Diacylglycerol acyltransferase (DGAT) in Crangon crangon and Pandalus montagui (Decapoda, Caridea) - Implications for lipid storage capacities and life history traits.

Lipids play essential roles in cell-structuring, cell-signaling, and as efficient metabolic energy stores. Lipid storage capacities determine life history traits of organisms and, thus, their ecological function. Among storage lipids, triacylglycerols (TAGs) are widespread in marine invertebrates. However, abilities to accumulate TAGs can vary even between closely related species, such as the caridean shrimps Crangon crangon and Pandalus montagui. The first species shows low TAG levels throughout the year in the main storage organ, the midgut gland, while the latter accumulates high TAG-levels, peaking in summer. TAGs synthesis is facilitated by the terminal step of the Kennedy-pathway, where the enzyme diacylglycerol-acyltransferase (DGAT) catalyzes the esterification of diacylglycerols with activated fatty acids. We investigated DGAT activity in the midgut gland using a fluorescent enzyme assay. Sequence information was extracted from whole transcriptome shotgun assembly data, that is publicly available on NCBI, and catalytic properties were deduced from molecular structure analysis. C. crangon showed significantly lower TAG synthesis rates than P. montagui, which explains the native TAG levels. Transcriptome data yielded several isoforms of DGAT enzymes in both species. C. crangon DGAT showed point mutations, which are capable of obstructing the catalytic capacity. The consequences are limited starvation resistance and, thus, presumably restricting C. crangon to a habitat with year-round sufficient food. In contrast, higher TAG synthesis rates presumably enable P. montagui to extend into northern subarctic habitats with limited food availability in winter. Moreover, the limited TAG synthesis and accumulation in the midgut gland may force C. crangon to direct energy into the ovaries, which results in multiple spawnings.

Responses of digestive enzyme profiles to various scenarios of food availability in newly-hatched Stage I phyllosoma larvae of the tropical spiny lobster Panulirus ornatus.

Stage I phyllosoma larvae of the spiny lobster Panulirus ornatus hatch in tropical oceanic waters with limited and variable food resources. To better understand how these larvae cope with food deprivation, this study examined specific (mU mg-1 protein) and total (mU larva-1) activities of major digestive enzymes (i.e., α-amylase, non-specific esterase, trypsin-like protease) during a series of ex situ experiments. Specifically, temporal changes in enzyme profiles were examined in three experiments simulating scenarios in which phyllosoma hatch and begin development in an environment where (1) prey is either continuously present or absent; (2) prey is initially present, but subsequently absent; (3) prey is initially absent, but subsequently present. Results indicated that the accessibility of suitable prey provides an overarching influence on digestive enzyme activities and substrate utilisation in Stage I phyllosoma of P. ornatus, with enzymatic responses to both intermittent prey availability and food deprivation being influenced by their nutritional history. In the absence of prey, larval digestive enzyme activities (mU larva-1) initially remained static from hatch but eventually declined with extended food deprivation. When prey became available, enzyme activities increased, with delayed access to prey having minimal impact on this enzymatic response. Furthermore, phyllosoma were able to adjust α-amylase and non-specific esterase activities (within 8 h) in response to the disappearance of prey, demonstrating adaptive changes to endure periods of food deprivation that were based on their nutritional history. The ability of phyllosoma to regulate enzyme activities from hatch is an important physiological strategy allowing them to survive in an environment characterised by highly variable zooplankton biomass and abundance, and explains why diets that differ greatly in nutrient composition have consistently been found effective for rearing phyllosoma in captivity.

Molecular aspects of lipid metabolism in the midgut gland of the brown shrimp Crangon crangon.

The brown shrimp, Crangon crangon, is well adapted to the variable environmental conditions in the southern North Sea. It is very abundant, has high reproduction rates, and holds a key position in coastal ecosystems. This species has very low lipid deposits in the midgut gland, suggesting that the main function of the midgut gland is metabolic turnover rather than energy storage. Based on seasonal gene expression studies and established transcriptome data, we investigated key components of lipid metabolic pathways. Gene expression of triacylglycerol lipase, phospholipase, and fatty acid desaturase were analyzed and compared with that of other digestive enzymes involved in lipid, carbohydrate, and protein catabolism. Our results suggest that gene expression of digestive enzymes involved in lipid metabolism is modulated by the lipid content in the midgut gland and is related to food availability. Brown shrimp seem to be capable of using cellular phospholipids during periods of food paucity but high energetic (lipid) requirements. Two of three isoforms of fatty acid binding proteins (FABPs) from the midgut gland involved in fatty acid transport showed specific mutations of the binding site. We hypothesize that the mutations in FABPs and deficiencies in anabolic pathways limit lipid storage capacities in the midgut gland of C. crangon. In turn, food utilization, including lipid catabolism, has to be efficient to fulfill the energetic requirements of brown shrimp.

