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Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses.

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.

Alkaline Phosphatase↗

Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis.

We cloned a cDNA encoding cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis (NsCtB). Nucleotide sequence of the isolated clone encoded a preproenzyme of 328 amino acids, comprising a 15-residue putative signal peptide, a 60-residue propeptide and the 253-residue mature enzyme. The mature NsCtB was 53% identical to human cathepsin B and conserved all the structural features characteristic of cysteine protease. The presence of an occluding loop in the mature region, a unique feature of cathepsin B, suggested the shrimp protein to be cathepsin B. Northern blot analysis revealed expression of NsCtB transcripts exclusively in the hepatopancreas tissues, suggesting a possible digestive role of this enzyme. An interesting feature of NsCtB was its remarkably high negative charge in comparison with other cysteine proteases, which was predicted to effectively locate and guide the positively charged residues of a substrate into the binding cleft. We also observed a repertoire of cysteine protease activities in the acidic milieu of shrimp hepatopancreas using synthetic substrates specific to various cathepsins. The activity profile revealed cathepsin B as the single most dominant enzyme with a specific activity comparable to that attributable to combined activities of other cathepsins. This activity could be blocked by E-64, a cysteine protease inhibitor, but not by Z-Phe-Tyr (t-Bu)-CHN(2), a specific inhibitor of cathepsin L.

Animals↗

Characteristics of NADH-dependent lipid peroxidation in sarcoplasmic reticulum of white shrimp, Litopenaeus vannamei, and freshwater prawn, Macrobrachium rosenbergii.

The iron-catalyzed NADH-dependent lipid peroxidation system in sarcoplasmic reticulum (SR) of cultured white shrimp, Litopenaeus vannamei and freshwater prawn, Macrobrachium rosenbergii was characterized. Production of thiobarbituric acid reactive substances was used to measure the activity of lipid peroxidation. In both species, the system preferred NADH to NADPH as the reducing agent. Lipid peroxidation activities of SR from both species increased when reaction temperatures increased from 6 to 26 degrees C. At 66 degrees C, the reaction was no longer NADH-dependent. Acidic pH amplified the lipid peroxidation activity. Sarcoplasmic reticular lipid peroxidation activity in white shrimp was always greater than in freshwater prawn. Fatty acid composition of SR lipids could be a major factor for this outcome. The proportion of n-3 highly unsaturated fatty acids, such as C20:5 and C22:6, in sarcoplasmic reticular lipids of white shrimp was twice of that in freshwater prawn. The results of this study provide important tools required for anti-oxidative nutrient study at sub-cellular level.

Animals↗

Utilization of shrimp shellfish waste as a substrate for solid-state cultivation of Aspergillus sp. S1-13: evaluation of a culture based on chitinase formation which is necessary for chitin-assimilation.

The utilization of shrimp shellfish waste as a substrate for solid-state cultivation of a filamentous fungus, Aspergillus sp. S1-13, was investigated. The organism was selected from among 220 isolates based on the productivity of its chitinolytic enzyme (chitinase), which might reflect microbial growth. The enzyme was produced only when the organism was grown on medium containing the shellfish waste. The addition of 58-65% water (w/w) to the medium was effective in enhancing production, and a certain amount of enzyme was observed in media of higher water content (up to about 75%). The initial pH and nitrogen source (ammonium sulfate) of the solid-state medium also affected the amount of enzyme. The amount of enzyme increased 2-fold in an optimum solid-state medium: 5 g of shrimp shellfish waste and 3 ml of basal medium (pH 5) containing 0.1% (NH4)2SO4 was inoculated with 4 ml of spore suspension; static cultivation at room temperature. The amount increased further (1.5-fold) when the cultivation was carried out at 37 degrees C, with 1.85 units of the enzyme formed from 1 g of shrimp shellfish waste. An analysis by ion-exchange column chromatography suggested the presence of at least two colloidal chitin-hydrolyzing enzymes and one p-nitrophenyl beta-D-N-acetylglucosaminide-hydrolyzing enzyme in an extract of the solid-state culture. The elution profile was similar to that obtained with a liquid culture filtrate.

Journal Article↗

Endocrine-mediated effects of UV-A irradiation on grass shrimp (Palaemonetes pugio) reproduction.

