Characterisation and expression of the biomineralising gene Lustrin A during shell formation of the European abalone Haliotis tuberculata.

The molluscan shell is a remarkable product of a highly coordinated biomineralisation process, and is composed of calcium carbonate most commonly in the form of calcite or aragonite. The exceptional mechanical properties of this biomaterial are imparted by the embedded organic matrix which is secreted by the underlying mantle tissue. While many shell-matrix proteins have already been identified within adult molluscan shell, their presence and role in the early developmental stages of larval shell formation are not well understood. In the European abalone Haliotis tuberculata, the shell first forms in the early trochophore larva and develops into a mineralised protoconch in the veliger. Following metamorphosis, the juvenile shell rapidly changes as it becomes flattened and develops a more complex crystallographic profile including an external granular layer and an internal nacreous layer. Amongst the matrix proteins involved in abalone shell formation, Lustrin A is thought to participate in the formation of the nacreous layer. Here we have identified a partial cDNA coding for the Lustrin A gene in H. tuberculata and have analysed its spatial and temporal expression during abalone development. RT-PCR experiments indicate that Lustrin A is first expressed in juvenile (post-metamorphosis) stages, suggesting that Lustrin A is a component of the juvenile shell, but not of the larval shell. We also detect Lustrin A mRNAs in non-nacre forming cells at the distal-most edge of the juvenile mantle as well as in the nacre-forming region of the mantle. Lustrin A was also expressed in 7-day-old post-larvae, prior to the formation of nacre. These results suggest that Lustrin A plays multiple roles in the shell-forming process and further highlight the dynamic ontogenic nature of molluscan shell formation.

Starch supplementation modulates amylase enzymatic properties and amylase B mRNA level in the digestive gland of the Pacific oyster Crassostrea gigas.

In the oyster Crassostrea gigas consumption-related traits, amylase properties and growth were found to be linked through genotypes that differed for polymorphism in the two amylase genes AMYA and AMYB. Modulation of AMYA mRNA level had already been observed in response to food availability, whereas the functional role of AMYB was still unknown. To improve knowledge about the regulation of amylase expression in C. gigas and the respective roles of the two genes, we made an assay of amylase expression at mRNA and enzymatic levels in the digestive gland of oysters that had received dietary supplements of starch. After 18 days, a significant increase of translatable mRNA for AMYB was observed, with a correlated increase in Michaelis-Menten constant Km values and a decrease in total amylase activity. This modulation is the first evidence of observable functioning of AMYB in digestive processes. Amylase B is suggested to display a higher Km than amylase A, offering a means of adapting to high substrate concentrations. The highest starch supplement level (10 mgL(-1)) induced alteration in oyster physiology. The 1 mgL(-1) treatment should be tested as a practical food supplement that could lead to growth benefits for oysters.

Association among growth, food consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas.

To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.

An amylase gene polymorphism is associated with growth differences in the Pacific cupped oyster Crassostrea gigas.

This study investigated the non-neutrality of genetic polymorphism in two alpha-amylase genes (AMYA and AMYB) in the oyster Crassostrea gigas. Bi-parental oyster families, bred to be polymorphic for markers in these genes, were monitored for growth and survival for 1 year under standard culture conditions in two French production sites. Within-family genotype frequencies indicated that the two amylase genes were closely linked (c. 1.7 cM). Within two of three families, significant differences in growth were observed between genotypes at one of the two production sites, suggesting that this polymorphism is not neutral and might be under selection because of its role in digestive function. Estimated daily yields were different between amylase genotypes, indicating the potential value of amylase markers in selective breeding programmes to improve oyster growth.

Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.

Molecular cloning and seasonal expression of oyster glycogen phosphorylase and glycogen synthase genes.

