Alain Van Wormhoudt

Adaptation of trypsin, chymotrypsin and α-amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda)

Abstract Adaptation of digestive enzymes to dietary proteins was studied in Penaeus vannamei at the level of enzyme activity, synthesis measured by an immunoquantitative assay and polymorphism estimated for enzymes by electrophoresis and for mRNA by RT-PCR amplification. An initial experiment was conducted over 20 days with dietary casein increasing between 25 and 48%, starch and the other constituants remaining unchanged. A significant dose response effect was established between trypsin and casein ranging from 25 to 40% in the diet. No apparent change in the isoform pattern was recorded. Chymotrypsin varied neither at the level of specific activities nor at the level of polymorphism. Amylase activities and amounts decreased while casein increased. Two major isoforms were detected for 25% casein in the diet and only one for 40% casein. In order to establish the level of polymorphism of the expressed amylase mRNAs, a 378 bp fragment was obtained by RT-PCR on total RNA. In the case of the 25% casein diet, two different fragments, which corresponded to two proteins differing by an acidic charge, were amplified while only one was amplified in the case of the 40% casein diet. A comparison of casein with other sources of protein, gelatin, squid meal and fish protein soluble concentrate at two different concentrations in the diets (25 and 40%, respectively) confirmed the stimulation of trypsin by casein. No dose response was measured with other protein sources: moreover, an inhibition of trypsin activity was measured with gelatin in the diet. Highest activities of chymotrypsin were measured with casein and squid meal diets and lowest activities with gelatin and FPSC diets. Concerning amylase, the same variations were measured. Moreover, all the 40% protein diets inhibited the expression of one isoform.

Metabolism and growth of juveniles of Litopenaeus vannamei: effect of salinity and dietary carbohydrate levels.

The present study was designed to understand how carbohydrate (CBH) and protein metabolism are related in the penaeid shrimp Litopenaeus vannamei. With this information, we obtained a comprehensive schedule of the protein-carbohydrate metabolism including enzymatic, energetic, and functional aspects. We used salinity to determine its role as a modulator of the protein-carbohydrate metabolism in shrimp. Two experiments were designed. The first experiment evaluated the effect of CBH-salinity combinations in growth and survival, and hemolymph glucose, protein, and ammonia levels, digestive gland glycogen, osmotic pressure, and glutamate dehydrogenase (GDH) of L. vannamei juveniles acclimated during 18 days at a salinity of 15 per thousand and 40 per thousand. The second experiment was done to evaluate the effect of dietary CBH level on pre- and postprandial oxygen consumption, ammonia excretion, and the oxygen-nitrogen ratio (O/N) of juvenile L. vannamei in shrimps acclimated at 40 per thousand salinity. We also evaluated the ability of shrimp to carbohydrate adaptation. We made phosphoenolpyruvate carboxykinase (PECPK) and hexokinase activity measurements after a change in dietary carbohydrate levels at different times during 10 days. The growth rate depended on the combination salinity-dietary CBH-protein level. The maximum growth rate was obtained in shrimps maintained at 15 per thousand salinity and with a diet containing low CBH and high protein. The protein in hemolymph is related to the dietary protein levels; high dietary protein levels produced a high protein concentration in hemolymph. This suggests hemolymph is able to store proteins after a salinity acclimation. Depending on the salinity, the hemolymph proteins could be used as a source of osmotic effectors or as metabolic energy. The O/N values obtained show that shrimp used proteins as a source of energy, mainly when shrimps were fed with low CBH. The role played by postprandial nitrogen excretion (PPNE) in apparent heat increase (AHI) (PPNE/AHI ratio) is lower in shrimps fed diets containing high CBH in comparison with shrimps fed diets containing low CBH levels. These results confirm that the metabolism of L. vannamei juveniles is controlled by dietary protein levels, affecting the processes involved in the mechanical and biochemical transformations of ingested food. A growth depression effect was observed in shrimps fed with low-CBH protein diets and maintained in 40 per thousand salinity. In these shrimps, the hemolymph ammonia concentration (HAC) was significantly higher than that observed in shrimps fed with low CBH and maintained in 15 per thousand salinity. That high HAC level coincided with lower growth rate, which suggests that this level might be toxic for juveniles of L. vannamei. Results obtained for GDH activity showed this enzyme regulated both HAC and hemolymph protein levels, with high values in shrimps fed with low CBH levels and maintained in 40 per thousand salinity, and lower in shrimps fed with high CBH and maintained in 15 per thousand salinity. These differences mean that shrimp with a high-gill GDH activity might waste more energy in oxidation of the excess proteins and amino acids, reducing the energy for growth. It was evident that L. vannamei can convert protein to glycogen by a gluconeogenic pathway, which permitted shrimp to maintain a minimum circulating glucose of 0.34 mg/ml in hemolymph. A high PECPK activity was observed in shrimps fed a diet containing low CBH level indicating that the gluconeogenic pathway is activated, as in vertebrates by low dietary CBH levels. After a change in diet, we observed a change in PEPCK; however, it was lower and seems to depend on the way of adaptation, because it occurred after 6 days when adapting to a high-CBH diet and with little change for the low-CBH diet.