Although much is known regarding photoperiodic effects on crustacean egg production, the effects of ultraviolet (UV) light on reproduction has not been investigated. Likewise, little is known concerning the interaction between UV and xenobiotic exposure on crustacean reproductive cycles. In this study, male and female grass shrimp, Palaemonetes pugio, were exposed to sublethal concentrations of endosulfan (200 ng/l and 400 ng/l ES) under both white fluorescent (WF) and UV-A (315-400 nm) light conditions for 50 days in laboratory bioassays. Female endocrine (vitellogenin, ecdysteroids, and cholesterol), reproductive (percent gravid, clutch size), and embryo (days to hatch, hatching success, and hatching survival) responses were assessed. UV-exposure alone caused a significant (>4-fold) increase in total Palaemonetes pugio female egg production over the course of 50 days. Exposure to ES and UV significantly lowered the percentage of gravid females relative to UV controls, whereas ES-exposed shrimp under WF lighting did not exhibit these trends. Although higher vitellogenin concentrations and lower ecdysteroid titers were correlated with increased female egg production, cholesterol titers only exhibited a dose-dependent change when exposed to ES. Embryos from females exposed to UV had significantly lower ecdysteroid titers and shorter hatching times but there were no differences in embryo vitellogenin concentrations, hatching success, or hatching survival. These results indicate that UV-A exposure has a pronounced effect on grass shrimp (Palaemonetes pugio) reproduction and is likely mediated through 5-hydroxytrptamine (5-HT)-related neuroendocrine pathways. The implications for decapod aquaculture and evaluating chronic contaminant effects are discussed.

Animals↗

Determination of chloramphenicol residues in shrimps by liquid chromatography-mass spectrometry.

A liquid chromatographic method with mass spectrometric detection and identification (LC-MS) is presented for the determination of chloramphenicol (CAP) in shrimp tissues. Homogenized shrimp samples were extracted with phosphate buffer (pH 7.0). Clean-up was carried out on a C(18) SPE cartridge. Chloramphenicol was determined by LC-MS-ESI in negative mode. The column used was a Symmetry Shield with a mixture of acetonitrile-water (25:75) as mobile phase. Shrimp samples were fortified at CAP levels between 0.2 and 50 ng g(-1) with 5D-CAP as internal standard. At these levels, accuracies lay between 101 and 110% and between-day reproducibilities were lower than 7.1%, expressed as the variation coefficient of the mean. Limit of decision (CCalpha) was 0.02 ng g(-1). Limit of quantitation (LOQ) was 0.2 ng g(-1).

Animals↗

Cathepsin B and glutathione peroxidase show differing transcriptional responses in the grass shrimp, Palaemonetes pugio following exposure to three xenobiotics.

The common molecular biology techniques, suppressive-subtractive hybridization (SSH) and semi-quantitative real-time PCR (SQRT-PCR), were used to identify differentially expressed genes in the grass shrimp, Palaemonetes pugio following exposure to three different xenobiotics. Lab-acclimated adult male grass shrimp were exposed to empirically derived 96-hr male-specific LC50 concentrations of fipronil (FP, a phenylpyrazole GABA disrupting pesticide), endosulfan (ES, a cyclodiene GABA disrupting pesticide), or cadmium (Cd), as well as a control (CC). An SSH gene expression library was constructed from surviving shrimp from the fipronil and control exposures. Clones obtained by SSH were identified by searching against the NCBI website. A total of 42 genes were identified that were up-regulated by FP exposure, and 47 that were down-regulated. A subset of the affected genes was tested with SQRT-PCR to verify responsiveness to fipronil, as well as to endosulfan and cadmium. Two genes showed strong and significant responses to the exposures: glutathione peroxidase was significantly up-regulated by all three exposures, while Cathepsin B was strongly responsive to the two pesticides, but not to cadmium.

Animals↗

Imidacloprid residues in Willapa Bay (Washington State) water and sediment following application for control of burrowing shrimp.

