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Molecular cloning and characterisation of a proteinase inhibitor, alpha 2-macroglobulin (alpha2-M) from the haemocytes of tiger shrimp Penaeus monodon.

An alpha 2-macroglobulin (alpha2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the alpha2-M cDNA consists of 4876 bp with an open reading frame (ORF) of 4494 bp, a 52 bp 5'-untranslated region, and a 327 bp 3'-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7 kDa with an estimated pI of 5.30. The P. monodon alpha2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate alpha2-Ms. Sequence comparison showed that alpha2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species alpha2-M showed that the overall structure is evolutionarily conserved and phylogenetic analysis revealed that P. monodon alpha2-M is closely related to other arthropod alpha2-M, and displays the highest similarity to M. japonicus alpha2-M. The alpha2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that alpha2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48 h post-peptidoglycan (PG) injection, but returned to the original values in 72 h post-PG injection.

Amino Acid Sequence↗

Shrimp carotenoids protect the developing rat cerebral cortex against the effects of ethanol on cortical spreading depression.

Cortical spreading depression is a neural phenomenon present in several animal species. Spreading depression features, like velocity of propagation, depends on several chemical and metabolic factors, as for example, anti-oxidants. Here we studied spreading depression-velocity changes in weaned rat-pups born from dams treated on a daily basis, either during gestation or lactation, with a carotenoid ethanolic extract (30 microg/kg/day) prepared from shrimp waste (heads). These pups were compared with age-mated ones, whose mothers were treated either with the vehicle (ethanol) or with distilled water. Compared to the distilled water-group (mean values, in mm/min, per hour of recording ranging from 3.02+/-0.26 to 3.15+/-0.27 [treatment during gestation; n=7], and from 3.03+/-0.25 to 3.22+/-0.30 [lactation; n=11]), ethanol-treated rats displayed higher spreading depression-velocities (from 3.74+/-0.06 to 3.82+/-0.08 [gestation; n=7], and from 4.26+/-0.32 to 4.33+/-0.34 [lactation; n=11]; p<0.05). Compared to the ethanol-group, carotenoid-treatment lead to lower spreading depression-velocities (p<0.05), ranging from 3.38+/-0.09 to 3.42+/-0.12, n=7 (gestation) and 3.58+/-0.13 to 3.62+/-0.17, n=12 (lactation). Carotenoid-treatment during lactation was shown to be significantly more effective than that during gestation (p<0.05), in lowering spreading depression-velocity. The results suggest a protective action of shrimp carotenoids against the ethanol effects on spreading depression. This protective effect could be related to the carotenoid antioxidant properties, as previously indicated by evidence showing spreading depression-effects of other antioxidants.

Animals↗

Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection.

White spot syndrome virus (WSSV) is a major shrimp pathogen causing large economic losses. In an attempt to identify the envelope proteins involved in virus infection, antisera against six WSSV envelope proteins were used in neutralization assays conducted in vivo. The results showed that the virus infection could be significantly delayed or neutralized by antibodies against three WSSV envelope proteins (VP68, VP281 and VP466). This neutralization was further confirmed by quantitative PCR. It could be concluded that the viral envelope proteins VP68, VP281 and VP466 played roles in WSSV infection to shrimp.

Animals↗

Recovery of carotenoids from shrimp waste in organic solvents.

Shrimp waste, which is produced in large quantities in the Indian seafood processing industries, is one of the important sources of natural carotenoids. Studies were carried out to assess the extractability of shrimp waste carotenoids in different organic solvents and solvent mixtures and to optimize the extraction conditions for maximum yield. A 50:50 mixture of isopropyl alcohol and hexane gave the highest (43.9 microg/g waste) carotenoid extraction yield compared to acetone, methanol, ethanol, isopropyl alcohol, ethyl acetate, ethyl methyl ketone, petroleum ether, and hexane individually and to a mixture of acetone and hexane. Extraction conditions such as percentage of hexane in the solvent mixture of isopropyl alcohol and hexane, ratio of solvent to waste and number of extractions was optimized using a statistically designed experiment. The optimized conditions for maximum yield of carotenoids were 60% hexane in solvent mixture, solvent mixture to waste ratio of 5:1 in each extraction and three extractions. A regression equation for predicting the carotenoid yield as a function of three processing variable (hexane % in solvent mixture, solvent-to-waste ratio and number of extractions) was derived by statistical analysis, and a model with predictive ability of 0.98 was obtained.

