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A pharmacological and behavioural study on beta, beta'-iminodipropionitrile (IDPN)-treated brine shrimp Artemia salina L.

The neurotoxic substance beta, beta-iminodipropionitrile (IDPN), which induces hyperkinesia in some mammals, was found to be neurotoxic to the brine shrimp Artemia salina L. (Crustacea, Anostraca) inducing discoordination of limb-movement and sedation of motility. Up to 9 metabolities of IDPN were detected in Artemia, including beta-alanine, cyanoacetic acid, and beta-aminopropionitrile (BAPN). Brine shrimp, especially nauplius (1 day), may be a useful test animal for the biological assay of IDPN.

Aging↗

Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon.

Penaeid shrimp aquaculture has suffered from many diseases, especially from viral origin such as white spot syndrome virus (WSSV). In an attempt to obtain antiviral-relevant proteins, two peptides with molecular masses at 73 and 75kDa were isolated from shrimp Penaeus monodon using affinity chromatography coupled with the purified WSSV or a fish iridovirus (Singapore grouper iridovirus, SGIV), and identified as hemocyanin by mass spectrometry. The results, using fish viruses capable of cell culture, showed for the first time that the hemocyanin had non-specific antiviral properties and no cytotoxicity against host cells.

Amino Acid Sequence↗

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph.

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca(2+)-dependent. The hemagglutinating activity of FmL was stable up to 55 degrees C and at pH 7.5-8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.

Animals↗

Preparation and functional evaluation of oligopeptide-enriched hydrolysate from shrimp (Acetes chinensis) treated with crude protease from Bacillus sp. SM98011.

Marine organisms are potentially an untapped source of drugs and value-added food production. Currently, Acetes chinensis is an underutilized shrimp species with low commercial value from the Bo Hai Gulf of China. In this paper, the shrimp was digested by a crude protease from Bacillus sp. SM98011 and filtered through a 3 kDa ultrafiltration membrane. Biological functions of the hydrolysate and ultrafiltrate were then assayed. The analyses showed that 41% of the oligopeptides in the ultrafiltrate had a molecular mass lower than 3 kDa. The antioxidant activities of the hydrolysate and ultrafiltrate were determined through the scavenger activity of the hydroxyl radical, with inhibitions of 42.38% and 67.95%, respectively. The hydrolysate and ultrafiltrate also had good Angiotensin-I-converting enzyme (ACE) inhibitory activity, with IC50 values of 0.98 mg/ml and 0.22 mg/ml, respectively. In addition, Chitin and chitosan were recovered from the hydrolytic sediment using a much smaller volume of strong acids and bases than is normally needed. With this method, we have shown that A. chinensis can be utilized to generate a high value-added product, and have revealed its hidden potential in the production of functional foods and ACE inhibitory peptides.

Amino Acids↗

Lime treatment of shrimp head waste for the generation of highly digestible animal feed.

In addition to approximately 20% ash, shrimp processing by-products contain 64% protein and chitin, both of which can be used to generate several valuable products. Chitin and chitosan production is currently based on several crustacean wastes, and at the present time the protein fraction is not being used. This paper describes the thermo-chemical treatment of shrimp processing wastes with lime to generate a protein-rich material with a well-balanced amino acid content that can be used as a monogastric animal feed supplement. The residual solid, rich in calcium carbonate and chitin, can still be used to generate chitin and chitosan through well-established processes.

Animal Feed↗

Inhibition of ovarian development by methyl farnesoate in the tadpole shrimp, Triops longicaudatus.

