications of these findings are discussed for the contribution of meiofauna to carbon cycling in marine sediments.

A number of studies have found high levels of mercury (Hg) in deep-sea organisms throughout the world's oceans, but the underlying causes are not clear as there is no consensus on the origin and cycling of Hg in the ocean. Recent findings suggested that Hg accumulation may increase with increasing forage depth and pointed to the deep-water column as the origin of most Hg in marine biota, especially its organic methylmercury (MeHg) form. In the present study, we determined the total mercury (THg) levels in 12 deep-sea fish species and a decapod crustacean and investigated their relationship with the species' nitrogen stable isotope ratio (δ(15)N) as an indicator of their trophic level, average weight and habitat depth. THg levels ranged from 0.27 to 4.42 μg/g w.w. and exceeded in all, except one species, the recommended 0.5 μg/g w.w. guideline value. While THg levels exhibited a strong relationship with δ(15)N values and to a lesser extent with weight, the habitat depth, characterized as the species' depth of maximum abundance (DMA), had also a significant effect on Hg accumulation. The fish species with a shallower depth range exhibited lower THg values than predicted by their trophic level (δ(15)N) and body mass, while measured THg values were higher than predicted in deeper-dwelling fish. Overall, the present results point out a potential risk for human health from the consumption of deep-sea fish. In particular, for both, the red shrimp Aristeus antennatus, which is one of the most valuable fishing resources of the Mediterranean, as well as the commercially exploited fish Mora moro, THg levels considerably exceeded the recommended 0.5μg/g w.w. limit and should be consumed with caution., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Ponto-Caspian mysid shrimp Limnomysis benedeni has rapidly invaded freshwater systems throughout Europe and is now found in extremely high abundances in invaded systems. However, very little is known about the trophic ecology of this mysid in invaded ecosystems, in particular the relative degree of herbivory, carnivory and detritivory of this potentially omnivorous species and where it derives its energy, i.e. via carbon fixed by algae inhabiting benthic or pelagic habitats or through allochthonous inputs. Here, we investigate the trophic ecology of L. benedeni in a recently established population in North-Western Germany using stable carbon and nitrogen isotopes. Our results suggest that in contrast to its previous classifications as a benthic or bentho-pelagic herbivore, L. benedeni is an omnivorous species, which can derive the bulk of its carbon from pelagic resources. Its trophic niche in different invaded ecosystems will be determined by multiple, system-dependent factors which have to be considered in order to predict the mysids' invasion potential.

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

The present study reports the level of total petroleum hydrocarbon (TPH) in 27 commercially important fish, crustaceans and cephalopods from Orissa coast, Bay of Bengal to provide the baseline data for oil contamination in marine biota. The results showed significant differences in the TPH concentration in different species. Of all fish, Carangoides malabaricus showed the maximum TPH level (av. 13.70 μg g(-1)). In cephalopods, Loligo recorded the maximum TPH (av. 14.87 μg g(-1)) while Metapenaeus dobsoni recorded the maximum TPH (av. 13.18 μg g(-1)) among crustaceans. The concentration of TPH recorded in the present study were higher than the reported values from other studies in Bay of Bengal but were comparable with those of other Indian coast and Gulf water. The species recording maximum concentration and giving significant correlation coefficient may be of interest in oil pollution monitoring and can be used as indicator of hydrocarbon pollution in the region., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Due to the over-harvesting and deterioration of wild populations, the ever-growing crustacean market is increasingly reliant on aquaculture, driving the need for better management techniques. Since most cultured crustacean species exhibit sexually dimorphic growth patterns, the culture of monosex populations (either all-male or all-female) is a preferred approach for gaining higher yields, with the ecological benefit of reducing the risk of invasion by the cultured species. Sexual manipulations may also render sustainable solutions to the environmental problems caused by the presence of invasive crustacean species with detrimental impacts ranging from aggressive competition with native species for food and shelter, to affecting aquaculture facilities and harvests and causing structural damage to river banks. Recent discoveries of androgenic gland (AG)-specific insulin-like peptides (IAGs) in crustaceans and the ability to manipulate them and their encoding transcripts (IAGs) have raised the possibility of sexually manipulating crustacean populations. Sexual manipulation is already a part of sustainable solutions in fish aquaculture and in the bio-control of insect pest species, and attempts are also being made to implement it with crustaceans. As recently exemplified in a commercially important prawn species, IAG silencing, a temporal, non-genetically modifying and non-transmissible intervention, has enabled the production of non-breeding all-male monosex populations that are the progeny of sexually reversed males ('neo-females'). IAG manipulations-based biotechnologies therefore have the potential to radically transform the entire industry. We review here how this proof of concept could be broadened to meet both aquacultural and environmental needs. We include the major cultured decapod crustacean groups and suggest a sustainable solution for the management of invasive and pest crustacean species. We also review the key considerations for devising a biotechnological approach that specifically tailors the molecular technological abilities to the management of each target group., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cave animals are excellent models to study the general principles of evolution as well as the mechanisms of adaptation to a novel environment: the perpetual darkness of caves. In this article, two of the major model systems used to study the evolution and development (evo-devo) of cave animals are described: the teleost fish Astyanax mexicanus and the isopod crustacean Asellus aquaticus. The ways in which these animals match the major attributes expected of an evo-devo cave animal model system are described. For both species, we enumerate the regressive and constructive troglomorphic traits that have evolved during their adaptation to cave life, the developmental and genetic basis of these traits, the possible evolutionary forces responsible for them, and potential new areas in which these model systems could be used for further exploration of the evolution of cave animals. Furthermore, we compare the two model cave animals to investigate the mechanisms of troglomorphic evolution. Finally, we propose a few other cave animal systems that would be suitable for development as additional models to obtain a more comprehensive understanding of the developmental and genetic mechanisms involved in troglomorphic evolution.