An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopenaeus vannamei juveniles

We are reporting results directed to explain the relation between carbohydrates (CHO), protein metabolism, and the energetic balance of Litopenaeus vannamei juveniles. The interaction of dietary CHO and salinity was measured to try to understand the relation between osmotic control and metabolism, both from a biochemical and energetic point of view. Two experiments were done. In the first experiment, shrimp were fed with 0%, 5%, 33%, and 61% CHO and maintained at 15‰ and 40‰ salinity. Glucose, lactate protein, hemocyanin, ammonia concentration, and osmotic pressure were measured in blood. Digestive gland glycogen (DGG) was measured also. In the second experiment, shrimp were fed with 0% and 38% dietary CHO and maintained at 15‰ and 40‰ salinity. From that shrimp, absorbed energy (Abs) was calculated as: Abs=respiration (R)+ammonia excretion (U) and production (P); assimilated energy (As) was calculated as the product of R×P. Osmotic pressure, hemocyanin, protein, lactate, and blood ammonia increased with the reduction in dietary CHO. In contrast, an increase in blood glucose was observed with an increase in dietary CHO. Digestive gland glycogen (DGG) increased following a saturation curve with a DGG maximum at 33% dietary CHO. Blood metabolites of fasting and feeding shrimp showed the same behavior. Energy balance results showed that shrimp maintained in low salinity and fed without CHO waste more energy in U production than for shrimp maintained in high salinity and fed with high CHO levels. Notwithstanding, the production efficiency was higher in shrimp fed without CHO than that observed in shrimp fed with high CHO independent of salinity. A scheme trying to integrate the relation between CHO and protein metabolism and the way in which both are modulated by salinity is presented. From published and present results, there are two factors that apparently control the use of high dietary CHO levels; α-amylase enzyme-dietary CHO level capacity and glycogen saturation in DG. Production of glucose is limited in shrimp because of saturation of α-amylase when shrimp are fed with diets above 33% CHO. This is the first control point of starch metabolism. The digestive gland is saturated with glycogen in shrimp fed with dietary CHO levels >33%. This is apparently the second control point of CHO metabolism that limits growth rate in such conditions. The high metabolic cost related to high CHO diets could explain why shrimp are well adapted to use protein as a source of energy.

Molecular cloning of hemocyanin cDNA from Penaeus vannamei (Crustacea, Decapoda): structure, evolution and physiological aspects

Hemocyanin is present as 2 subunits in the hemolymph of Penaeus vannamei. Isolated from a hepatopancreas cDNA library of this penaeid shrimp, the cDNA chain (2095 bp) corresponds to a full length hemocyanin messenger as determined by Northern hybridization, with a short 5′ untranslated region (17 bp), an open reading frame (1989 bp counting initiation and termination codons) coding for a signal peptide (13 residues) and a mature hemocyanin (648 amino acids), and a 3′ untranslated region (89 bp) followed by the polyadenylated track. It is the first time that the existence of a hydrophobic signal peptide is shown in arthropod hemocyanin. Two primary N‐terminal sequences are determined and a 3‐fold increase of mRNA content, measured in the hepatopancreas during the premoult stages, is reported. The low level of polymorphism shown by P. vannamei hemocyanin, along with its weak percentage identity with counterparts and its similarity with hemocyanin from Panulirus interruptus, suggests that this arthropod hemocyanin may be a primitive subunit that has evolved independently, following gene duplication.