Metabolic responses to metal pollution in shrimp Crangon affinis from the sites along the Laizhou Bay in the Bohai Sea.

Marine environment in the Laizhou Bay is potentially contaminated by metals from industrial discharges. In this study, metal concentrations in shrimps Crangon affinis indicated that two typical sites (S6283 and S5283) close to Longkou and Zhaoyuan cities along the Laizhou Bay have been contaminated by metals, including Cd, As, Cu, Ni, Co, and Mn. In particular, Cd and As were the main metal contaminants in S6283. In S5283, however, Cu was the most important metal contaminant. The metabolic responses in the shrimps indicated that the metal pollution in S6283 and S5283 induced disturbances in osmotic regulation and energy metabolism and reduced anaerobiosis, lipid metabolism, and muscle movement. However, alteration in the levels of dimethylglycine, dimethylamine, arginine, betaine, and glutamine indicated that the metal pollution in S5283 induced osmotic stress through different pathways compared to that in S6283. In addition, dimethylamine might be the biomarker of Cu in shrimp C. affinis.

Impact of metal pollution on shrimp Crangon affinis by NMR-based metabolomics.

Both cadmium and arsenic are the important metal/metalloid pollutants in the Bohai Sea. In this work, we sampled the dominant species, shrimp Crangon affinis, from three sites, the Middle of the Bohai Sea (MBS), the Yellow River Estuary (YRE) and the Laizhou Bay (LZB) along the Bohai Sea. The concentrations of metals/metalloids in shrimps C. affinis indicated that the YRE site was polluted by Cd and Pb, while the LZB site was contaminated by As. The metabolic differences between shrimps C. affinis from the reference site (MBS) and metal-pollution sites (YRE and LZB) were characterized using NMR-based metabolomics. Results indicated that the metal pollutions in YRE and LZB induced disturbances in osmotic regulation and energy metabolism via different metabolic pathways. In addition, a combination of alanine and arginine might be the biomarker of Cd contamination, while BCAAs and tyrosine could be the biomarkers of arsenic contamination in C. affinis.

Effect of seasonal and experimental temperature on de novo synthesis of fatty acids in C. crangon.

The intensity of in vivo lipogensis was measured and in this purpose, the radioactivity of incorporation of tritium into fatty acids (FAs) in tissues of C. crangon was determined. De novo synthesis of FAs was five times higher in hepatopancreas than in muscle in summer period but not much higher in autumn. The higher FAs synthesis was recorded at 25 °C, both for hepatopancreas and muscle, and the summer was higher than the autumn in the hepatopancreas and in the muscles of the opposite situation was observed. The higher amounts of SFAs in hepatopancreas from autumn, when in experimental conditions the ambient temperature C. crangon changed from 6 °C to the experimental higher temperature. When content of PUFAn-3 declined dramatically (Autumn 1 h, 25 °C). In contrast, at a lower temperature, the amount of polyunsaturated FAs is much higher than at 25 °C (Autumn 1 h 6 °C).

Absence of Ca2+-induced mitochondrial permeability transition but presence of bongkrekate-sensitive nucleotide exchange in C. crangon and P. serratus.

Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca(2+)-induced permeability transition in the presence of a profound Ca(2+) uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca(2+)-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca(2+)-induced permeability transition. Ca(2+) uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca(2+)-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca(2+)-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena.

Selenium reduces the retention of methyl mercury in the brown shrimp Crangon crangon.

Methyl mercury accumulated at the top of aquatic food chains constitutes a toxicological risk to humans and other top predators. Because the methyl mercury enters the aquatic food chains at the lower trophic levels, uptake and elimination processes at these levels affect the methyl mercury content at the higher levels. Selenium modulates the biokinetics of mercury in aquatic organisms in fairly complex ways, increasing mercury retention in some aquatic mammals, but decreasing methyl mercury retention in fish. However, it is not known if selenium modulates methyl mercury accumulation at lower trophic levels in aquatic food chains. Here, we show that selenium administered via the food augments the elimination of methyl mercury from marine shrimp and that the effect is dose-dependent, demonstrable down to natural selenium concentrations in aquatic food items. Selenite, seleno-cystine, and seleno-methionine exert this effect but selenate does not. Our results suggest that the selenium naturally present at the lower trophic levels in marine food chains may play an essential role as a modifier of methyl mercury accumulation at these levels, thereby potentially also affecting biomagnification of methyl mercury toward the higher trophic levels in the aquatic food chains.