To investigate the control at the mRNA level of glycogen metabolism in the cupped oyster Crassostrea gigas, we report in the present paper the cloning and characterization of glycogen phosphorylase and synthase cDNAs (Cg-GPH and Cg-GYS, respectively, transcripts of main enzymes for glycogen use and storage), and their first expression profiles depending on oyster tissues and seasons. A strong expression of both genes was observed in the labial palps and the gonad in accordance with specific cells located in both tissues and ability to store glucose. Cg-GPH expression was also found mainly in muscle suggesting ability to use glycogen as readily available glucose to supply its activity. For seasonal examinations, expression of Cg-GYS and Cg-GPH genes appeared to be regulated according to variation in glycogen content. Relative levels of Cg-GYS transcripts appeared highest in October corresponding to glycogen storage and resting period. Relative levels of Cg-GPH transcripts were highest in May corresponding to mobilization of glycogen needed for germ cell maturation. Expression of both genes would likely be driven by the oyster's reproductive cycle, reflecting the central role of glycogen in energy storage and gametogenic development in C. gigas. Both genes are useful molecular markers in the regulation of glycogen metabolism and reproduction in C. gigas but enzymatic regulation of glycogen phosphorylase and synthase remains to be elucidated.

Structure of amylase genes in populations of Pacific Cupped oyster ( Crassostrea gigas): tissue expression and allelic polymorphism.

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC(37)) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation ( F(st) between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.

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.

Molecular cloning of a cDNA encoding alpha-glucosidase in the digestive gland of the shrimp, Litopenaeus vannamei.

The complete sequence of the 3-kb cDNA and the 5' genomic structure are reported for the gene encoding the shrimp alpha-glucosidase. Alpha-glucosidase cDNA was isolated from a shrimp digestive gland cDNA library. The 2830-base pair cDNA contains an open reading frame that encodes 919 amino acids. The shrimp alpha-glucosidase cDNA shows a high level of identity with that of the human sucrase-isomaltase, human maltase-glucoamylase, and human acid lysosomal alpha-glucosidase, indicating that the protein shares the same structural domains. The similarities among these proteins are found as clusters and characterize the glycosyl hydrolase family 31. To our knowledge, this is the first report to describe a satellite sequence in the 5' genomic structure before the TATA box in an invertebrate sequence.

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.

Amylase mRNA expression in Crassostrea gigas during feeding cycles.

A Crassostrea gigas digestive gland copy DNA (cDNA) library constructed in the lambda phage ZapII (Stratagene, La Jola, USA) was screened with an amylase heterologous proble. 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 Pecten maximus 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.

Secretagogues and Growth Factors in Fish and Crustacean Protein Hydrolysates.

: The search for new molecules in fish protein hydrolysates is of great interest in animal feeding as it is in aquaculture, fertilizer, cosmetic, and pharmacologic domains. Different sources of hydrolysates such as shrimp waste (Pandalus borealis), cod (Gadus morhua) head, and head and viscera of sardine (Sardina pilchardus), obtained after hydrolysis or autolysis, were tested on fibroblast cell cultures and by gastrin radioimmunoassay. The level of hydrolysis seems to play an important role in the presence of biological peptides. Elution profile on a gel filtration Sephadex G-50 column was used to estimate the degree of hydrolysis of the fractions studied. Growth-factor-like activities were found in less-hydrolyzed fractions. Conversely, the most-hydrolyzed fractions showed gastrin and cholecystokinin immunoreactivity.

Heparin-binding molecules with growth factor activities in regenerating-tissues of the starfish Asterias rubens.

Regenerating-tissues of the starfish Asterias rubens were studied for the presence of growth factors liable to stimulate the proliferation of fibroblast and epithelial cells (3T3, BHK21 and Hela cells). As a first attempt to isolate growth factors, the extracts were fixed on heparin-affinity column and were eluted by 1-1.2 M NaCl. After separation on a Vydac C18 HPLC column. a fraction that stimulates the proliferation of fibroblast cells was isolated. It contained four different peptides, separated by electrophoresis, and for which the amino acid composition and molecular mass were determined. All the peptides were lysine rich and one presented an amino-acid composition comparable to basic-fibroblast growth factor (b-FGF) while its molecular weight was higher.

Polymorphism and evolution of collagenolytic serine protease genes in crustaceans.

