Different early weaning protocols in common sole (Solea solea L.) larvae: implications on performances and gene expression of trypsinogen, chymotrypsinogen and amylase

A study on common sole (Solea solea L.) larvae was carried out in order to assess the potential of different early weaning time protocols onto commercial microdiet before the end of metamorphosis. Four days post hatching (dph) larvae were randomly allocated in twelve flat bottom (0.64 m2) 280 L square tanks (2500 larvae tank-1) connected to a recirculating system. Four experimental weaning time protocols (WPs) were used in triplicate. One was used as control group in which larvae were fed live feed (Artemia nauplii and subsequently metanauplii) until 27 dph (WP-27) and weaned with dry feed. The others were provided only with dry feed (WP-4); live feed until 13 dph (WP-13) and live feed until 18 dph (WP-18) followed by weaning onto the same commercial diet used in WP-27 (AgloNorse Ewos, Norway). The trial lasted until 33 dph. Larvae were sampled at 4, 13, 19 and 33 dph for weight determination and at 4, 14, 19, 26, 33 dph for length. Metamorphosis degree was assessed at 14, 19, 26 and 33 dph while a salinity stress test was conducted at 16 and 33 dph. Relative gene expression of trypsinogen 1, chymotrypsinogen and amylase were evaluated at 4, 8, 13, 18, 27, 33 dph by quantitative real time PCR using the relative standard curve method. One-way ANOVA with Tukey’s post test were used to analyze data (P≤0.05). At the end of the trial, larvae in WP-4 showed lower survival rate, lower growth and a delay in the onset of metamorphosis when compared to the other groups. At the same time, treatments WP-13 and WP-18 showed significantly lower weight and length when compared to WP-27 whereas metamorphosis development and survival was not influenced. Salinity stress test values were influenced by treatment WP-4 at 16 dph where larvae showed an higher cumulative mortality index in comparison with the others. Trypsinogen and chymotrypsinogen mRNA levels showed a rapid increase in all the treatments between 4 and 8 dph. Afterwards, trypsinogen expression displayed a stable and homogeneous fluctuating pattern for WP-4 and WP-27, whereas WP-13 and WP-18 showed a peak at 18 and 27 dph respectively, which corresponds to the sampling time after weaning. A similar peaking was also observed for chymotrypsinogen in the same treatments but in this case a further up-regulation trend was also present from 27 to 33 dph. Amylase mRNA increased moderately throughout the study in all treatments. In conclusion, an early weaning at 13 and 18 dph using a commercial diet in common sole larvae affects growth performance but not survival rate and metamorphosis development. The lack of live food (WP-4) resulted in a consistently decrease of all the performances although at the end of the trial 18% of larvae still survived with a positive growth trend. Generally, the two proteolytic enzymes display parallel ontogeny increasing rapidly in the early larval stages while amylase was up-regulated over all the trial. The transcripts analyzed were not greatly influenced by feeding protocols and larval size. Study financed by Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZLER, Italy) and project Aquagenomics CDS2007-00002 (MICINN, Spain).