A systemic study on the natural radionuclides such as (210)Po and (210)Pb in the environmental matrices and biota of Ennore Creek has been undertaken to establish a baseline data on the radiation profile of Ennore Creek environment. The environmental samples such as water, sediment, and biota (seaweeds, molluscs, crustaceans, and fishes) have been subjected to analyses. It has been observed that the concentration of (210)Po and (210)Pb in the water samples of Ennore Creek as 2.7 and 1.63 m Bq L(-1), respectively. The activity concentration of (210)Po and (210)Pb in the sediment sample was 17.9 and 28.9 Bq kg(-1), respectively. The bivalve mollusk Perna viridis have been identified to accumulate higher concentrations of (210)Po and (210)Pb suggesting that they could serve as bioindicator of radionuclides in the Ennore Creek environment. The committed effective dose for human beings was found at 81.13-216.8 and 2.1-297.2 μSv year(-1) for (210)Po and (210)Pb, respectively.

Studies on the digestive secretions in aquatic animals can elucidate certain aspects of their nutritive physiology. The aim of the present study was to compare the digestive lipase and phospholipase activities in ten marine species belonging to four classes following the taxonomic classification of marine organisms. All aquatic digestive tissues tested are equipped with lipase and phospholipase activities, assuming the hydrolysis of fat-rich food. The lipolytic activities determined in the pancreases of cartilaginous fishes were greater than those in bony fishes, molluscs and crustaceans. This finding might be explained by the strong digestive utilization of fat-rich macronutrients by these carnivorous fishes. A trend of activities and stabilities at different pH and temperatures for crude lipases and phospholipases from these aquatic animals suggests that the optimum pH and temperature for marine lipases are species dependent. Interestingly, the sardine caecal lipase and phospholipase were found to be mostly stable in a broad range of acidic pH values. The maximum activities of lipolytic enzymes from the hepatopancreases of Hexaplex trunculus (molluscs) and Carcinus mediterranus (crustaceans) were found to be 50 and 60 °C, respectively, whereas the optimal temperature of lipolytic enzymes for the other species was classically around 40 °C. Thermoactivity of molluscs' lipolytic preparations makes them potential candidates in industrial applications. Among digestive glands studied, only pancreas (cartilaginous fish) contained the classically known colipase. Regarded as the most primitive living jawed vertebrates, cartilaginous fishes represented by sharks and rays could be considered as the oldest vertebrates possessing a complex digestive system like that of mammals.