Two genomic DNA fragments encoding crustacean collagenolytic serine protease genes show coding fragments that span 1522-1526 base pairs and contain seven exons encoding the complete amino acid sequence of two enzymes, CHYA and CHYB. As in serine protease genes from other organisms, the region coding for the residues around the active site is split by two introns. Although the introns differ from those of other organisms in size and nucleotide sequence, their number and location are more or less the same as found in mammalian chymotrypsin or elastase genes that evolved lately, but different for trypsin genes. Meanwhile, the junction that occurs between the propeptide and the maturation site is only found in the shrimp genes. This is also the case for the junction located 13 amino acids after the active site aspartic acid in these genes. Between 40 and 50 copies of the genes are reported by Southern analysis. Seven different genes within ChyA Pv family present 0-6% base changes, whereas five different genes belonging to ChyB Pv family show changes of up to 27% in the short studied portion of exon 4. This last family presents a mosaic organization of the coding parts, which are also expressed in the hepatopancreas of the shrimp as the variant PVC5 cDNA.

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.

Genomic organisation and polymorphism of a crustacean trypsin multi-gene family.

The coding sequences of three trypsin genes, obtained by polymerase chain reaction (PCR), were determined in Penaeus vannamei (Crustacea, Decapoda). These genes were characterised by two short introns, which occur at a position quasi-conserved as the first two introns in vertebrate counterparts. Belonging to three different families, two of the genes are expressed in the digestive gland. A 5' RACE-PCR amplification of hepatopancreatic mRNA, together with the presence of short 5' extensions, confirmed that the third gene family is not expressed in this tissue. The second intron in the genes expressed in the hepatopancreas presents a 5' splice site consensus, beginning with a GC motive which is reported for the first time in trypsin genes and is of phase I in mammals. A high copy number was determined for these genes. Several restricted PCR were performed to describe the polymorphism of these sequences. Five genes were partially sequenced for each family and five genes coding the different, previously described cDNAs were recovered. These sequences also confirm that the third family resembles a mosaic of type I and type II gene families. A high degree of polymorphism in the introns (54-59% identity) among the three families is observed, but mutations in corresponding introns inside each of the families are low (3-6%).

alpha-Glucosidase from the hepatopancreas of the shrimp, Penaeus 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.

Amylase on Pecten maximus (Mollusca, bivalves): protein and cDNA characterization; quantification of the expression in the digestive gland.

The digestive enzyme alpha-amylase in Pecten maximus has been purified from the digestive gland, where it is present as two isoforms, In order to gain information on its structure and regulation, a digestive gland cDNA library, constructed in lambda phage Zap II (Stratagene, La Jolla, Calif., U.S.A.), was screened with a shrimp alpha-amylase cDNA probe. Only 0.02% of the clones were positive, and the longest clone, having a size of 1700 bp and identical to that of the mRNA, was fully sequenced. It contains the complete cDNA coding frame for one of the amylase isoforms of P. maximus. The deduced protein sequence is 508 amino acids long, with a putative 18 amino acid, highly hydrophobic signal peptide and a mature enzyme of 489 residues. The molecular weight corresponds to 54,500 Da and the calculated isoelectric point is 6.76. Locations of conserved sequences confirms the high level of similarity with the other members of the family.

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 and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle.

Trypsin is the most abundant protease in Crustacea. This enzyme was purified from the digestive gland of Penaeus vannamei, revealing three major isoforms (molecular weights 31-32 kDa) and several minor components. Five cDNAs encoding five isoforms of trypsin were detected by two successive screenings of an amplified cDNA library from the digestive gland of P. vannamei. The longest isolated and sequenced cDNA encoded a preproenzyme of 255 amino acids containing a putative precursor peptide of 14 residues and a highly hydrophobic signal sequence of 14 amino acids. Amino acid sequence alignments revealed a high degree of identity between the trypsin from P. vannamei and that from crayfish (74%) and an equal level of sequence similarity to that from mammals and insects (approximately 40). Dot blot hybridization and subsequent analysis of the variation in trypsin-specific activities revealed that mRNA expression is at a maximum during early premoult (D1), declining sharply in late premoult (D2-D3). The specific activity of trypsin also followed this pattern, suggesting the regulation of trypsin biosynthesis is, at least in part, transcriptional. The characterization of trypsin cDNA from P. vannamei provides the first description of a putative zymogen sequence in a crustacean species, enabling us to elucidate the regulatory mechanism of trypsin synthesis in these important marine organisms.