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Ontogenetic changes in biochemical composition during larval and early postlarval development of Lepidophthalmus louisianensis, a ghost shrimp with abbreviated development.

Changes in growth and biochemical composition during the transition from egg through zoea to decapodid in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935), were documented in terms of dry weight, lipid classes, fatty acid composition, and carbon to nitrogen (C:N) ratios. Larvae of the ghost shrimp were mass-reared in the laboratory (28 degrees C; 20% S) from hatching to the decapodid stage. latroscan lipid class analysis revealed that major lipid classes in recently produced eggs were phospholipids (80.8 +/- 1.3%) and triglycerides (16.0 +/- 1.1%), which decreased during the incubation period. Polar lipids (zoea 1: 77.4 +/- 1.7%; zoea II: 77.5 +/- 2.1%; decapodid: 80.0 +/- 1.7%) and neutral lipids, of which free fatty acids (zoea I: 10.5 +/- 2.7%; zoea II: 13.1 +/- 5.2%; decapodid: 7.8 +/- 2.1%) were dominant, represented the major lipid classes in the zoeal and decapodid stages. Triglycerides were present in small amounts. The predominant fatty acids of L. louisianensis eggs, zoeae and decapodids were palmitic (16:0), stearic (18:0), eicosapentaenoic (20:5omega3), oleic (18:1omega9), and arachidonic (20:4omega6). Elemental composition of eggs, larvae, and the decapodid stage revealed conspicuous changes in the C:N ratio, with N being relatively stable during larval development but C decreasing during the decapodid stage. These data suggest independence of newly hatched L. louisianensis on external energy resources. This combined with the ability to incorporate saturated fatty acids into polar lipids provides a selective advantage for fast development of new tissue and growth, characteristic of decapod crustacean larvae with lecithotrophic development.

Animals↗

Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay.

Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In the immunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning and characterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.

Amino Acid Sequence↗

Effects of inorganic mercury on the respiration and the swimming activity of shrimp larvae, Pandalus borealis.

In order to test the sensitivity of respiration (physiological and potential) to mercury (Hg) contamination, larval shrimp Pandalus borealis were exposed to inorganic Hg (0-160 ppb) for 27 h in the laboratory. Oxygen consumption rates (RO2), potential respiration (determined by respiratory electron transfer system activity, ETSA), protein content, and swimming activity for zoeae III and zoeae V stages were measured. For both zoeae stages, ETSA and protein content remained constant after 27 h exposure to 160 ppb Hg whereas RO2 and swimming activity decreased. This study revealed the impact of different Hg levels and different exposure times on RO2 of shrimp larvae. After 10 h exposure to 160 ppb Hg, the RO2 decreased by 43 and 49% in zoeae III and zoeae V stages, respectively. Exposure time of 27 h to 80 ppb Hg and higher, induced paralysis in nearly 100% larvae. Surprisingly, the paralysed larvae displayed almost 50% of the control's RO2. The results showed that Hg disturbs a part of the respiration process without modifying the maximum activity of the enzymes involved in the ETSA assay. Therefore, the ETSA assay can not be used as a sublethal bioanalytic probe to detect Hg in short-term exposures. The decline of the RO2/ETSA ratios reported here, indicates an inability of contaminated larvae to adapt their metabolism to physiological stress caused by Hg.

Animals↗

In vitro susceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps in Northwestern Mexico.

The sensitivity of 144 isolates of Vibrio spp isolated from shrimp was compared using common antibiotics and those used in the shrimp industry. The in vitro susceptibility of the isolates was studied using amikacin, ampicillin, carbenicillin, cephalothin, cefotaxime, ceftriaxone, chloramphenicol, gentamicin, netilmicin, nitrofurantoin, pefloxacin, trimethoprim-sulphamethoxazole, enrofloxacin, oxytetracycline and florfenicol. The relationship between the minimum inhibitory concentration and the disk inhibition zone was also studied for some antibiotics.

Animals↗

Preparation of spent brewer's yeast beta-glucans with a potential application as an immunostimulant for black tiger shrimp, Penaeus monodon.

Spent brewer's yeast was autolysed and the insoluble yeast cell wall fraction obtained was used as a raw material for the preparation of brewer's yeast beta-glucan (BYG). A simple alkaline extraction was applied and optimized. The BYG with significantly (P</=0.05) high carbohydrate and glucan contents of 92% and 51% (w/w), respectively and with a low protein content of 1.6% (w/w) was obtained by a single alkaline extraction using 5 volumes of 1.0 N NaOH at 90 degrees C for 1 h. In vitro, this glucan significantly (P</=0.05) enhanced phenoloxidase (PO) activity of black tiger shrimp hemolymph as compared to controls without added glucan. Also in vivo, an oral administration of 0.2% (w/w) in diets for 3 days significantly (P</=0.05) increased the PO-activity of the shrimp. The potential immunostimulating properties of a commercial and other yeast-derived products were also evaluated for comparative purposes.

