EARLY ONTOGENY OF SELECTED DIGESTIVE ENZYMES IN LARVAL SOUTHERN FLOUNDER (Paralichthys lethostigma)

The southern flounder (Paralichthys lethostigma) is a commercially and recreationally important species in the Gulf of Mexico.  Stock enhancement programs throughout the state of Texas seek to supplement wild production in order to sustain a healthy population.  In order to effectively raise healthy larvae, the development of their digestive tract must be better understood.  The present study was conducted to characterize the ontogeny of several digestive enzymes as a function of fish age.  

Eggs were collected from natural spawns of captive southern flounder broodstock at the hatchery of the CCA Marine Development Center in Flour Bluff, Texas.  Starting at 3 days post hatch (dph), larvae were fed with enriched rotifers (Brachionus sp.) and were then transitioned onto enriched Artemia nauplii at 20 dph.  Larvae were collected on 0, 3, 5, 7, 9, 11, 13, 15, 18, 21, and 24 days post hatch.  After collection, larvae were rinsed on an appropriately sized mesh and immediately frozen with liquid nitrogen.  Larvae were homogenized in cold 50 mM Tris-HCl, 20 mM CaCl₂ buffer and the supernatants were stored at -80°C until further analysis.  The amount of protein in each sample was determined by the Bradford method using bovine serum albumin as a standard.

The larvae were analyzed in triplicate for the following digestive enzymes from each sampling day:  pepsin using hemoglobin as a substrate, trypsin using N-a-benzoyl-DL-arginine 4-nitroanilide hydrochloride as a substrate, chymotrypsin using 5 mM-N-benzoyl-L-tyrosine ethyl ester as a substrate, aminopeptidase using L-leucine p-nitroanilide as substrate, α-amylase using soluble starch as a substrate, lipase using sodium cholate hydrate and β-naphthyl-caprylate as a substrate, and acid/alkaline phosphatases using 4-nitrophenylphosphate as a substrate.  Enzyme responses were measured spectrophotometrically and expressed as units of enzyme per mg sample.  One unit is defined as the increase of 0.01 units of absorbance/minute.  

Several of the enzymes measured, such as lipase, alkaline phosphatase, acid phosphatase, and trypsin, were present at hatching.  Pancreatic enzymes (trypsin, α-amylase, lipase), phosphatase enzymes and aminopeptidase are thought to help enable the larvae to absorb the yolk sac.  Amylase appeared to peak around first feeding, 5 dph, at which point the absorbance/mg sample decreased significantly.  Aminopeptidase was first evident at 3 dph and the absorbance/mg sample continued to increase until 15 dph, when it decreased slightly.  These results provide further characterization of digestive tract development in southern flounder larvae and may be used to help refine feeding protocols for this species.