Digestive Proteinases of Artemesia longinaris (Decapoda, Penaeidae): characterization and dietary relationship

FERNÁNDEZ GIMENEZ, ANALIA VERÓNICA; DÍAZ, ANA CRISTINA; VELURTAS, SUSANA MARIA; FENUCCI, JORGE LINO

Mazatlán, Sinaloa, México

Simposio; VIII International Symposium on Aquatic Nutrition; 2006

Universidad Autónoma de Nuevo León

DIGESTIVE PROTEINASES OF ARTEMESIA LONGINARIS (DECAPODA, PENAEIDAE): CHARACTERIZATION AND DIETARY RELATIONSHIP. Fernández Gimenez,A.V.; Díaz, A.C.; Velurtas, S.M. and Fenucci,J.L. Departamento de Ciencias Marinas, Facultad Ciencias Exactas y Naturales, Universidad Nacional Mar del Plata, CONICET. CIC. Funes 3350, B7602AYL, Mar del Plata, Argentina. FAX 55-223-4753150. jfenucci@mdp.edu.ar key words Crustacea, nutrition, proteinases Introduction. The prawn Artemesia longinaris has a distribution along the South American coast, from 23°S to 43°S and is one of the most valuable species in the Argentine markets. Proteins in feeds modulate the activity of proteinases in penaeid shrimp. Understanding the mode of regulation of midgut gland enzymes is important for rational use of protein feed ingredients using for formulated feeds. Previous experiments in this species demonstrated good survival and growth under culture conditions, determined the nutritional requirements, and characterized the digestive proteinases in relation to the molting cycle (Fernández Gimenez et al 2002). The present study describes the effect of different protein sources in formulated feeds on the proteinases activity in the midgut gland of prawn A. longinaris. Materials and Methods. Three isoproteic and isolipidic formulated feeds (Table 1) were designed containing different amounts of meat and bone meal (feed 2), and soybean meal (feed 3) as substitution of fish meal (feed 1) (protein 38.8%; lipids 7.7%). Table 1. Ingredient composition of formulated feeds. Ingredient Formulated feed (g/100g dry feed) 1 2 3 Fish meal (24.5% crude protein) 48 27 27 Meat and bone meal (42.0% crude protein) - 23 - Soybean meal (41.8% crude protein) 17 17 23 Protein squid mantle - - 10 Manioc starch 20 20 22 Wheat 9.50 7.50 12.50 Fish oil 2 2 2 Fish soluble 2 2 2 Lecithin 0.5 0.5 0.5 Cholesterol 0.5 0.5 0.5 Vitamin supplement 0.5 0.5 0.5 The animals were obtained from coastal waters of Mar del Plata, Argentina (38°S). Feeding trials were carried out on juveniles (1.3±0.51 g initial weight) held in 150 l glass aquaria (33‰ salinity, 18°C, pH 7, 11:14 h photoperiod). Each feed was tested in three replicate groups of 8 prawns during three weeks. Midgut gland extracts from wild prawns (as control group) and from each treatment, were homogenized and assayed to quantify the proteinase activitiy using azocasein and specific substrates for trypsin and chymotrypsin activity. Classes of proteinase were identified by SDS-polyacrylamide gel electrophoresis; specific inhibitors for trypsin (TLCK), chymotrypsin (TPCK) and serin proteinases (PMSF and SBTI) were used. Results and Discussion. Total proteolytic activity was not significantly different among the wild prawn and the three treatments (0.29 to 0.44 abs min-1 mg-1). A. longinaris fed with different formulated feeds exhibited differences in trypsin activity related to the control. Chymotrypsin activity was highest for prawns fed with feed supplemented with meat and bone meal. Trypsin and chymotrypsin specific activity (abs min-1 mg-1) was lowest in wild prawns, meanwhile chymotrypsin activity was significant highest in prawns fed with meat and bone meal feed. This adaptation after feeding was observed in Litopenaeus vannamei (Muhlia-Almazán et al. 2003). TLCK and SBTI reduced azocasein hydrolysis between 36.8% (feed 2) and 81.7% (feed 3) (Table 2). TPCK did not decrease hydrolytic activity. Tabla 2. Effect of inhibitors on proteinase activity. Inhibitor/ Feed Percentage inhibition TLCK SBTI PMSF Wild 75.93±10.893a 28.63±3.623 a 25.50±4.928 a 1 59.30±4.122 a 49.76±1.940b not determined 2 36.78±3.111 b 41.57±1.662c not determined 3 81.73±11.66 a 76.96±2.370d 14.25±7.390 a Protein and activity bands were compared and common patterns were observed. Specific inhibitors and zymograms of A. longinaris midgut gland extracts determined the composition and molecular weight of the fraction with activity. Six active bands distributed from 16.6 to 53.7 kDa were detected; four trypsins (16.60, 18.20, 21.90, 25.10 kDa) and one chymotrypsin (53.70 kDa) were found. Conclusions. It have been determined an adaptation of trypsin and chymotrypsin to the protein quality in feeds. No clear relationship between different sources of protein in feeds and enzymes were established. References. Fernández Gimenez, A.V.; García-Carreño, F.L.; Navarrete del Toro, M.A. and Fenucci, J.L. 2002. CBP 132B: 593-598. Muhlia-Almazán, A.; García-Carreño, F.; Sánchez-Paz, J.; Yepiz-Plascencia, G. and Peregrino-Uriarte, A. CBP 135B: 373-383.