Administration, Oral↗

Heterologous expression in Pichia pastoris and single-step purification of a cysteine proteinase from northern shrimp.

A distinct cysteine proteinase (NsCys) of northern shrimp Pandalus borealis belonging to cathepsin L subgroup of the papain superfamily has been overexpressed as a precursor form (proNsCys) in Pichia pastoris. We adopted a simple and quick procedure to generate an expression cassette by constructing a donor vector harboring proNsCys followed by recombination with an acceptor vector in a way so that the proNsCys gene was placed downstream of the methanol-inducible AOX1 promoter and alpha-mating factor signal sequence gene. In addition, we used glycerol complex medium that supported high growth of yeast before induction while induction was carried out in minimal methanol medium thereby facilitating the secreted protein to be purified with a single size-exclusion chromatography. The recombinant enzyme was purified in two enzymatically active fractions: both corresponding to mature NsCys with, however, the major one comprising two molecular species of NsCys which had their severed prodomain non-covalently attached. The overall yield was about 100 mg of crude or 60 mg of purified recombinant enzyme comprising both mature and prodomain-attached forms of NsCys per liter of yeast culture. The recombinant NsCys was biologically active as observed by gelatin zymography and its ability to cleave Z-Phe-Arg-MCA, a synthetic substrate for cathepsin L. The development of the system reported here provides a cost-effective and easy to manipulate expression system to obtain large quantities of fully functional shrimp enzyme that will enable the functional characterization of this unique enzyme for both research and industrial purposes.

Amino Acid Sequence↗

Development of an anti-vitellin ELISA for the assessment of reproduction in the ridgeback shrimp, Sicyonia ingentis.

To investigate the reproductive regulation of the ridgeback shrimp, Sicyonia ingentis, vitellin (Vn) synthesis was studied. Using gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Vn was found to have a molecular mass of 322 kDa and to be composed of three subunits of 182, 91 and 85 kDa. Purified Vn was used to prepare anti-Vn antiserum, which was used to develop an enzyme-linked immunosorbent assay (ELISA) with a dynamic range of 0.3-300 ng. The ELISA was used to measure hemolymph levels of yolk proteins. The mean hemolymph concentrations in fresh caught animals ranged from 256 (+/-36.6 S.E.M.) to 1073 (+/-87.6 S.E.M.) mg/ml in stage 2 and 4 animals, respectively. The ELISA was also used to determine the effects of steroid hormone injections in adult non-reproductive female shrimp. One milligram injections of progesterone, 17alpha-hydroxyprogesterone or 17beta-estradiol were administered for three consecutive days to individual females. There were no changes in hemolymph vitellogenin levels during the successive 7-day period following the first injection of any steroid.

17-alpha-Hydroxyprogesterone↗

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.

Amino Acid Sequence↗

Molecular characterization of the bifunctional VHDL-CP from the hemolymph of white shrimp Penaeus vannamei.

A very high-density lipoprotein (VHDL) purified from the hemolymph of the white shrimp Penaeus vannamei is shown to be identical to the clotting protein (CP) previously reported from the same organism based on size, subunits and N-terminal amino acid sequence. The approximately 440-kDa protein, a homodimer of approximately 200-kDa subunits, was present in KBr gradient fractions ranging in density from 1.155 to 1.212 g/ml. Samples of VHDL after purification by strong cation exchange chromatography were subjected to electrophoresis on native polyacrylamide gels. Lipids associated with the VHDL were detected by Sudan Black and Oil Red O staining and comprise 9-15% of the purified protein. Circular dichroism of VHDL-CP indicates that the alpha-helix content of the VHDL-CP is 32%, while beta-sheets correspond to 33%, closely resembling the secondary structure of CP from the shrimp Penaeus monodon and, remarkably, the secondary structure of very high-density lipophorin E (VHDLpE) from the tobacco hornworm, Manduca sexta.

Amino Acid Sequence↗

Expression of hemocyanin and digestive enzyme messenger RNAs in the hepatopancreas of the Black Tiger Shrimp Penaeus monodon.

In order to define the cellular site of synthesis for hemocyanin and digestive enzymes in the decapod hepatopancreas, we studied the expression of messenger ribonucleic acids (RNAs) for these molecules in the epithelium lining hepatopancreas tubules. In situ hybridisation of gene probes for the digestive enzymes amylase, cathepsin-L, cellulase, chitinase-1 and trypsin to tissue sections of the shrimp hepatopancreas confirmed that the F-cells lining tertiary, secondary and primary ducts are the sites of synthesis for digestive enzyme messenger RNA (mRNA). The F-cells also contained mRNA for the hemocyanin gene. This finding raises important questions on the mechanism by which mature hemocyanin accumulates in the shrimp hemolymph. Our in situ hybridisation studies further showed that Penaeus monodon F-cells remain transcriptionally active for digestive enzyme mRNAs during periods of starvation.