Pesticides have been used in the Willapa Bay estuary in western Washington State to control the exotic invasive plant species Spartina alterniflora (cordgrass) and the native species of burrowing shrimp (Callianassa sp.; Upogebia sp.) that affect oyster production. Carbaryl, the only registered insecticide for control of burrowing shrimp, has not been extensively studied in the Willapa Bay. However, carbaryl use has been severely restricted, and alternatives likely to have less severe environmental impacts are being sought. Imidacloprid applied directly to exposed sediments when the tide is out is efficacious for burrowing shrimp control but lacks studies of its behavior in the estuary. For this study, imidacloprid dissipation was monitored as the tide was rising in Willapa Bay. Over 99% of applied material dissipated from small plots within 24 h, but residues near the analytical detection limit were found in sediments 28 days later. At a distance of 152 m along a transect from the plot in the direction of tidal flow, imidacloprid residues in water peaked within 10 min after initiation of tidal flow. Within 30 min, imidacloprid residues were not detected, nor were residues detected in the water any time over the next month after application. Carbaryl residues in water were also monitored, and they exhibited the same rise and fall at the 152 m distance from the experimental plot as did the imidacloprid residues. However, carbaryl levels significantly above the detection limit were still present in water over the next month after application. The rapid dissipation of imidacloprid from water was hypothesized to be due to extensive dilution by the tide. The hypothesis was tested in batch equilibration sorption studies with radiolabeled imidacloprid and Willapa Bay sediment. Sorption distribution coefficients were <1 mL/g, and hysteresis was not observed during two desorption cycles, suggesting that imidacloprid was widely dispersed to extremely low levels soon after application.

Adsorption↗

Characteristics of salt-fermented sauces from shrimp processing byproducts.

A salt-fermented sauce from shrimp processing byproducts (heads, shells, and tails) was prepared and characterized. Three types of sauces were prepared; sauce C, with 30 g of salt/100 g of byproduct (high salt); sauce E, with 30 g of salt and 0.2 g of sodium erythorbate (high salt); and sauce L, with 20 g of salt, 0.2 g of sodium erythorbate, 6 g of sorbitol, 0.5 mL of lactic acid, and 5 mL of ethanol (low salt). Sauces C and E showed higher exopeptidase activities than sauce L, whereas sauce L showed the highest endopeptidase activity. After 3 months of fermentation, the amino N content of sauce increased from 150-200 to 500-600 mg/100 g and the nonprotein nitrogen content increased from 300 to 950-1050 mg/100 g. Volatile basic nitrogen content increased significantly from 18 to 60 mg/100 g. The total carotenoids retained in sauces C, E, and L were 26.3, 76.2, and 73%, respectively, thus indicating that the addition of sodium erythorbate to sauces E and L retarded oxidation. Water activities of sauces C, E, and L were 0.753, 0.751, and 0.773, respectively. According to the omission test, the taste of sauces was influenced by the content of free amino acids, mainly glutamic acid and aspartic acid. All three sauces examined showed a 35% higher total amino acid content than commercial salt-fermented shrimp sauces. Therefore, shrimp processing byproducts may lend themselves to the preparation of high-quality salt-fermented sauces.

Adenosine Triphosphate↗

Presence and dehydration of ikaite, calcium carbonate hexahydrate, in frozen shrimp shell.

Ikaite, calcium carbonate hexahydrate, has by means of X-ray diffraction analyses of frozen samples been identified as the mineral component of the white spots formed in the shell of frozen shrimp during storage. When the shrimp thaw and the shell material is dried and kept at room temperature, ikaite rapidly transforms into a mixture of anhydrous calcium carbonate forms. X-ray diffraction analyses and Raman spectra of synthetic ikaite as well as the dehydration product confirm the assignments, and the rate constant for dehydration is approximately 7 x 10(-)(4) s(-)(1) at ambient temperature. Differential scanning calorimetry showed that dehydration of synthetic ikaite is an entropy-driven, athermal process and confirms that a single first-order reaction is rate-determining. Ikaite is found to be stable in aqueous solution at temperatures below 5 degrees C and in the shell of frozen shrimps but decomposes on thawing to form anhydrous calcium carbonates.

Animals↗

Use of potato disc and brine shrimp bioassays to detect activity and isolate piceatannol as the antileukemic principle from the seeds of Euphorbia lagascae.

EtOH extracts of the seeds of Euphorbia lagascae inhibited crown gall tumors on potato discs, were active against 3PS (in vivo) and 9PS (in vitro) mouse leukemia, and were toxic to brine shrimp. Using the potato disc and brine shrimp bioassays to monitor fractionation, piceatannol (3,4,3',5'- tetrahydroxystilbene ) was isolated as the 3PS active principle. Aesculetin (6,7-dihydroxycoumarin) was isolated in large quantity, was weakly cytotoxic in 9KB , but was inactive in the other bioassay systems. Sucrose was also isolated. A combination of the simple crown gall and brine shrimp bioassays can thus be used both to detect and to isolate plant antitumor substances, minimizing the need for extensive antitumor testing in vivo.

Animals↗

Establishment of cell culture systems from penaeid shrimp and their susceptibility to white spot disease and yellow head viruses.

