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Chimeric polypeptide of thymidine kinase and thymidylate kinase of shrimp white spot syndrome virus: thymidine kinase activity of the recombinant protein expressed in a baculovirus/insect cell system.

The unique chimeric organization of the white spot syndrome virus (WSSV) tk-tmk gene encodes a protein which has significant homology to both cellular-type thymidine kinase (TK) and cellular-type thymidylate kinase (TMK), but the functional activity of this protein has not been demonstrated. Because TK is usually expressed only at very low levels in host cells, in this study, the coding region of WSSV tk-tmk was expressed in an insect/baculovirus expression system. The His-tagged recombinant WSSV TK-TMK was purified by affinity chromatography, and its enzyme activity was characterized by steady-state kinetics. The recombinant WSSV TK-TMK catalyzed the phosphorylation of thymidine to form thymidine monophosphate (TMP), but we found no evidence that it was able to catalyze the further phosphorylation of TMP to form thymidine diphosphate (or thymidine triphosphate). This TK activity is sensitive to feedback inhibition by thymidine triphosphate. In addition to thymidine, of the nine other substrates tested, including acyclovir, ganciclovir, and 5-(2-bromovinyl)-2'-deoxyuridine, only 2'-deoxyuridine and 5-bromo-2'-deoxyuridine could also serve as substrates. These data suggest that the enzymatic characteristics of the recombinant WSSV TK-TMK are similar to those of the eukaryotic cytosolic TKs. We also found that TK activity increased as infection advanced in the integument and gills of experimentally infected shrimp, suggesting its functional involvement during WSSV infection.

Animals↗

Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus.

The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5'-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2-4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized.

Amino Acid Sequence↗

Nucleotide variation and molecular structure of the heterochromatic repetitive AluI DNA in the brine shrimp Artemia franciscana.

It has been suggested that DNA bending could play a role in the regulation of gene expression, chromosome segregation, specific recombination and/or DNA packaging. We have previously demonstrated that an AluI DNA family of repeats is the major component of constitutive heterochromatin in the brine shrimp A. franciscana. By the analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments we verified that the repetitive AluI DNA shows a stable curvature that determines a solenoidal geometry to the double helix. This particular structure could be of relevant importance in conferring the characteristic heterochromatic condensation. In this paper we evaluate how the point mutations that occurred during the evolution of the AluI sequence of A. franciscana could influence the sequence-dependent tridimensional conformation. The obtained data underline that, in spite of the high sequence mutation frequency (10%) of the repetitive DNA, the general structure of the heterochromatic DNA is not greatly influenced, but rather there is a substantial variation of the copy number of the repetitive AluI fragment. This variation could be responsible for the hypothetical function of the constitutive heterochromatin.

Animals↗

Pigment biogenesis in freshwater shrimp ventral nerve chord chromatophores.

The possible biogenesis of two pigment granule types present in the monochromatic, brown chromatosomes enveloping the ventral nerve chord of the freshwater palaemonid shrimps Macrobrachium acanthurus, M. heterochirus and M. olfersii is examined by transmission electron microscopy in thin section and freeze fracture replicas. Prominent, membrane limited granules are suggested to have their origin in a complex, juxtanuclear, smooth endoplasmic reticulum labyrinth, continuous with the nuclear envelope. Amembranous, lipocarotenoid granules possibly derive from the external surface of the smooth endoplasmic reticulum. Nuclear envelope and SER membranes contain numerous 11 nm diameter intramembranous particles while pigment granule membranes exhibit fewer particles. A dictyosomal origin for the lipocarotenoid granules is discounted. Granulogenesis is suggested to be a continuous process in crustacean chromatophores.

Animals↗

Rhabdom changes in the shrimp, Palaemonetes.

The fine structure of the principal compound eye of the shrimp, Palaemonetes, was studied under conditions of light and dark adaptation. Ommatidium the situation in other decapod crustaceans. Light and dark adapted eyes differ in that the rhabdom changes its shape; morphological evidence suggests a possible sequence of events involving production, utilization, and degradation of photoreceptor membrane, a discontinuous process occurring only during changes from light to dark and dark to light. A hypothesis of membrane turnover is proposed.

Animals↗

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana.

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adjacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH2F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.

Animals↗

Effects of metals on early life stages of the brine shrimp, Artemia: a developmental toxicity assay.

The need for simple, fast and inexpensive tests to study metal pollution in the marine environment has become more pressing as utilization of coastal waters increases. To address this concern, the influence of four metals, cupric sulfate, lead nitrate, zinc sulfate and nickel sulfate, on emergence and hatching of the brine shrimp, Artemia, has been assessed. Occurrence of these easily recognized developmental milestones provides a convenient method to assay metal effects on development. Copper and lead were about equally toxic, reducing the rate and extent of Artemia development at or below concentrations of 0.1 microM. Zinc was somewhat less toxic than copper and lead, while nickel was the least toxic. Emerging Artemia are much more sensitive to metals than are larvae and adults. Furthermore, in contrast to results obtained with larvae and adults, the toxicity of lead is high when prelarval stages are considered. It is apparent from the findings that use of early stages of Artemia is an alternative to the examination of slower growing animals for the study of metal pollution in coastal marine waters.

