Development of the digestive tract, trypsin activity and gene expression in eggs and larvae of the bullseye puffer fish Sphoeroides annulatus

Hatchery-produced Sphoeroides annulatus were studied from fertilized egg until day 32 post-hatch to examine the digestive tract development and to evaluate its digestive capacity during the larval period. Fish larvae were progressively fed microalgae, rotifers, Artemia nauplii and a formulated microdiet. Digestive tract development, trypsin activity, and trypsinogen gene expression in fish samples were analyzed by histology, histochemistry and reverse-transcription coupled to the polymerase chain reaction (RT–PCR) respectively. The intestine and liver started to develop on day 1 after hatch, followed by the pancreas. The mouth opened at day 4 after hatch, which was the start of rotifer feeding. Trypsinogen gene expression was detected very early in development, starting in the fertilized egg, showing a sharp increase in eggs at 75 h after fertilization, and then a gradual increase after hatching as the larvae developed. Trypsin activity by histochemistry was first detected at day 2 post-hatch, and maximum expression and activity were observed at days 16 to 24 after hatch, which corresponded to the period of Artemia nauplii feeding. No gastric glands were observed during the whole period of study. From day 28 onwards the fish were fed with a formulated microdiet, at this time both trypsin synthesis and activity decreased, suggesting a more important role for other enzymes in the digestion process.