Monolayer cultures were established from ovary, heart, lymphoid tissue and peripheral hemocytes of penaeid shrimps including Penaeus monodon, P. japonicus and P. penicillatus. The most favorable conditions for the culture of penaeid shrimp cells in vitro was in CMRL and L-15 tissue culture media when used within an osmolarity range of 620--760 mmol/kg. The optimal maintenance temperature was 25 degrees C for tissues of P. japonicus and 28 degrees C for tissues of P. monodon and P. penicillatus. Among the four tissues tested, lymphoid tissue, or 'Oka organ', was superior to the other tissues for the formation of confluent cell monolayers. Cell cultures from lymphoid tissue and ovary have been subcultured up to three times. When peripheral hemocytes and heart were cultured, a maximum survival of 4 days was obtained. In contrast, cell cultures derived from ovary and lymphoid tissue were maintained alive for at least 20 days in appropriate culture systems. Neither confluent cell sheet nor adherence of cells was obtained in cultivation of hepatopancreas using the present culture systems. The results obtained from the present study also revealed that ovary extract, muscle extract and lobster hemolymph enhanced the survival of the cultured cells of penaeid shrimp in vitro. When the 'Oka organ' cell monolayer was incubated with either white spot disease virus (WSDV) or yellow head virus (YHV), no cytopathic effect (CPE) was obtained. However, at 5--7 days after establishment, significant CPE (a few foci) was observed in cell monolayers derived from WSDV- and YHV-infected Oka tissue. By electron microscopy, virions of WSDV and YHV were observed in the nuclei and cytoplasm of cultured cells. The CPE foci developed further with increased incubation time.

Animals↗

Determination of indole in shrimps by differential-pulse voltammetry in an emulsified medium.

A method has been developed to determine indole in shrimps by differential-pulse voltammetry at a Pt electrode in an emulsified medium, after extraction into hexane - ethyl acetate (49 + 1). The standard additions method was used in order to minimise the matrix effect. Recoveries at the concentration levels of 11.71 and 0.94 micrograms g-1 of shrimp were 82 and 75%, respectively. The relative standard deviations were 3.5 and 4.0%, respectively. The accuracy of the proposed method was verified by comparing the results obtained from the analysis of shrimp samples allowed to decompose at room temperature for 1, 3 and 4 d, with those obtained from a spectrophotometric method by applying Student's t-test.

Animals↗

Function and cellular localization of farnesoic acid O-methyltransferase (FAMeT) in the shrimp, Metapenaeus ensis.

The isoprenoid methyl farnesoate (MF) has been implicated in the regulation of crustacean development and reproduction in conjunction with eyestalk molt inhibiting hormones and ecdysteroids. Farnesoic acid O-methyltransferase (FAMeT) catalyzes the methylation of farnesoic acid (FA) to produce MF in the terminal step of MF synthesis. We have previously cloned and characterized the shrimp FAMeT. In the present study, recombinant FAMeT (rFAMeT) was produced for bioassay and antiserum generation. FAMeT is widely distributed in shrimp tissues with the highest concentration observed in the ventral nerve cord. Interestingly, an additional larger protein in the eyestalk also showed immunoreactivity to anti-FAMeT serum. FAMeT was localized in the neurosecretory cells of the X-organ-sinus gland complex of the eyestalk. As shown by RT-PCR, FAMeT mRNA is constitutively expressed throughout the molt cycle in the eyestalk and the ventral nerve cord. To show that our cloned gene product had FAMeT activity, we demonstrated that expressed rFAMeT gene product catalyzed the conversion of FA to MF in a radiochemical assay. The ubiquitous distribution of FAMeT suggests that this enzyme is involved in physiological processes in addition to gametogenesis, oocyte maturation and development and metamorphosis of the shrimp. We hypothesize that FAMeT directly or indirectly (through MF) modulates the reproduction and growth of crustaceans by interacting with the eyestalk neuropeptides as a consequence of its presence in the neurosecretory cells of the X-organ-sinus gland.

Animal Structures↗

Invertebrate miRNA pva-small RNA-11881/pva-miR-11881 as a potential RNA-based therapeutic against white spot syndrome virus in infected shrimp.

