Digestive enzymes activity during ontogenetic development and effect of starvation in Japanese flounder, Paralichthys olivaceus

Abstract

Japanese flounder larvae and early juveniles were reared for 39 days after hatching (DAH) in order to determine pancreatic enzymes activities (trypsin and neutral lipase) using fluorogenic substrates during ontogeny and to compare enzymes activities of starved fish at different developmental stages. Larvae were fed L-type rotifers Brachionus plicatilis from 3 to 28 DAH, Artemia nauplii from 14 to 39 DAH and then gradually switched to pelleted feed from 25 DAH. Temperature was kept at 18 °C and photoperiod was 12L : 12D. Three starvation trials were made: from 2 to 8 DAH (premetamorphic larvae), 23–29 DAH (metamorphic) and 31–39 DAH (metamorphic and postmetamorphic). Fluorometric determinations on individual larvae were made using specific substrates for trypsin and lipase activity. Trypsin and neutral lipase activities were found in 2 DAH larvae (0.18 ± 0.09 U μg protein − 1 and 25.12 ± 7.36 nmoles 10 − 3 4MU μg protein − 1 min − 1 , mean ± SD, respectively), just at the time of first feeding. The evolution of both enzymes activities showed a profile marked by decreases during settlement (27–36 DAH). There is a significant increment ( P < 0.05) on specific trypsin activity at 15 DAH, corresponding with the beginning of Artemia feeding and a significant decrease of lipase activity at 6 DAH. In the first starvation experiment, larval mortality was 100% after 6 days. In the second trial a starvation-feeding experiment was done with two and four days starved larvae. Survival at day 6 was 50% and 25%, respectively. On the third trial post-metamorphic larvae were starved 5 days, survival at day 6 was 81%. As a general trend, enzymes activities get significantly lower ( P < 0.05) in starved fish. On two day starved–fed fish, lipase and trypsin activities levels took two days to reach control levels; on the other hand four days starved–fed fish showed significant differences two days after feeding again. During the last trial (31–39 DAH) lipase and trypsin activities went down, coinciding with juvenile settlement. These results show that trypsin and also neutral lipase activity measurements could be used for determining the critical periods during ontogeny and the nutritional status of larvae.