A. Van Wormhoudt

Influence of dietary carbohydrate on the metabolism of juvenile Litopenaeus stylirostris

The effect of dietary carbohydrates (CBH) on glucose and glycogen, digestive enzymes, ammonia excretion and osmotic pressure and osmotic capacity of Litopenaeus stylirostris juveniles was studied. The increase of CBH, ranging between 1 and 33%, stimulates activities of alpha-amylase and alpha-glucosidase in the hepatopancreas. High levels of glucose in hemolymph and of glycogen in the hepatopancreas were reached at the highest level of dietary CBH; however, the kinetics of accumulation is different. Shrimps fed with low level of CBH needed 3 h to reached glucose peak, whereas only 1 h is necessary for high CBH levels. A saturation curve was observed in glycogen level and alpha-amylase activity with maximum values in shrimp-fed diets containing 21% CBH. This level could be used to be included as a maximum shrimp dietary CBH level. Pre-prandial glycogen levels were observed in shrimp fed a diet containing 1% CBH, indicating an important gluconeogenesis, which affected the protein metabolism. The present results show that a diet containing 10% CBH may not be enough to cover the CBH requirement, which could be satisfied by dietary protein content. The low osmotic capacity observed in shrimp fed on a diet containing 10% CBH coincided with a relatively low post-prandial nitrogen excretion which reflects a low concentration of amino acids circulating in hemolymph, which affected the osmotic pressure and the osmotic capacity. These results reflect the high plasticity of shrimp species to use protein to obtain metabolic energy from food and its limited capacity for processing dietary CBH.

Cloning and expression of cathepsin L-like proteinases in the hepatopancreas of the shrimp Penaeus vannamei during the intermolt cycle.

Cysteine protease activities have been characterized with benzyloxycarbonyl-lysinep-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimpPenaeus vannamei. An optimum pH of 5.1 has been measured. To characterize these cysteine proteases, a hepatopancreas cDNA library was screened by hybridization to a Norway lobster cysteine protease cDNA fragment. Two cDNAs encodingP. vannamei cysteine protease precursors have been cloned and sequenced. The encoded polypeptides have 326 and 322 amino acid residues, respectively, each consisting of partial signal sequences (15 and 10 residues), a pro-region (93 and 94 residues), and a mature enzyme polypeptide (218 residues). Cys25, His159 and Asn175 form the catalytic triad in the putative active site of the mature enzymes. Compared with invertebrate cysteine proteases (Homarus andFasciola), each of the two shrimp enzymes shows 70 and 52% amino acid sequence identity, respectively; 63% identity is shown with rat cathepsin L. Northern hybridization analysis showed the same size for the different cysteine protease transcripts in hepatopancreas tissue (approximately 1.1 kb). During intermolt cycles, variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA.

Purification, biochemical characterization and N-terminal sequence of a serine-protease with chymotrypsic and collagenolytic activities in a tropical shrimp, penaeus vannamei (crustacea, decapoda)

1. Two chymotrypsin variants, with collagenolytic activities, were purified from the hepatopancreas of Penaeus vannamei using radioactive protein as the substrate. 2. These proteases are very close as far as amino acid composition, molecular weight, inhibitors studies and specificity against small synthetic substrates are concerned. 3. N-terminal amino acid sequences of both variants are identical and are very close to other known crustacean serine proteases.

Chitinolytic enzymes in the integument and midgut-gland of the shrimpPalaemon serratus during the moulting cycle

Chitinase andN-acetyl-β-D-glucosaminidase activity were quantified inPalaemon serratus (Pennant) integument and midgut gland during the moulting cycle. Studies were performed on specimens collected near Concarneau, France, in July 1989. The changes in specific activity are different in the two organs: in the midgut gland enzymatic activity is high throughout the whole moulting cycle with a weak peak at the early premoult Stage D1′, whereas in the integument the activity of both enzymes is very low throughout post- and intermoult stages and rises only after premoult Stage D1′. The highest specific activity is reached in D1‴ for chitinase and somewhat earlier (D1″) forN-acetyl-β-D-glucosaminidase. The increase in specific chitinolytic activity coincides with an increase in ecdysteroids.

Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea decapoda): Quantitative and qualitative changes

During the embryogenesis of Palaemon serratus profound changes in ecdysteroid concentrations were found. Ecdysteroid concentrations increase at the appearance of the Y-organ and a slight decrease is observed just before hatching. Two peaks of ecdysteroid concentration occur between these two events if Palaemon is reared at 19 degrees, but only one broad maximum is observed after rearing at 11-12 degrees. The pattern of ecdysteroids changes during embryogenesis; the ecdysone level decreases, while the 20-OH-ecdysone concentration and the amount of high polarity products increase. During the embryonic stage C the ratio of 20-OH-ecdysone to ecdysone was shown by HPLC analysis to steadily increase. After enzymatic hydrolysis of high-polarity products the main RIA-positive material elutes from reversed-phase HPLC columns as 20-OH-ecdysone.