2-Propanol↗

Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus.

A natural agglutinin from the serum of the Indian white shrimp Fenneropenaeus (Penaeus) indicus was purified to electrophoretic homogeneity by a single-step affinity chromatography on N-acetylglucosamine-Sepharose 6B. The expression of hemagglutinating (HA) activity of F. indicus agglutinin (FIA) was independent of the presence of divalent cations and insensitive to their chelators. FIA gave a single symmetrical peak in its native form with a molecular mass estimate of 200 kDa on gel filtration in HPLC, and SDS-PAGE under reducing conditions revealed that it is a homo-oligomer of a 27-kDa subunit protein. The pattern of reactivity of FIA against anti-FIA rabbit serum in immunodiffusion and immunoelectrophoretic analysis provided additional evidence for its purity and homogeneity. HA-inhibition studies documented exclusive specificity of FIA for acetyl groups in carbohydrates independently of the presence of these groups at the C-2 or C-5 position and its stereochemical arrangement in the axial or equatorial orientation. The unique ability of FIA to recognize acetyl groups was also explicitly demonstrated with sialo- and asialo-glycoproteins. Strikingly, FIA also interacted equally with amino acids and chemicals containing acetyl groups, thereby unambiguously demonstrating the exquisite specificity of FIA for an acetyl group, irrespective of the presence of this group in carbohydrate or noncarbohydrate ligands. The susceptibility of HA activity of FIA to inhibition by lipopolysaccharides from diverse gram-negative bacteria as well as its ability to selectively agglutinate several bacterial species isolated from infected shrimps implicate a potential role of this humoral agglutinin of F. indicus in the host immunodefense reactions against microbial invaders.

Acetylation↗

Metabolic profiles of penaied shrimp: dietary lipids and ovarian maturation.

The major impediment to the culture of penaeid shrimp in captivity in the United States has been an inability to obtain ovarian maturation and spawning. Lipid profiles of tissues (gonads, hepatopancreas, and tail muscle) of Penaeus setiferus caught at sea have shown that cholesterol is the dominant sterol and that polyunsaturated fatty acids known to be essential in man comprise a significant portion of the fatty acid fraction. A prioprietary marine ration contains cholesterol, but is devoid of polyunsaturated fatty acids. Ovarian maturation and spawning were obtained when the shrimp diet was supplemented with an annelid rich in lipids containing these compounds. The biochemical significance of these findings is discussed.

Animal Nutritional Physiological Phenomena↗

Effect of light on the feeding rates of pelagic and littoral mysid shrimps: a trade-off between feeding success and predation avoidance.

It has been shown that freshwater pelagic mysid shrimps capture zooplankton at a higher rate in light than in darkness. This has been suggested to be due to facilitation of visual predation on evasive zooplankton prey. To test this hypothesis with Baltic mysid shrimps, and to see whether pelagic (migrating) and littoral (non-migrating) mysids differ in this respect, we compared the feeding rates of Mysis mixta and Praunus flexuosus on the copepod Acartia spp. As light conditions change radically from the beginning of summer to late autumn at the Baltic latitudes, we conducted the experiments during three different times of the year to determine if there was a seasonal response to light in mysids. The feeding rates of pelagic mysids were significantly higher in total darkness than in light. In contrast, the feeding rates of littoral mysids did not differ in the dark and the light in the three different seasons. The decreased feeding of pelagic mysids under well-lit conditions may be an adaptation to avoid visual predation by pelagic fish. In contrast, littoral mysids, which live in the well-lit layer throughout the day, do not show a similar response. The fact that light did not increase feeding in either of the mysid species indicates that these mysid species do not use vision in capturing prey, but rely mainly on mechano-reception.

Journal Article↗

The 1.9 A crystal structure of heat-labile shrimp alkaline phosphatase.

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature. Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer. SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzyme's net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.

Alkaline Phosphatase↗

The contribution of ecdysis to the fate of copper, zinc and cadmium in grass shrimp, Palaemonetes pugio Holthius.