Methyl farnesoate (MF), a putative crustacean hormone, is the immediate precursor of insect juvenile hormone III (JHIII) in the biosynthetic pathway. We examined whether MF, shown to inhibit adult metamorphosis in several crustacean species, is a juvenilizing factor in the tadpole shrimp, Triops longicaudatus. Oocyte production was chosen as a parameter for measuring reproductive development. MF was administered to juveniles by ingestion via biological vector (Artemia nauplii), MF-coated food pellets, and MF liposome food pellets. Artemia were incubated in 30 microl of 5 microg/ml MF. The MF-coated and MF liposome pellets were prepared with MF concentrations ranging between 0.1 microg/g and 10 microg/g MF by weight. Groups of tadpole shrimp were treated with these vectors from the time of hatching for 5 or 10 days in laboratory and field studies. The treatment groups of all the MF vectors showed reductions in oocyte production. Lower concentrations of MF (0.75 microg/g-3.8 microg/g MF) appeared to have a physiological effect on fecundity, but higher concentrations (10 microg/g MF) reduced somatic growth. MF-coated pellets (1 microg/g MF) administered to adults (after 5 days) caused no difference in oocyte production. The observed reductions of fecundity and the disparity of results between MF treatment on juveniles and adults suggest that MF may regulate ovarian development.

Animals↗

Adaptive patterns of osmotic and ionic regulation, and the invasion of fresh water by the palaemonid shrimps.

To evaluate trends in the osmoregulatory behavior of neotropical, palaemonid shrimps, we investigated osmotic and ionic regulatory patterns in five species of Palaemon or Macrobrachium. The species' life histories depend on saline water to differing degrees, their habitats ranging from the marine/intertidal (P. northropi), through estuaries (P. pandaliformis) to coastal, freshwater streams (M. olfersii, M. potiuna) and inland, continental river systems (M. brasiliense). Hemolymph osmolality, chloride, sodium and magnesium concentrations were measured in shrimps exposed to experimental media ranging from fresh water (<0.5 per thousand ) to concentrated seawater (42 per thousand ) for up to 10 days. The marine and estuarine Palaemon species exhibit well-developed hyper/hypo-osmotic, sodium and chloride regulatory capabilities in mid-range salinities, tending to hyperconform in low salinities. The freshwater Macrobrachium species show variable hyperosmotic, sodium and chloride regulatory capacities, tending to hypoconform or unable to survive at higher salinities. All species hyper-regulate magnesium in fresh water, but hyporegulate strongly in saline media. Palaemonids from the saline habitats show the strongest osmoregulatory capabilities, and fresh water may have been gradually invaded by ancestral species with similar regulatory capacity. However, this regulatory plasticity has been lost to varying degrees in extant freshwater species.

Adaptation, Physiological↗

Increase of uricogenesis in the kuruma shrimp Marsupenaeus japonicus reared under hyper-osmotic conditions.

Tissues of kuruma shrimp Marsupenaeus japonicus Bate (5.7+/-1.1 g) reared in salinities of 18, 26, 34 and 42 were examined for levels of nucleotide-related compounds, ammonia, urea and uric acid, and activities of xanthine dehydrogenase (XDH), xanthine oxidase (XOD) and uricase. Levels of total nucleotide-related compounds, including xanthine and hypoxanthine, in gill increased directly with salinity, whereas these same levels in hepatopancreas were inversely related with salinity. Hemolymph ammonia, urea and uric acid levels, and epidermal ammonia, urea and uric acid levels increased directly with salinity, whereas hepatopancreas ammonia and uric acid and gill uric acid levels were inversely related to salinity. Activities of XDH and XOD in hepatopancreas increased directly with salinity level, whereas no significant difference of uricase activity in hepatopancreas was observed among the four salinities. It is concluded M. japonicus exhibited uricogenesis and uricolysis, and an increase of uricogenesis occurred for the shrimp under hyper-osmotic conditions (salinity of 42). Uric acid produced in the hepatopancreas was transported and accumulated in the epidermis, and removed along with the spongy connective tissue at the time of molting.

Ammonia↗

Shell protein characteristics and vitellogenin-like proteins in brine shrimp Artemia franciscana exposed to municipal effluent and 20-hydroxyecdysone.