CATHEPSIN L GENE ORGANIZATION IN CRUSTACEANS

The gene structure of a cathepsin L of the shrimp Penaeus vannamei has been determined by the polymerase chain reaction. It comprises six exons of various lengths spanning a total of 1792bp. This architecture is homologous to that of rat cathepsin L, three conserved sites of intron position have been effectively identified, with the exception of the third intron break-point located immediately after the cysteinyl active site. In contrast, no similarity is observed with Drosophila or Plasmodium cathepsin L-like gene organizations. This gene expresses a major cathepsin L enzyme in the hepatopancreas. The last intron is polymorphic, suggesting the presence of at least three different genes.

Chymotrypsin gene expression during the intermolt cycle in the shrimpPenaeus vannamei (Crustacea; Decapoda)

InPenaeus vannamei, chymotrypsin is present as two isoenzymes in the hepatopancreas. The enzyme has been localized in F-cells by immunocytochemistry using a specific antibody. By in situ hybridization, with a 510 pb cDNA probe encoding for the first 170 amino acids of the shrimp chymotrypsin, mRNA was localized in the same cells. Gene expression was followed during the intermolt cycle by measuring changes in specific activity in crude extracts, and by the estimation of mRNA levels by Northern blots using the same probe. The increase in specific activity in premolt is preceded in early premolt by an increase in the amount of chymotrypsin mRNA. A second increase is observed in postmolt, suggesting a different mode of regulation of gene expression.

Amylase mRNA expression in Crassostrea gigas during feeding cycles.

Abstract A Crassostrea gigas digestive gland copy DNA (cDNA) library constructed in the λ phage ZapII (Stratagene, La Jola, USA) was screened with an amylase heterologous probe. To get access to the complete cDNA, a polymerase chain reaction extension was conducted using DNA extracted from the phages. The complete cDNA sequence is 1688 base pairs (EMBL = Y08370). The deduced protein sequence is 519 aminoacids long with a 19 aminoacid signal peptide. Similarity with Pectenmaximus amylase is 72%. A 3-day nutrition experiment with a cyclic algal food supply was carried out. Amylase enzyme activities and mRNAs were individually measured on five animals, nine times a day. Messenger RNAs were quantified by dot hybridization using the previously characterized cDNA as probe. Variation of amylase mRNA was observed, in relation with the level of activity of the enzyme. Coordinated changes in RNA and enzyme levels suggested a possible transcriptional regulation of amylase in C. gigas as in vertebrates.

Changes and characteristics of the crustacean hyperglycemic hormone (CHH material) in Palaemon serratus pennant (crustacea, decapoda, natantia) during the different steps of the purification

Abstract 1. 1. The importance of the MTGX2 sinus gland complex in the production and the storage of CHH material has been demonstrated by bioassays using extracts of the different eyestalks structure. 2. 2. The purification of the hyperglycemic material (CHH) was carried out in Palaemon serratus by two procedures, starting from total eyestalk extracts or from extirpated sinus glands. 3. 3. There is evidence for the presence of 3 different forms of the hyperglycemic active fractions expressed by their electrophoretic mobility and molecular weight. 4. 4. The main fraction has a molecular weight of around 8000 and a r m of 0.59 in Davis electrophoresis but small peptides (around 2000 mol. wt) are also hyperglycemic if injected in eyestalkless shrimps.

In vitro protein synthesis and α amylase activity in F cells from hepatopancreas ofPalaemon serratus (Crustacea; Decapoda)

In crustaceans, all the steps in the assimilation of food take place in the hepatopancreas. To facilitate the study of this organ, a method for the dissociation of cell types was developed. The hepatopancreas of the prawnPalaemon serratus was mechanically dissociated and the cells separated by Percoll density-gradient centrifugation. The E and R cells had similar densities of around 1.05 g/ml. The F cells were separated into two distinct fractions with densities of 1.075 and 1.082 g/ml. The B cells sedimented at a density of 1.12 g/ml. The ratio between the two populations of F cells was found to vary during the intermolt cycle while B cells disappeared after the molt. When the density gradient fractions were incubated with3H-leucine, incorporation was highest in the F cell fractions. Measurements of α-amylase activity, indicated that the two populations of F cells may be derived from the same cell type.