Animals↗

Toxicity of Fusarium sambucinum Fuckel sensu lato to brine shrimp.

In a screening test seven Fusarium strains out of 17 proved to be toxic towards brine shrimp. Among these, four F. sambucinum strains as well as three F. venenatum isolates caused toxic effects. The chemical screening by TLC analysis revealed the presence of sambucinol and diacetoxyscirpenol in the extracts of the toxic isolates of F. venenatum 64537.

Animals↗

Some biological properties of a rhabdovirus isolated from penaeid shrimps.

Some of the relevant biological properties of a rhabdovirus isolated from penaeid shrimps (RPS) were examined. The virus replicated in an established fish cell line, epithelioma papulosum cyprini (EPC) which allowed for the development of a quantitative plaque assay protocol. Virus replication was not inhibited by the DNA antagonist, 5-bromo-2'-deoxyuridine (20 micrograms/ml). Virus infectivity was sensitive to 20% ethyl ether, low pH, and to 37 degrees C. The virus showed marked lability to repeated freezing and thawing and storage at -10 degrees C, but was stable at -70 degrees C for several weeks. The virus particle to infectious unit ratio in EPC was found to be 30.

Animals↗

Adaptation of a quantitative programme for the X-ray analysis of solubilized tissue as microdroplets in the transmission electron microscope: application to the moult cycle of the shrimp Crangon crangon (L).

A quantitative programme for X-ray microanalysis is used in a non-standard manner on solubilized tissue which has been spiked with cobalt and sprayed as microdroplets on electron microscope grids. During the procedure the count time and the concentration of cobalt is related to the peak integral and, from the relative efficiencies, the concentrations of other elements are computed from the peak integrals. Absorption is taken into account but the X-ray background is not used to estimate the total mass and the beam current is not measured. The method is applied to the hepatopancreas and blood from individual shrimps, Crangon crangon, to give the concentrations of sodium, magnesium, silicon, phosphorus, sulphur, potassium and calcium at different stages of the moult cycle. In the hepatopancreas the absolute and relative quantities of phosphorus, sulphur and other elements change in phase with the moult cycle. This situation must be linked with fluctuations in levels of metabolic activity and may affect the metal-binding capacity of the tissue which is known to fluctuate. The hepatopancreas accumulates lipid and phosphorus during the intermoult period, but the level of phospholipid phosphorus remains as a constant proportion of the tissue wet weight. The gland does not store calcium for hardening the new exoskeleton after ecdysis. Magnesium is a more important and variable component and could be linked with metabolic activity. The blood composition remains more stable. However, sulphur concentration is high and variable and this may, to some extent, reflect changes in the concentration of taurine. The concentration of copper increases towards the end of the moult cycle and decreases during moulting; opposite changes occur in the hepatopancreas.

Animals↗

Sterols of the lobster (Homarus americanus) and the shrimp (Pandalus borealis).

In this study we have analyzed the sterol compositions of two continental shelf species of crustacea, the lobster (Homarus americanus) and the shrimp (Pandalus borealis). Cholesterol was found to be the most abundant sterol in these two species with smaller amounts of desmosterol, 24-methylcholesterol, 24-ethylcholesterol, 24-methylenecholesterol and 22-dehydrocholesterol.

Animals↗

Non-methylene-interrupted and omega4 dienoic fatty acids of the white shrimp Penaeus setiferus.

The total lipid fatty acids from the white shrimp Penaeus setiferus were found to contain several unusual dienoic fatty acid species. These included two methylene-interrupted species: delta 11, 14-C18:2 (18:2omega 4) and delta 13, 16-C20:2 (20:2omega4). Also found were several non-menthylene-interrupted dienoic fatty acids including delta 7, 11 and delta 7, 13-C20:2, delta 7, 13-C21:2, delta 7, 13, delta 7, 15, delta 9, 13, delta 9, 15,, and delta 7, 17-C22:2. Many minor C20:2 non-mentylene-interrupted dienes were found but could not be unequivocally characterized.

Animals↗

Neural factors influence the degeneration of muscle fibers in the chelae of snapping shrimps.

The asymmetric pincer and snapper claws in the snapping shrimp differ in external morphology and musculature. The snapper is a massive claw used for displays and defense; the pincer is small and slender, used for feeding and burrowing. The snapper has only slow muscle fibers; the pincer has both slow and fast. Removal or denervation of the snapper claw induces transformation of the contralateral pincer to a snapper type of claw at the subsequent molt. A removed claw regenerates as a pincer type, as long as the innervation of the remaining claw is intact. Fast muscle fibers, found exclusively in the pincer claw, normally degenerate completely within 10 d after the moult, which transforms the pincer to a snapper. Morphological transformation of the pincer following removal of the snapper claw can occur even if the pincer claw is denervated. Denervation of the pincer, however, delays degeneration of the fast fibers, increasing the estimated half-time of muscle degeneration, for 4.4 +/- 0.2 to 19.5 +/- 0.8. d after the transforming moult. Neural influences therefore are involved both in the determination of the morphology of the claw and in the induction of degenerative changes during the remodeling of an existing claw.