Depuration through ecdysis by grass shrimp, Palaemonetes pugio, was examined by exposure to a sublethal mixture of copper, zinc and cadmium for 72 h, followed by placement in uncontaminated water to molt. Percent eliminated with the exuviae varied for each metal; of the total intermolt body burden, 11% Cu, 18% Zn and 26% Cd was associated with the exuviae. Cu concentrations of intermolt exoskeletons were significantly higher than of the exuviae of post-ecdysis shrimp suggesting that Cu contained in the exoskeleton was reabsorbed before molting. Exuvial Cd concentration was not significantly different than the concentration of the intermolt exoskeleton, suggesting that most Cd in the exoskeleton was depurated with the exuviae. Although Zn whole-body burdens were lower after a molt, Zn losses were most likely due to excretion because exuvial concentrations were significantly lower than in the intermolt exoskeleton. Cu, Cd and Zn concentrations in exuviae shed in metal-enriched water were significantly higher due to adsorption than exuviae produced in uncontaminated water.

Animals↗

Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens.

BACKGROUND: Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil. OBJECTIVE: The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens. METHODS: Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence. RESULTS: Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates. P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION: Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.

Adolescent↗

A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1,3-glucan.

A beta-glucan-binding protein (BGBP) has been identified in brown shrimp plasma by using a polyclonal antiserum against a BGBP from the freshwater crayfish. The protein was purified by immunoaffinity chromatography, and its molecular and biological properties described. Brown shrimp BGBP is a monomeric protein with a molecular mass of 100 kDa, similar to those described for other crustacean BGBPs. This protein is capable of enhancing the prophenoloxidase (proPO) system activation induced by laminarin. Both amino acid composition and N-terminal sequence are markedly similar to those of crayfish BGBP.

Adjuvants, Immunologic↗

Dot-blot nitrocellulose enzyme immunoassays for the detection of white-spot virus and yellow-head virus of penaeid shrimp.

Dot-blot nitrocellulose enzyme immunoassays (DB-NC-EIA) were developed for the detection of white-spot virus (WSV) and yellow-head virus (YHV) in infected shrimp. The assays utilized HRP-conjugated virus-specific antibodies to detect virus antigen present in gill homogenates of infected shrimp spotted onto nitrocellulose membrane. The assays are by far the simplest and most rapid detection methods available for WSV and YHV.

Animals↗

The time-course change of nitrogenous excretion in the Kuruma shrimp Penaeus japonicus following nitrite exposure.

Penaeus japonicus (12.83 +/- 1.24 g) which were exposed individually to 0.002 (control), 0.076, 0.362, 0.719 and 1.433 mM nitrite at 30 ppt of salinity were examined for hemolymph ammonia, urea and nitrite, and nitrogenous excretion after 3, 6, 12, 24 and 48 h, respectively. Hemolymph nitrite and hemolymph urea increased directly with ambient nitrite and exposure time, whereas hemolymph ammonia was inversely related to ambient nitrite and exposure time. Specific excretions of total-N (total nitrogen), ammonia-N, urea-N and organic-N (organic nitrogen) increased directly with ambient nitrite and exposure time. The contribution of ammonia-N excretion and urea-N excretion in the total-N excreted by the control shrimp was 41.7-90.8 and 2.8-10.5%, respectively. The contribution of ammonia-N in the total-N excreted by P. japonicus decreased to 10.0 and 3.8%, when they were exposed for 24 h to 0.076 and 1.433 mM nitrite, respectively. The contribution of urea-N excretion increased to 58.7 and 21.4%, and the organic-N excretion increased to 31.3 and 74.8% in the total-N excreted by the shrimp following 24 h exposure to 0.076 and 1.433 mM nitrite, respectively. It was concluded that P. japonicus following 24 h exposure to nitrite as low as 0.076 mM increased its ammonia-N excretion by a factor of 1.9, its urea-N excretion by 200, and its organic-N excretion by 37, as compared to those in the control solution.

Ammonia↗

Combined effect of antimicrobial coating and gamma irradiation on shelf life extension of pre-cooked shrimp (Penaeus spp.).

The present study was conducted to evaluate the combined effect of low-dose gamma irradiation and antimicrobial coating on the shelf life of pre-cooked shrimp (Penaeus spp.). Antimicrobial coatings were obtained by incorporating various concentrations of thyme oil and trans-cinnamaldehyde in coating formulations prepared from soy or whey protein isolates. Coated shrimps were stored at 4 +/- 1 degrees C under aerobic conditions and were periodically evaluated for aerobic plate counts (APCs) and Pseudomonas putida. Sensory evaluations were performed for appearance, odor, and taste using a hedonic test. Results showed that gamma irradiation and coating treatments had synergistic effects (p < or = 0.05) in reducing the APCs and P. putida with at least a 12-day extension of shelf life. Without irradiation, the inhibitory effects of the coating solutions were closely related to the concentration of thyme oil and trans-cinnamaldehyde. No detrimental effects of gamma irradiation on organoleptic parameters (appearance, odor, and taste) were observed. However, incorporation of thyme oil and trans-cinnamaldehyde reduced the acceptability scores for taste and odor.