Molecular cloning of a cDNA that encodes a serine protease with chymotryptic and collagenolytic activities in the hepatopancreas of the shrimp Penaeus vanameii (Crustacea, Decapoda)

Two clones were isolated by screening a shrimp hepatopancreas cDNA library with a DNA fragment obtained by PCR amplification using two oligonucleotides based on the partial protein sequence of Penaeus vanamei chymotrypsin purified earlier. One of these clones, PVC 7 contains a complete cDNA coding for a serine protease. The deduced amino acid sequence shows the existence of a 270 residue‐long preproenzyme containing a highly hydrophobic signal peptide of 14 amino acids. This suggests the existence of a putative zymogen form of the enzyme containing a 30 amino acid‐long peptide which is cleaved to give a mature protein of 226 residues. A highly preferred codon usage is observed for this protein. The other obtained cDNA was found to encode the less predominant variant of the protein. Sequence alignments show that shrimp chymotrypsin is highly homologous with crab collagenase (77% homology taking into account the same amino acid at the same position, and 83% homology taking into account amino acids wall conserved function) and that it is more similar to mouse trypsin (41% homology of strictly conserved amino acids) than to hornet chymotrypsin (35% homology).

CLONING AND SEQUENCING ANALYSIS OF THREE AMYLASE CDNAS IN THE SHRIMP PENAEUS VANNAMEI (CRUSTACEA DECAPODA) : EVOLUTIONARY ASPECTS

InPenaeus vannamei, α-amylase is the most important glucosidase and is present as at least two major isoenzymes which have been purified. In order to obtain information on their structure, a hepatopancreas cDNA library constructed in phage lambda-Zap II (Strategene) was screened using a synthetic oligonucleotide based on the amino acid sequence of a V8 staphylococcal protease peptide ofP. vannamei α-amylase. Three clones were selected: AMY SK 37 (EMBL sequence accession number: X 77318) is the most complete of the analyzed clones and was completely sequenced. It contains the complete cDNA sequence coding for one of the major isoenzymes of shrimp amylase. The deduced amino acid sequence shows the existence of a 511-residue-long pre-enzyme containing a highly hydrophobic signal peptide of 16 amino acids. Northern hybridization of total RNA with the amylase cDNA confirms the size of the messenger at around 1,600 bases. AMY SK 28, which contains the complete mature sequence of amylase, belonged to the same family characterized by a common 3′ terminus and presented four amino acid changes. Some other variants of this family were also partially sequenced. AMY SK 20 was found to encode a minor variant of the protein with a different 3′ terminus and 57 amino acid changes.Phylogenetic analysis established with the conserved amino acid regions of the (β/α) eight-barrel domain and with the total sequence ofP. vannamei showed close evolutionary relationships with mammals (59–63% identity) and with insect α-amylase (52–62% identity). The use of conserved sequences increased the level of similarity but it did not alter the ordering of the groupings. Location of the secondary structure elements confirmed the high level of sequence similarity of shrimp α-amylase with pig α-amylase.

Digestive enzyme activities of Penaeus notialis during reproduction and moulting cycle

The degree of ovarian maturation and the specific activities in hepatopancreas and stomach of amylase, trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase were determined at different stages of the moulting cycle of wild female pink shrimp Penaeus notialis. Ovarian maturation appeared from intermoult stage C1–C2 to early premoult stage D1. The highest values for amylase in stomach and hepatopancreas ocurred at stages D1 and B2 to C3–C4, respectively. Total proteolytic activity in the hepatopancreas was highest in stages B2 to C3–C4, and in the stomach the highest value was observed in D1. For the endopeptidases, the highest value was for trypsin TAME in stomach during stage D1 and in the hepatopancreas at stage C1–C2. The exopeptidases showed peaks in hepatopancreas in stages C1–C2 and C3–C4. Leucine aminopeptidase showed the lowest proteolytic values and peaks in stomach were observed at stages C3–C4 and D2.