We developed biomarkers to monitor the endocrine-disrupting potential of contaminants and municipal effluents in aquatic arthropods. Artemia fransciscana shrimp were cultured and exposed to increasing concentrations of 20-hydroxyecdysone (20HE) and solid phase (C-8) municipal effluent extract (MEE) for 48 h at 20 degrees C. The levels of vitellogenin (Vtg)-like proteins, alkali-labile phosphates in total proteins and acetylcholinesterase (AChE) activity were determined in soft tissues. The levels of acid-soluble, alkali-soluble (sclerotin), neutral-soluble (arthropodin) proteins and chitin were determined to characterize the maturation state of shells. Both 20HE and the municipal effluent extract readily increased the total activity of acetylcholinesterase, alkali-labile phosphates in proteins and vitellogenin-like proteins in brine shrimp. In shells, 20HE and the effluent extract increased the proportion of chitin in shells and acid-soluble proteins but were not statistically significant for the latter. The proportion of sclerotin was increased by 20HE but was not changed by the effluent extract. The proportion of arthropodin was decreased by both 20HE and the effluent extract. Correlation analysis revealed that, as expected, the proportion of acid-soluble protein for biomineralisation and chitin levels were positively correlated and the proportion of arthropodin and sclerotin were negatively correlated in shells exposed to 20HE. Principal component analysis revealed that the proportion of chitin, arthropodin, sclerotin and acid-soluble proteins explained 63% of the responses. Shell protein and carbohydrate contents, controlled by the molting hormone 20HE, could be used as efficient markers for ecdysial properties of various contaminants. The municipal effluent appears to contain ecdysial-like chemicals that are capable of producing changes in shell protein composition that are similar to 20HE.

Acetylcholinesterase↗

Reproduction obstacles for the female green neon shrimp (Neocaridina denticulata) after exposure to chlordane and lindane.

The purpose of this study was to investigate the effects of chlordane and lindane on reproduction obstacles and endocrine disruption in female green neon shrimp (Neocaridina denticulata). Individuals of N. denticulata, a common inhabitant of freshwater systems in Taiwan, was exposed to different levels of chlordane (1 and 10 ngl(-1)) and lindane (0.1 and 1 microgl(-1)). The reproductive ability and reproductive hormone levels were observed after exposure. According to our findings, an increase in estrogen, induction of a vitellogenin-like protein, and changes in reproductive performance were observed in both chlordane- and lindane-treated shrimp. Thus, it was concluded that chlordane and lindane may cause some reproduction obstacles and disruption of endocrine functions in N. denticulata.

Animals↗

High-performance liquid chromatography method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate.

This study presents an HPLC method for the simultaneous quantification of retinol, alpha-tocopherol, and cholesterol in shrimp waste hydrolysate lipid fraction. The method includes microscale saponification and extraction with n-hexane. Liposoluble vitamins and cholesterol were quantified by HPLC with UV detection (HPLC-UV), on a 25 cm x 0.46 cm SS Exil ODS 5 microm column, mobile phase 68:28:4 (v/v/v) methanol:acetonitrile:water; flow rate 1.4 ml/min; column temperature 36 degrees C. The detection was operated using two channels of a diode-array spectrophotometer, 325 nm for retinol and 208 nm for alpha-tocopherol and cholesterol. With these conditions, the overall recovery was 95.7, 100.8, and 98.0% for retinol, alpha-tocopherol, and cholesterol, respectively. The method precision (relative standard deviation) was 1.83% for retinol, 2.32% for alpha-tocopherol, and 1.98% for cholesterol. This method was used to quantify the cited analytes in the hydrolysate obtained during lactic acid fermentation of shrimp waste. This hydrolysate may be a valuable supplement of nutrients in fish production.

Animals↗

A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities.

A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.

Amino Acid Sequence↗

Expression and characterisation of tiger shrimp Penaeus monodon penaeidin (mo-penaeidin) in various tissues, during early embryonic development and moulting stages.