Animals↗

Aeromonas species in fish, fish-eggs, shrimp and freshwater.

Aeromonas spp. are common contaminants of fish and seafood. They also are ubiquitous in the water environment. Aeromonas spp. were identified in 27 (93%) of 29 fish, in 17 (100%) fish-egg, in two (16%) of 12 shrimp samples and in 23 (100%) freshwater samples. In total, 117 Aeromonas strains were isolated from 69 positive samples, several samples having had two or three Aeromonas species. Included in this were also 26 mesophilic Aeromonas strains isolated in association with the study on fish diseases. The distribution of the species into 13 known hybridization groups (HGs) were studied by phenotypic and molecular methods. Ribopattern analysis of SmaI digested DNA was used for the identification of HGs. The predominant HG in fish, fish-eggs and freshwater samples was A. hydrophila HG 3 because 63% (22/37), 28% (16/57) or 80% (16/20) of the strains, respectively, were in HG 3. A. hydrophila HG 2 was also common in fresh fish samples but was not identified in fish-egg samples. HG 7 was common in fish samples studied for fish diseases and in freshwater samples. Strains which were not allotted to any HGs were common (19 of 143 strains). A. hydrophila HG 1, A. caviae HG 4, A. veronii subspecies sobria or subspecies veronii HG 8/10 known to be associated with human diarrhea were uncommon in all samples. The three strains isolated from frozen shrimp during two suspected food-borne outbreaks were A. hydrophila HG 2 and HG 3.

Aeromonas↗

Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV).

We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

Amino Acid Sequence↗

Tolerance to, and avoidance of, hypoxia by the penaeid shrimp (Metapenaeus ensis).

Aquatic hypoxia caused by eutrophication may lead to mass mortality of valuable living resources such as fish and shrimp. However, there is little information on the hypoxic tolerance of penaeid shrimp, and whether they are able to avoid hypoxia. In laboratory experiments, LC50, LT50 and heart beats per minute were determined for juvenile Metapenaeus ensis at 0.5, 1.0, 2.0 and 6.0 mg O2 l(-1). The 8-h LC50, for DO was 0.77 mg O2 l(-1), while the LT50 at 0.5 mg O2 l(-1) was 399 min. Heart beat rate significantly declined when DO fell below 1.0 mg O2 l(-1). When confronted with a gradient of dissolved oxygen, M. ensis were able to avoid hypoxic areas and move to oxygenated water. M. ensis appeared to be sensitive to hypoxia, and their ability to detect and avoid hypoxia may enhance their survival in habitats where hypoxia may occur.

Adaptation, Physiological↗

The use of the chicken embryo screening test and brine shrimp (Artemia salina) bioassays to assess the toxicity of fumonisin B1 mycotoxin.

Chicken embryos and brine shrimp naulpii were utilized in short-term toxicity bioassays to assess their sensitivity to the mycotoxin fumonisin B1 (FB1). Fertile chicken eggs (Cobb x) were dosed with FB1 on day 2 of incubation by the injection of 100 microliters of aqueous solution into the air space of each egg. Eggs were incubated with mechanical rotation until hatch, at which time mortality was assessed. Probit transformation of the mortality data produced a linear line of best fit (P < 0.05), from which an LD50 of 52 micrograms FB1/egg, equivalent to a concentration of 1.3 microns hatched in artificial seawater and exposed to FB1 in an optimized 96-well plate assay with a 48 hr mortality endpoint. Probit transformation of the mortality data resulted in an LC50 of 1.7 microns FB1, or 1.2 micrograms FB1/ml. Thus, at the cellular level, both bioassays appeared sensitive to FB1; however, from the standpoint of use as a screening assay, the chicken embryo bioassay is limited by the relatively high dose of FB1 required per egg. It is anticipated that the design and simplicity of the brine shrimp bioassay will accommodate screening for FB1 toxicity in contaminated samples.

Animals↗