Amylases↗

Molecular characterization of vitellin from the ovaries of the white shrimp Penaeus (Litopenaeus) vannamei.

Vitellin (Vt) was purified from ovary extracts of mature females of the white shrimp Penaeus vannamei using Sepharose CL-4B and Q-Sepharose columns. Native Vt had an apparent molecular weight of 388 kDa as detected in Native-PAGE, bound the lipophilic dye Oil Red O and had a total lipid content of approximately 43.8%. Under reducing and denaturing conditions (SDS-PAGE), Vt is composed of three major subunits of 87, 78 and 46 kDa, although minor bands of 65, 61 and 31 kDa are also detected. The 87- and 78-kDa polypeptides were strongly recognized by Penaeus semisulcatus anti-Vt polyclonal and Penaeus monodon anti-Vt monoclonal antibodies. Furthermore, the N-terminal amino acid sequence of the 78-kDa polypeptide is very similar to Penaeus japonicus vitellogenin (Vg) and P. semisulcatus Vt, with an identity of 76%. Circular dichroism indicates that the beta-helix content of Vt is 25% while beta-sheets correspond to 37 and 14% of unordered secondary structure. These values are similar to insect microvitellogenin. Vt has an emission fluorescence maximum at 329 nm, comparable to the shrimp high-density lipoprotein/beta-glucan binding protein (HDL/BGBP).

Amino Acid Sequence↗

Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei.

We investigated the effect of starvation as a stimulant of the digestive system on digestive proteinase activities in the white shrimp Penaeus vannamei. The starved organisms were sampled periodically according to the molting stage and compared with a continuously fed group. Molting stage was included as an independent variable. Most analyzed variables, except for trypsin, were more affected by starvation than by molting, indicating that starvation is a stimulant that masks the effect of molting and showing that food or alimentary stress is more conspicuous than physiological ones. We found that starvation is a stimulant that surpasses the effect of molting, and because it affects the activity of digestive proteinases, studies of starving organisms in combination with tools of molecular biology, can be a helpful working model in the understanding of mechanisms of regulation of digestive enzyme activity. In the starved organisms, trypsin and chymotrypsin activities were similar, suggesting dependence of one to the other. Changes in proteolytic activities and the number of protein bands in electrophoresis showed evidence of synthesis regulation in the midgut gland of white shrimp.

Chymotrypsin↗

Joint action of elevated ambient nitrite and nitrate on hemolymph nitrogenous compounds and nitrogen excretion of tiger shrimp Penaeus monodon.

Penaeus monodon (12.13+/-1.14 g) exposed individually to six different nitrite and nitrate regimes (0.002, 0.36 and 1.46 mM nitrite combined with 0.005 and 7.32 mM nitrate), at a salinity of 25 ppt, were examined for hemolymph nitrogenous compounds and whole shrimp's nitrogen excretions after 24 h. Nitrogen excretion increased directly with ambient nitrite and nitrate. Hemolymph nitrite, nitrate, urea and uric acid levels increased, while hemolymph ammonia, oxyhemocyanin and protein were inversely related to ambient nitrite. Exposure of P. monodon to elevated nitrite in the presence of 7.32 mM nitrate did not alter hemolymph nitrite, ammonia, uric acid, oxyhemocyanin and protein levels, but caused an increase in hemolymph nitrate and a decrease in hemolymph urea as compared to exposure to elevated nitrite only. Following exposure to elevated nitrite, nitrite was oxidized to nitrate and P. monodon showed uricogenesis and uricolysis. The shrimp also used strategies to avoid joint toxicities of nitrite and metabolic ammonia by removing ammonia or reducing ammonia production under the stress of elevated nitrite.

Ammonia↗

Superoxide dismutase as modulator of immune function in American white shrimp (Litopenaeus vannamei).

The immunomodulatory action of superoxide dismutase (SOD) and its possible use as an indicator of immune responses in American white shrimp (Litopenaeus vannamei) were studied. Juvenile shrimp were immersed in aerated beta-glucan and sulfated polysaccharide solutions for 6 h. SOD activity in haemocytes and muscle was quantified to evaluate whether beta-glucan and sulfated polysaccharide induce immunostimulatory activity. Haemocytes and muscle showed similar increased levels of SOD activity (1.5- and 1.4-fold that of control, respectively). Total haemocyte count decreased within the first 24 h after challenge with immunostimulants, but total haemocyte count and total soluble haemocyte protein increased over normal values after 48-120 h. Single immunostimulation with beta-glucan and sulfated polysaccharide is sufficient to generate an increase in the antioxidant activity of L. vannamei SOD.