Small RNAs and microRNAs (miRNAs) play diverse roles in host virus interactions and hold promise for therapeutic applications. An uncharacterized shrimp miRNA with potent activity against white spot syndrome virus (WSSV), a major double-stranded DNA pathogen in aquaculture, was identified and characterized. Among the 1,239 differentially expressed unannotated small RNAs in Penaeus vannamei hemocytes, one of the most strongly downregulated candidates, termed pva-small RNA-11881 or pva-miR-11881, was predicted to target multiple WSSV genes. A pva-small RNA-11881/pva-miR-11881 isomir that originates from the 5' untranslated region of a host lipase 3-like gene was identified. Its primary transcript contains Drosha and Dicer processing sites, and the precursor exhibits canonical pre-miRNA features. In vivo administration of its primary transcript, pva-pri-miR-11881, significantly reduced WSSV copy number and improved shrimp survival. Mechanistically, pva-miR-11881 directly suppresses crucial WSSV genes WSSV004, WSSV164, and WSSV419 and modulates the host immune response against WSSV infection by enhancing phenoloxidase activity, thereby reducing apoptosis and necrosis, and promoting caspase-1-mediated cell death. These findings reveal that the pva-miR-11881 in P. vannamei holds strong potential as a biotherapeutic agent for managing viral diseases in shrimp.

Animals↗

Crustacean immunity. Antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge.

We report here the isolation from plasma of two penaeid shrimp species of novel peptides/polypeptides with exclusive antifungal activities. A set of three molecules was purified with molecular masses at 2.7 kDa (Penaeus vannamei), 7.9 kDa, and 8.3 kDa (Penaeus stylirostris). Primary structure determination was performed by a combination of Edman degradation and mass spectrometry. The peptides display 95-100% sequence identity with a C-terminal sequence of hemocyanin, indicating that they are cleaved fragments of the shrimp respiratory protein. Specific immunodetection of the hemocyanin-derived (poly)peptides revealed that experimental microbial infections increase their relative concentration in plasma as compared with nonstimulated animals. Thus, the production of antifungal (poly)peptides by limited proteolysis of hemocyanin could be relevant to a shrimp immune reaction that would confer a new function to the multifunctional respiratory pigment of crustaceans.

Amino Acid Sequence↗

Effects of chlordane and lindane on testosterone and vitellogenin levels in green neon shrimp (Neocaridina denticulata).

The purpose of this study was to investigate the acute toxicity of chlordane and lindane as well as their endocrine disruption effect on green neon shrimp (Neocaridina denticulata), a common habitant in freshwater system of eastern Asia and Hawaii. First, the organisms were exposed to chlordane and lindane to estimate the 96-h LC(50)(96-h median lethal concentration). Then, levels of testosterone and vitellogenin in hemolymph of N. denticulata after exposure to sublethal concentrations of chlordane (1 ng/L and 10 ng/L) and lindane (0.1 microg/L and 1 microg/L) were also examined. The 96-h LC(50) values obtained from the results of acute exposure were 127.03 (130.11-122.35) ng/L and 9.36 (8.00-10.96) microg/L for chlordane and lindane, respectively. Furthermore, reductions of testosterone concentration were observed in both chlordane- and lindane-treated shrimps, whereas induction of vitellogenin-like protein was only apparent in chlordane-treated shrimps. Thus, it is concluded that chlordane and lindane may probably show some disruption endocrine functions on N. denticulata.

Animals↗

Molecular cloning and functional characterization of crustapain: a distinct cysteine proteinase with unique substrate specificity from northern shrimp Pandalus borealis.

A cDNA clone encoding a cysteine proteinase of the papain superfamily has been isolated from the hepatopancreas of northern shrimp Pandalus borealis (NsCys). NsCys shares the highest identity of 64% with a cathepsin L-like cysteine proteinase from lobster, and its identity to the well-characterized mammalian cathepsins S, L, and K falls within a narrow range of 54-59%. However, it differs from each of these cathepsins in certain key residues including, for example, the unique occurrence of tryptophan and cysteine residues at the structurally important S2 subsite. Consequently, NsCys produced in Pichia pastoris appears to be distinct in various physicokinetic properties. The recombinant enzyme is active and stable over a wide range of pH values, and its substrate specificity is unusual, as demonstrated by its poor affinity for phenylalanine residues. Instead, it shows the highest specificity for proline residues, a property similar to cathepsin K. Unlike cathepsin K, however, NsCys cleaves valine residues more efficiently than leucine. Similar results were obtained with the natural peptide substrate glucagon. The shrimp proteinase is further distinguished by its potent collagenolytic activity, resulting in a cleavage pattern reminiscent of bacterial collagenase. To distinguish such unique structural and enzymatic properties, we propose the trivial name "crustapain" for the shrimp proteinase, indicating that it is a papain-like cysteine proteinase from a crustacean species.

Amino Acid Sequence↗