Diana Martinez-Alarcon

Is digestive cathepsin D the rule in decapod crustaceans

Cathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Transcriptome analysis of the midgut gland of the brown shrimp Crangon crangon indicates high polymorphism in digestive enzymes

Tolerance of organisms towards heterogeneous and variable environments is highly related to physiological flexibility. An effective strategy to enhance physiological flexibility is the expression of polymorphic enzymes. This seems to be the case in the brown shrimp Crangon crangon. It shows high reproduction rates, feeds opportunistically on endo- and epibenthic organisms, and is apparently well adapted to variable environmental conditions. Previous electrophoretic studies revealed a high level of polymorphism and no consistent phenotype of digestive enzymes between individuals. In order to understand the underlying biochemical processes, we carried out a transcriptome-based study of digestive enzymes of C. crangon. Detailed sequence analyses of triacylglycerol lipase, phospholipase A2, alpha amylase, chitinase, trypsin and cathepsin L were performed to identify putative isoforms. The number of isoforms, and thus the degree of polymorphism varied among enzymes: lipases and carbohydrases showed higher numbers of isoforms in enzymes that besides their extracellular function also have diverse intracellular functions. Furthermore, cysteine proteinases showed a lower polymorphism than serine proteinases. We suggest that the expression of enzyme isoforms improves the efficiency of C. crangon in gaining energy from different food sources.