Erick Perera

Polymorphism and partial characterization of digestive enzymes in the spiny lobster Panulirus argus.

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804).

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways

SUMMARY The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency.

LARGE SCALE ASSESSMENT OF RECRUITMENT FOR THE SPINY LOBSTER, PANULIRUS ARGUS, AQUACULTURE INDUSTRY

[Investigations to study the long and complex larval cycle of the spiny lobster, Panulirus argus in artificial shelters, on the barrier reef and in sea grass meadows, became efficient programs for catching large numbers of pueruli and juveniles at some locations of the Cuban archipelago. These data support a study to obtain a better understanding of the recruitment variability, of the mortality rates during the juvenile phase, and to evaluate the potential alternative for growout of pueruli and juveniles to marketable sizes. The intensity of larval recruitment is much higher in the area close to the edge of the shelf (32.2 pueruli/collector) and diminishes toward the shallow coastal waters (1.2 pueruli/collector). The total cost to collect 1,116 pueruli was around US

Lobster (Panulirus argus) Hepatopancreatic Trypsin Isoforms and Their Digestion Efficiency

1656 (US

New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket

1.50/puerulus), and 400 juveniles require around US

Trypsin isozymes in the lobster Panulirus argus (Latreille, 1804): from molecules to physiology

1697 (US

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus

4.14/juvenile) in five days. In the seaweed collectors, there is an absence of predators and diseases, a good level of food (a high variety of invertebrates, fish eggs and fish larvae, Foraminifera, and filamentous algae), and a gregarious behaviour in a structurally complex tassel may increase individual growth rates. The natural mortality rate in the juvenile nursery area has been estimated at 0.42 year −1 , so that 58% was estimated to survive and be recruited to the fishery. The results of our studies indicate that juvenile removals from the wild have a negative impact on pre-recruits and subsequent catches. Improvement of the techniques to collect pueruli is needed to catch a large number of these, because the current objective of the program in Cuba is to measure puerulus recruitment and to predict catches. Any strategy of commercial investment for the growout of pueruli and post-pueruli to marketable sizes requires accurate seasonal catch-and-effort data, and knowledge of the population parameters, all needed to assist in preventing overfishing. Las investigaciones para estudiar el largo y complejo ciclo larval de la langosta Panulirus argus en refugios artificiales, en la barrera de arrecifes y el seibadal marino, se convirtieron en programas eficaces para capturar un elevado numero de puerulos y juveniles en algunas localidades del archipielago cubano. Estos datos apoyan el estudio para lograr una mejor compresion de la variabilidad del reclutamiento, las tasas de mortalidad durante la fase juvenil, y para evaluar el potencial alternativo de engorde de puerulus y juveniles a las tallas comerciales. La intensidad del reclutamiento larval es mucho mayor en el area cercana al borde de la plataforma (32,2 puerulos/colector) y disminuye hacia la costa en aguas someras (1,2 puerulos/colector). El costo total para colectar en cinco dias 1.116 puerulos es de alrededor de 1656 US

A Holistic View of Dietary Carbohydrate Utilization in Lobster: Digestion, Postprandial Nutrient Flux, and Metabolism

(1,50/puerulo US

The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus

) y 400 juveniles es de 1697 US