A penaeidin family, mo-penaeidin was cloned from the haemocytes of tiger shrimp Penaeus monodon using genomic polymerase chain reaction (PCR) by gene specific primers. Analysis of nucleotide sequence revealed that this mo-penaeidin consists of 1348 bp containing one intron (680 bp) and two exons (210 and 458 bp). It has an open reading frame (ORF) of 222 p, which encodes a protein of 74 amino acids including a signal peptide of 19 amino acids. The calculated molecular mass of the mature protein (55 amino acids) is 6.059 kDa with an estimated pI of 9.3. The deduced amino acid sequence of mo-penaeidin has similarity to that of penaeidin from Fenneropenaeus chinensis (73%), Farfantepenaeus paulensis (66%), Litopenaeus schmitti (53-67%), L. stylirostris (50-67%), L. setiferus (50-62%), L. vannamei (44-66%), and Marsupenaeus japonicus (33%), respectively. Phylogenetic tree analysis indicated that penaeidin (including mo-penaeidin, penaeidin, and penaeidin 5, 2, 3k, 3c1) of P. monodon is distinct from penaeidin 1, penaeidin 2, penaeidin 3 and penaeidin 4 of other penaeid shrimps. The mo-penaeidin mRNA was detected in various tissues including ovary and mandibular organ. The mo-penaeidin mRNA was present in one cell to postlarva stage with higher level at nauplius I (9h post hatching) and higher expression during the intermoult stage indicating an early innate immunity and different immunity at moulting stage.

Amino Acid Sequence↗

The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: cloning, distribution and expression.

Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185bp with a 660bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with white spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured.

Amino Acid Sequence↗

Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection.

To better understand the pathogenesis of white spot syndrome virus (WSSV) and to determine which cell pathways might be affected after WSSV infection, two-dimensional gel electrophoresis (2-DE) was used to produce protein expression profiles from samples taken at 48 h post-infection (hpi) from the stomachs of Litopenaeus vannamei (also called Penaeus vannamei) that were either specific pathogen free or else infected with WSSV. Seventy-five protein spots that consistently showed either a marked change (>50%) in accumulated levels or else were highly expressed throughout the course of WSSV infection were selected for further study. After in-gel trypsin digestion followed by LC-nanoESI-MS/MS, bioinformatics databases were searched for matches. A total of 53 proteins were identified, with functions that included energy production, calcium homeostasis, nucleic acid synthesis, signaling/communication, oxygen carrier/transportation, and SUMO-related modification. 2-DE results were shown to be consistent with relative EST database data from a previously developed EST database of two Penaeus monodon cDNA libraries. For seven selected genes, 2-DE and EST data were also compared with transcriptional time-course RT-PCR data. This study is the first global analysis of differentially expressed proteins in WSSV-infected shrimp, and in addition to increasing our understanding of the molecular pathogenesis of this virus-associated shrimp disease, the results presented here should be useful both for identifying potential biomarkers and for developing antiviral measures.

Animals↗

Effects of methamidophos on acetylcholinesterase activity, behavior, and feeding rate of the white shrimp (Litopenaeus vannamei).

The toxicity of methamidophos on the shrimp Litopenaeus vannamei was evaluated using acetylcholinesterase (AChE) activity, behavior, and feeding rate as effect criteria. The biochemical characterization of the soluble cholunesterase (ChE) present in both muscle and eye tissues of L. vannamei was performed in a first phase of the study. In both tissues, almost full inhibition of enzyme activity by eserine sulfate was found, indicating that the measured activity is mainly from ChE and not from other esterases. The highest rate of substrate hydrolysis was found when acetylthiocholine was used as substrate. To evaluate the effects of methamidophos on L. vannamei AChE, behavior, and feeding rate, shrimps were exposed for 24h to several sublethal concentrations of methamidophos. Significant effects of the pesticide on behavior and AChE were found, with behavior being a more sensitive endpoint than AChE inhibition. Feeding rate was not a sensible endpoint under conditions tested.