Organotins in North Sea brown shrimp (Crangon crangon L.) after implementation of the TBT ban.

The organotin (OT) compounds tributyltin (TBT) and triphenyltin (TPhT) are potent biocides that have been used ubiquitously in antifouling paints and pesticides since the mid-1970s. These biocides are extremely toxic to marine life, particularly marine gastropod populations. The European Union therefore took measures to reduce the use of TBT-based antifouling paints on ships and ultimately banned these paints in 2003. Despite sufficient data on OT concentrations in marine gastropods, data are scarce for other species such as the North Sea brown shrimp (Crangon crangon), a dominant crustacean species in North Sea inshore benthic communities. The present study provides the first spatial overview of OT concentrations in North Sea brown shrimp. We have compared these data with historical concentrations in shrimp as well as with sediment concentrations. We have also addressed the effect on the shrimp stock and any human health risks associated with the OT concentrations found. TBT and TPhT in shrimp tail muscle ranged from 4 to 124 and from 1 to 24 µg kg(-1) DW, respectively. High levels are accumulated in estuarine areas and are clearly related with sediment concentrations (biota-sediment accumulation factor ~10). Levels have decreased approximately 10-fold since the ban took effect, coinciding with a recovery of the shrimp stock after 30 years of gradual regression. Furthermore, the OT levels found in brown shrimp no longer present a human health risk.

The heterodimeric ecdysteroid receptor complex in the brown shrimp Crangon crangon: EcR and RXR isoform characteristics and sensitivity towards the marine pollutant tributyltin.

Decapod crustaceans are characterized by multiple ecdysteroid receptor (EcR) and retinoid-X-receptor (RXR) isoforms, which likely exhibit variant dimerization and transactivation interactions. In the brown shrimp C. crangon we cloned C-terminally truncated CrcEcR and CrcRXR isoforms and isoforms exhibiting deletions within the hinge region. For the former, in silico modeling of the CrcEcR indicated that, where the conserved helices H10 and H11 of the ligand-binding domain (LBD) are missing, an alternative C-terminal α-helix repairs the ligand-binding pocket (LBP). The truncated CrcRXR isoforms lack a major part of the LBD (H4-H12), thereby compromising ligand binding and dimerization. Through an in vitro ecdysteroid responsive reporter assay, we showed that these natural receptor variations do not impair receptor functioning but probably alter the receptor dimerization preferences. By the same in vitro assay, using full-length CrcEcR and CrcRXR, the effect of tributyltin (TBT) on ecdysteroid-induced transactivation was evaluated. The transactivation by 10nM PonA was reduced with 64% by 20 nM TBT. In silico modeling confirmed that TBT fits in the full-length CrcRXR-LBD. Furthermore, semi-quantitative PCR indicated altered expression of CrcEcR and CrcRXR isoforms after in vivo acute exposure to TBT, especially in the ovaries.

The brown shrimp (Crangon crangon L.) ecdysteroid receptor complex: cloning, structural modeling of the ligand-binding domain and functional expression in an EcR-deficient Drosophila cell line.

cDNAs encoding ecdysteroid receptor (EcR) and retinoid X receptor (RXR) were cloned and sequenced from brown shrimp Crangon crangon (Crustacea: Decapoda), a common faunal species and commercially important in the North-West European coastal waters. A 3D model of the ligand-binding domain (LBD) of EcR was created and docking of ponasterone A (PonA) was simulated in silico. Finally, we report the transfection of expression plasmids for these receptors in the mutant Drosophila L57-3-11 cell line. Through an ecdysteroid responsive reporter assay we clearly prove the functionality of shrimp ecdysteroid receptor in the transfected L57-3-11 cell line. Our results indicate that the Drosophila L57-3-11 cell line and in silico LBD modeling can be used to study the function of crustacean ecdysteroid receptors and be applied to assess endocrine disrupting effects on non-target crustacean species.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

Bioaccumulation of the new antifoulant medetomidine in marine organisms.