Animals↗

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (Crustacea, Decapoda).

The production of antimicrobial peptides represents a first-line host defense mechanism of innate immunity that is widespread in nature. Only recently such effectors were isolated in crustacean species, whereas numerous antimicrobial peptides have been characterized from other arthropods, both insects and chelicerates. This review presents findings on a family of antimicrobial peptides, named penaeidins, isolated from the shrimp Penaeus vannamei. Their structure and antimicrobial properties as well as their immune function will be discussed through analyses of penaeidin gene expression and peptide distribution upon microbial challenge.

Amino Acid Sequence↗

Tissue-Specific Expressed Sequence Tags from the Black Tiger Shrimp Penaeus monodon.

: Expressed sequence tag data were generated from complementary DNA libraries created from cephalothorax, eyestalk, and pleopod tissue of the black tiger shrimp (Penaeus monodon). Significant database matches were found for 48 of 83 nuclear genes sequenced from the cephalothorax library, 22 of 55 nuclear genes from the eyestalk library, and 6 of 13 nuclear genes from the pleopod library. The putative identities of these genes reflected the expected tissue specificity. For example, genes for digestive enzymes were identified from the cephalothorax library and genes involved in the visual and neuroendocrine system from the eyestalk library. A few sequences matched anonymous EST or genomic sequences, and others contained mini-satellite or microsatellite repeat sequences. The remainder, 31 from the cephalothorax library, 25 from the eyestalk library, and 5 from the pleopod library, were sequences of high nucleotide complexity with no matches in any database searched and thus may represent novel genes.

Journal Article↗

Heavy metal toxicity and differential effects on the hyperglycemic stress response in the shrimp Palaemon elegans.

Agricultural and industrial activities cause heavy metal pollution of the aquatic environment. The sensitivity of crustaceans to heavy metals is well documented. However, the hormonal and metabolic target of physiological functions affected by sublethal toxicity and stress responses have been scarcely investigated. Exposure of Palaemon elegans to increasing concentrations of heavy metals dissolved in artificial sea water resulted in an order of toxicity tested by LC(50) for 96 h in intact and eyestalkless animals in which Hg is the most toxic, followed by Cd, Cu, Zn, and Pb. Eyestalkless animals were found to be more sensitive than intact individuals. Heavy metals affect the blood glucose levels, yet manipulative stress does not. The intermediate sublethal concentrations of Hg, Cd, and Pb produced significant hyperglycemic responses within 3 h, while the highest concentrations elicited no hyperglycemia in 24 h. In contrast, animals exposed to Cu and Zn showed hyperglycemia even at high concentrations. This difference in response between Cu or Zn and the nonessential heavy metals Cd, Hg, or Pb can probably be explained by the physiological roles of the former in crustaceans and by tolerance adaptations. Involvement of the crustacean hyperglycemic hormone (cHH) was tested by routine bioassay on eyestalkless individuals; each group was injected with a two-eyestalk-equivalent extract from control animals or from shrimp exposed to high concentrations of Cd, Hg, Pb, or low concentrations of Cu or Zn. All showed a hyperglycemic response within 2 h. In contrast, extracts of eyestalk removed from animals that had developed a full hyperglycemic reaction after exposure to low concentrations of Hg, Cd, Pb, or high concentrations of Cu and Zn were depleted of cHH as shown by the attenuation of the response after injection of the extracts into eyestalkless animals. This generalized and predictable sublethal response can be used as a quantitative physiological biomarker for water quality monitoring assessment.

Animals↗

Critical factors in chitin production by fermentation of shrimp biowaste.

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-1 beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction.

Acetic Acid↗

A novel, highly viscous polysaccharide excreted by an alteromonas isolated from a deep-sea hydrothermal vent shrimp.

A deep-sea, mesophilic, aerobic, and heterotrophic microorganism, able to produce an extracellular polysaccharide, was isolated from a shrimp collected near an active hydrothermal vent of the Mid-Atlantic Ridge. On the basis of phenotypic and phylogenetic analyses and DNA/DNA relatedness, this strain could be assigned to the species Alteromonas macleodii as a variant of the fijiensis subspecies. It was selected for its ability to exhibit a swarming mucoid phenotype on specific media. The bacterium secreted, under laboratory conditions, an extremely viscous exopolysaccharide consisting of glucose, galactose as neutral sugars, and glucuronic, galacturonic acids as uronic acids, along with pyruvate and acetate as main substituents.

Alteromonas↗