Amylase polymorphism in crustacea decapoda: electrophoretic and immunological studies

Alpha-amylase polymorphism was studied in 40 species of crustaceans belonging to four infraorders of Decapoda. Specific activities of alpha-amylases from hepatopancreas extracts of the same species were also measured. Very low specific activities were measured for Carcinus, Munida, Nephrops and Palinurus, whilst high activities were measured for shrimps and crabs. Amylases from one species of each of the four infraorders of Decapoda were purified by affinity chromatography. Very few differences in the specific activities of the pure enzymes were detected. The amylase content of crude extracts ranges between 0.1% of total protein, from the hepatopancreas in Carcinus maenas and Eupagurus bernhardus, to 1% Penaeus vannamei and Procambarus clarkii. The apparent molecular weight, determined by denaturating electrophoresis, was 55 kDa for Eupagurus and Procambarus amylases but only about 30 kDa for Carcinus and Penaeus amylases. Using immunodiffusion, rocket immunoelectrophoresis and enzyme inhibition with antibodies against Palaemon serratus α-amylase, we have shown that all the Decapod α-amylases are immunologically related but that they are different from those of Copepods, Bacillus or pig pancreatic amylases. The use of these data for phylogenetic analysis is discussed.

Characterization and Expression of Glutamate Dehydrogenase in Response to Acute Salinity Stress in the Chinese Mitten Crab, Eriocheir sinensis

Background Glutamate dehydrogenase (GDH) is a key enzyme for the synthesis and catabolism of glutamic acid, proline and alanine, which are important osmolytes in aquatic animals. However, the response of GDH gene expression to salinity alterations has not yet been determined in macro-crustacean species. Methodology/Principal Findings GDH cDNA was isolated from Eriocheir sinensis. Then, GDH gene expression was analyzed in different tissues from normal crabs and the muscle of crabs following transfer from freshwater (control) directly to water with salinities of 16‰ and 30‰, respectively. Full-length GDH cDNA is 2,349 bp, consisting of a 76 bp 5′- untranslated region, a 1,695 bp open reading frame encoding 564 amino acids and a 578 bp 3′- untranslated region. E. sinensis GDH showed 64–90% identity with protein sequences of mammalian and crustacean species. Muscle was the dominant expression source among all tissues tested. Compared with the control, GDH expression significantly increased at 6 h in crabs transferred to 16‰ and 30‰ salinity, and GDH expression peaked at 48 h and 12 h, respectively, with levels approximately 7.9 and 8.5 fold higher than the control. The free amino acid (FAA) changes in muscle, under acute salinity stress (16‰ and 30‰ salinities), correlated with GDH expression levels. Total FAA content in the muscle, which was based on specific changes in arginine, proline, glycine, alanine, taurine, serine and glutamic acid, tended to increase in crabs following transfer to salt water. Among these, arginine, proline and alanine increased significantly during salinity acclimation and accounted for the highest proportion of total FAA. Conclusions E. sinensis GDH is a conserved protein that serves important functions in controlling osmoregulation. We observed that higher GDH expression after ambient salinity increase led to higher FAA metabolism, especially the synthesis of glutamic acid, which increased the synthesis of proline and alanine to meet the demand of osmoregulation at hyperosmotic conditions.

Alpha‐glucosidase from the hepatopancreas of the shrimp, Penaus vannamei (Crustacea‐Decapoda)

Penaeus vannamei is an omnivorous species, and it can be assumed that a high level of carbohydrates is necessary for growth. Alpha-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates. Using acarbose affinity chromatography, a glycosylated alpha-glucosidase with a molecular mass of approximately 105 kDa was isolated for the first time from the hepatopancreas of the shrimp. Exhibiting an optimal catalytic activity in the temperature range from 40 degrees C to 50 degrees C at pH 6, the purified enzyme hydrolyses alpha 1-4 bonds and liberates glucose from different oligo and polysaccharides. By contrast to other known glucosidases, no alpha 1-6 glucose link with hydrolysis has been observed. This could explain the different rates of growth in shrimp aquaculture with starches from various origins. The amino-acid composition, together with the partial sequence of a hydrolytic peptide, shows a high degree of similarity to the alpha-glucosidases reported for various organisms including yeast and fungi and may help determine the phylogeny of the family.