Adjuvants, Immunologic↗

The retinoids of seven species of mantis shrimp.

Eyes of stomatopod crustaceans, or mantis shrimps, contain the greatest diversity of visual pigments yet described in any species, with as many as ten or more spectral classes present in a single retina. In this study, the eyes of seven species of mantis shrimp from three superfamilies of stomatopods were examined for their content of retinoids. Only retinal and retinol were found; neither hydroxyretinoids nor dehydroretinoids were detected. The principal isomers were 11-cis and all-trans. The eyes of most of these species contain stores of 11-cis retinol, principally as retinyl esters, and in amounts in excess of retinal. Squilla empusa is particularly noteworthy, with over 5000 pmoles of retinol per eye.

Animals↗

The morphology of the dorsal eye of the hydrothermal vent shrimp, Rimicaris exoculata.

The bresiliid shrimp, Rimicaris exoculata, lives in large masses on the sides of hydrothermal vent chimneys at two sites on the Mid-Atlantic Ridge. Although essentially no daylight penetrates to depths of 3500 m, very dim light is emitted from the hydrothermal vents themselves. To exploit this light, R. exoculata has evolved a modified compound eye on its dorsal surface that occupies about 0.5% of the animal's body volume. The eye's morphology suggests that it is extremely sensitive to light. The cornea of the dorsal eye is smooth with no dioptric apparatus. The retina consists of two wing-shaped lobes that are fused across the midline anteriorly. The rhabdomeral segments of the 7000 ommatidia form a compact layer of photosensitive membrane with an entrance aperture of more than 26 mm2. Within this layer, the volume density of rhabdom is more than 70%. Below the rhabdomeral segments, a thick layer of white diffusing cells scatters light upward into the photoreceptors. The arhabdomeral segments of the five to seven photoreceptors of each ommatidium are mere strands of cytoplasm that expand to accommodate the photoreceptor nuclei. The rhabdom is comprised of well-organized arrays of microvilli, each with a cytoskeletal core. The rhabdomeral segment cytoplasm contains mitochondria, but little else. The perikaryon contains a band of mitochondria, but has only small amounts of endoplasmic reticulum. There is no ultrastructural indication of photosensitive membrane cycling in these photoreceptors. Vestigial screening pigment cells and screening pigment granules within the photoreceptors are both restricted to the inner surface of the layer of the white diffusing cells. Below the retina, photoreceptor axons converge in a fanshaped array to enter the dorsal surface of the brain. The eye's size and structure are consistent with a role for vision in shrimp living at abyssal hydrothermal vents.

Animals↗

Characterization of shrimp shell deproteinization.

The aim of this paper was to contribute to the interpretation of the mechanism of shrimp shell deproteinization. We used amino acid analysis to quantify the amount of proteins remaining in chitin. NaOH 1 M was added to a demineralized shrimp shell powder with a solution-to-solid ratio of 15 mL/g at ambient temperature. Because of the limited precision of the technique, after 24 h the protein content measured by elemental analysis had to be considered as negligible. However, with the use of amino acid analysis, it was still possible to determine with precision this content down to 0.25%. We also showed that among the peptides remaining linked to chitin after deproteinization, acidic amino acids were always in proportion higher than alkaline ones, but the balance between the two kinds of residues increased in favor of the latter with time. The kinetic study of the deproteinization clearly revealed a three-step mechanism with very different rate constants. The variation of these constants with temperature was used to calculate the energies of activation and the frequency factors of collision, thus allowing us to propose a new interpretation of the mechanism of deproteinization.

Amino Acids↗

Alpha-chitin nanocrystals prepared from shrimp shells and their specific surface area measurement.

Alpha-chitin was isolated from shrimp shells. The chitin was subjected to extensive treatments of acid hydrolysis and mechanical disruption to yield nanocrystals. The goal of this article is to characterize alpha-chitin nanocrystals produced from shrimp shells in regard to crystallite properties and the specific surface area of the chitin nanoparticles. X-ray diffraction data indicate an increase in chitin crystallinity after hydrolysis, as less-ordered chitin domains are digested. Line broadening data were used to measure crystallite size and particle size in the hydrolyzed chitin nanocrystals. Dye adsorption with Congo red was used to measure the specific surface area of the particles, indicating values near 350 m2/g. This value was supported with calculations derived from X-ray crystallite size measurements. Particle surface area measurements were compared with similarly prepared cellulose nanocrystals.

Acids↗