Biofouling is a huge problem globally and new alternative antifoulants are presently being investigated. One candidate is medetomidine, a commonly used sedative in veterinary medicine, which has been shown to effectively prevent settlement of barnacles. The purpose of this study was to measure uptake, elimination and bioconcentration of medetomidine in Mytilus edulis, Abra nitida, Crangon crangon and periphyton communities to evaluate the risk of bioaccumulation in the marine environment. Bioconcentration factors (BCF) and bioaccumulation factors (BAF) were used to assess the bioaccumulation. The calculations of these factors were based on the distribution of the radiolabelled medetomidine. BCF for C. crangon was 2.8 while M. edulis had a BCF of 134 and the periphyton communities' BCF was 1195 l/kg fresh weight (FW). The concentration of medetomidine in the animals reached steady state after 24-48 h for all test systems except for A. nitida, which never stabilised enough to calculate a bioaccumulation factor (BAF). Elimination from the organism's tissues was rapid for three of the test systems with half-lives between 1 and 24 h. A. nitida had a half-life of 96-120 h. This study demonstrates that the bioconcentration and bioaccumulation of medetomidine differs between aquatic organisms and that microalgal communities in the form of periphyton have the highest bioconcentration factor of the organisms tested.

Sediment quality assessment in the Gulf of Gdansk (Baltic Sea) using complementary lines of evidence.

Sediments from Polish coastal environments were classified by a quality assessment approach that took into account trace metal and organic micropollutant concentrations, grain-size distribution, and organic carbon content. Generally, no benthic organisms were found at sites where sediments were classified as heavily polluted. However, areas characterized by a moderate contamination showed a variable composition of the benthic community and changing bioaccumulation patterns; therefore, no single species found in the Gulf of Gdansk could be considered representative of the whole benthic environment. Although sediment monitoring must be considered a suitable tool to detect hot-spot pollution areas in coastal and inland waters, it should be complemented by bioaccumulation measurements to evaluate the actual risk posed by contaminants to benthic organisms. This "biological information" allows a better appreciation of the real benthic infaunal community exposure to chemicals and can usefully complement the existing sediment quality guidelines.

Chitin purification from shrimp wastes by microbial deproteination and decalcification.

Chitin was purified from Penaeus monodon and Crangon crangon shells using a two-stage fermentation process with anaerobic deproteination followed by decalcification through homofermentative lactic acid fermentation. Deproteinating enrichment cultures from sewage sludge and ground meat (GM) were used with a proteolytic activity of 59 and 61 mg N l(-1) h(-1) with dried and 26 and 35 mg N l(-1) h(-1) with wet P. monodon shells. With 100 g wet cells of proteolytic bacteria per liter, protein removal was obtained in 42 h. An anaerobic spore-forming bacterium HP1 was isolated from enrichment GM. Its proteolytic activity was 76 U ml(-1) compared to 44 U ml(-1) of the consortium. Glucose was fermented with Lactobacillus casei MRS1 to lactic acid. At a pH of 3.6, calcium carbonate of the shells was solubilised. After deproteination and decalcification of P. monodon or C. crangon shells, the protein content was 5.8% or 6.7%, and the calcium content was 0.3% or 0.4%, respectively. The viscosity of the chitin from P. monodon and C. crangon was 45 and 135 mPa s, respectively, whereas purchased crab shell chitin (practical grade) had a viscosity of 21 mPa s, indicating a higher quality of biologically purified chitin.

Formation of semicarbazide (SEM) in food by hypochlorite treatment: is SEM a specific marker for nitrofurazone abuse?

Semicarbazide (SEM) is considered to be a characteristic protein-bound side-chain metabolite of the banned veterinary drug nitrofurazone. It is therefore used as a marker for nitrofurazone abuse. Recently, there has been concern about other sources of SEM in tissue samples, which are not linked to the illegal use of nitrofurazone. The present studies have shown that SEM can occur naturally, e.g. in algae, shrimps and eggs, and is formed from natural substances, e.g. arginine and creatine. A significant formation of SEM was observed in samples treated with hypochlorite commonly used in food processing for disinfection or bleaching. SEM was formed in different kinds of nitrogen compound-containing samples (0.3-20 microg kg(-1)) after treatment with 1% active chlorine. It was detected in the mg kg(-1) range after hypochlorite treatment (0.015% active chlorine) of creatine. Lower levels were also formed from creatinine, arginine and urea. SEM present in hypochlorite-treated carrageenan proved mostly to occur in the tissue-bound form. Therefore, differentiation between SEM from nitrofurazone abuse and SEM originating from natural constituents (due to hypochlorite treatment) seems not to be unambiguously possible.