A total of 250 chitinolytic bacteria from 68 different marine samples were screened employing enrichment method that utilized native chitin as the sole carbon source. After thorough screening, five bacteria were selected as potential cultures and identified as; Stenotrophomonas sp. (CFR221 M), Vibrio sp. (CFR173 M), Phyllobacteriaceae sp. (CFR16 M), Bacillus badius (CFR198 M) and Bacillus sp. (CFR188 M). All five strains produced extracellular chitinase and GlcNAc in SSF using shrimp bio-waste. Scanning electron microscopy confirmed the ability of these marine bacteria to adsorb onto solid shrimp bio-waste and to degrade chitin microfibers. HPLC analysis of the SSF extract also confirmed presence of 36-65 % GlcNAc as a product of the degradation. The concomitant production of chitinase and GlcNAc by all five strains under SSF using shrimp bio-waste as the solid substrate was optimized by 'one factor at a time' approach. Among the strains, Vibrio sp. CFR173 M produced significantly higher yields of chitinase (4.8 U/g initial dry substrate) and GlcNAc (4.7 μmol/g initial dry substrate) as compared to other cultures tested. A statistically designed experiment was applied to evaluate the interaction of variables in the biodegradation of shrimp bio-waste and concomitant production of chitinase and GlcNAc by Vibrio sp. CFR173 M. Statistical optimization resulted in a twofold increase of chitinase, and a 9.1 fold increase of GlcNAc production. These results indicated the potential of chitinolytic marine bacteria for the reclamation of shrimp bio-waste, as well as the potential for economic production of chitinase and GlcNAc employing SSF using shrimp bio-waste as an ideal substrate.

This study determined the concentrations of eleven metals in the blue crab, Callinectes danae, from nine sites in the Santos Estuarine System of Sao Paulo State, Brazil. The results were compared to guidelines established in the United States, Europe and Brazil for the safety of human consumers. Muscles of blue crabs were removed by dissection and concentrations of Al, Cd, Co, Cr, Cu, Fe, Hg, Mn Ni, Pb and Zn were determined. In general, the concentrations of metals were low, and the crabs were regarded as safe for human consumption. Crabs from a single site (site 4) exceeded the guidelines established by the United States and Europe, but not Brazil, for Pb, with a mean tissue concentration of 1.725 μg g(-1). With the exception of Al, Fe and Ni, significant differences were noted between sites in the concentrations of each metal in crab tissue.

The concentrations of 16 perfluorinated compounds (PFCs) were measured in surface water and organisms from Baiyangdian Lake. Perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid, and perfluorobutanoic acid (PFBA) were the major PFC species in the water at 6.8-56.8, 0.1-17.5 and 3.0-14.6 ng/L, respectively. The PFC contamination in Baiyangdian Lake was significantly impacted by the inflow from Pinghe River and Fuhe River. There was no significant correlation between the concentrations of PFCs and the trophic levels in aquatic organisms. There were no significant PFC risks in Baiyangdian Lake according to the risk assessment of PFCs in water.

Crab wastes are employed for simultaneous production of chitin and L(+)-lactic acid by submerged fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source. Response surface methodology was applied to design the culture media considering demineralization. Fermentations in stirred tank reactor (2L) using selected conditions produced 88% demineralization and 56% deproteinization with 34% yield of chitin and 19.5 gL(-1) of lactic acid (77% yield). The chitin purified from fermentation displayed 95% degree of acetylation and 0.81 and 1 ± 0.125% of residual ash and protein contents, respectively.

The blue crab Callinectes sapidus has been used as an experimental model organism for the study of regulation of cardiac activity and other physiological processes. Moreover, it is an economically and ecologically important crustacean species. However, there was no previous report on the characterization of its neuropeptidome. To fill in this gap, we employed multiple sample preparation methods including direct tissue profiling, crude tissue extraction and tissue extract fractionation by HPLC to obtain a complete description of the neuropeptidome of C. sapidus. Matrix-assisted laser desorption/ionization (MALDI)-Fourier transform mass spectrometry (FTMS) and MALDI-time-of-flight (TOF)/TOF were utilized initially to obtain a quick snapshot of the neuropeptide profile, and subsequently nanoflow liquid chromatography (nanoLC) coupled with electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem MS analysis of neuropeptide extracts was conducted for de novo sequencing. Simultaneously, the pericardial organ (PO) tissue extract was labeled by a novel N,N-dimethylated leucine (DiLeu) reagent, offering enhanced fragmentation efficiency of peptides. In total, 130 peptide sequences belonging to 11 known neuropeptide families including orcomyotropin, pyrokinin, allatostatin A (AST-A), allatostatin B (AST-B), FMRFamide-like peptides (FLPs), and orcokinin were identified. Among these 130 sequences, 44 are novel peptides and 86 are previously identified. Overall, our results lay the groundwork for future physiological studies of neuropeptides in C. sapidus and other crustaceans., (Copyright © 2012 Elsevier Inc. All rights reserved.)