Acetylcholinesterase↗

Distribution of trace elements in tissues of two shrimp species from the Persian Gulf and roles of metallothionein in their redistribution.

This study investigates the distribution of three trace elements (Cd, Cu and Zn) in tissues (exoskeleton, abdominal muscle and hepatopancreas) of two shrimp species (Penaeus merguiensis and Penaeus semisulcatus). Special emphasis in this study was placed on evaluating the effects of storage conditions on redistribution of the elements and moreover the potential roles of metallothionein in tissue redistribution. The specimens were collected from the northern part of Persian Gulf during September 2001 and January 2002. The concentrations of metals in the tissues were measured using an Inductively Coupled Plasma-Atomic Emission Spectrophotometer (ICP-AES) and an ICP-Mass Spectrometer (ICP-MS). Metallothionein levels were determined using Differential pulse polarography. Results show a similar hierarchy for Cd and Cu concentrations among different tissues as follows: hepatopancreas>exoskeleton>muscle; and for Zn, the hepatopancreas again contained the highest levels, though there were not any significant differences in Zn concentrations between muscle and exoskeleton. Significant differences were found between the species in the case of Cd and Zn concentrations. Cd and Zn levels in P. semisulcatus were markedly higher than in P. merguiensis. In general, there were highly significant differences among the storage conditions from accumulation the selected trace elements and metallothionein in both species point of view, but in most cases, no significant differences could be observed among the fresh, refrigerated and frozen specimens of P. semisulcatus. In regards to Zn and Cu accumulation in edible (muscle) tissues of P. merguiensis, optimum conditions for frozen storage is -30 degrees C as ice-frozen, and in terms of short-term storage, 1-day refrigeration is recommended. The redistribution of Zn and Cu during the storage period can be attributed to the role of metallothionein in their transport from organ(s) to muscle during storage. The variations in pH values measured in the muscle of P. merguiensis in different storage conditions showed a similar trend with metallothionein, Zn and Cu. In contrast, it appears that cadmium is transported from muscle to the other tissues. Exoskeleton is probably one of the recipient tissues of cadmium. The inter-elemental relationships were assessed for each species, separately. A significant positive relationship was observed between the concentrations of Zn and metallothionein in P. semisulcatus, whereas in P. merguiensis, Cd concentrations decreased significantly with increasing metallothionein. Highly significant differences between the sexes in Cu and Zn contents could be detected. The differences between the sexes in the levels of Zn and Cu were significantly greater in P. semisulcatus. Highly significant size-related differences were found in the levels of Cd, Cu and metallothionein in the selected tissues of the shrimp species. Comparison between the mean concentrations of the elements in muscle tissue and existing guidelines indicate that in nearly all cases, the concentrations were well below permissible levels for human consumption. The only exception was observed regarding mean values of Cd, which was somewhat greater than some of the recommended levels.

Animals↗

Performance of a constructed wetland treating intensive shrimp aquaculture wastewater under high hydraulic loading rate.

A water treatment unit, mainly consisting of free water surface (FWS) and subsurface flow (SF) constructed wetland cells, was integrated into a commercial-scale recirculating aquaculture system for intensive shrimp culture. This study investigated performance of the treatment wetlands for controlling water quality. The results showed that the FWS-SF cells effectively removed total suspended solids (55-66%), 5-day biochemical oxygen demand (37-54%), total ammonia (64-66%) and nitrite (83-94%) from the recirculating water under high hydraulic loading rates (1.57-1.95 m/day). This led to a water quality that was suitable for shrimp culture and effluent that always satisfied the discharge standards. The area ratios of wetlands to culture tank being demonstrated (0.43) and calculated (0.096) in this study were both significantly lower than the reported values. Accordingly, a constructed wetland was technically and economically feasible for managing water quality of an intensive aquaculture system.

Animals↗