Gene expression in Siberian sturgeon larvae in response to different rearing temperatures

Introduction Most of the species of Sturgeons, such as Siberian sturgeon (Acipenser baerii), are currently on the list of endangered species, due to overfishing, pollution and habitat degradation. Aquaculture plays an important role on providing an alternative source of caviar and meat, as well as on the conservation of the natural stocks. Farmed fish, in general, is continuously subjected to stress factors, such as handling, weighing, confinement, crowding, grading, transportation and water quality variations. Environmental temperature is one of the main factors affecting fish development and physiology. Sturgeon species present a specific optimum temperature range for regular larval development and ontogenesis, and this represents one of the critical aspects that are of great importance in hatchery management (Gisbert and Williot, 2002). It has been demonstrated that physiological stress may have serious negative consequences on growth (Wendelaar Bonga, 1997). This experimental work was focused on the rearing of sturgeon larvae at three different temperatures. The aims of the study are, thus, the evaluation of: 1) the influence of rearing temperature on yolk-sac larvae development; 2) the expression of some genes involved both in the stress response and growth, starting from hatching to the yolk sac absorption phase. Materials and Methods The study was carried out at the Experimental Animal Research and Application Centre of the University of Milan, and approved by the Ethic Committee of the structure (OPBA_20_2016). Siberian sturgeon eggs were incubated at 16.1 ± 0.2°C; after hatching one experimental group was maintained at 16°C (16.4 ± 0.2 °C), while other two groups were subjected to 19°C (19.3 ± 0.2) and 22°C (21.9 ± 0.2 °C) until complete yolk-sac absorption. Sampling time points were: hatching, schooling and complete yolk sac absorption phase. Samples were frozen at -80°C until use. RNA was extracted from two specimen after hatching and three specimen of the three groups and retrotranscribed to cDNA using commercial kits. Genes coding for HSP70, HSP90a, HSP90b, Myogenin (myog) and IGF1 proteins were analysed. GAPDH and RPL6 were used as reference genes. Primers for quantitative PCR were designed starting from A. baerii transcriptome (Song et al., 2016). Quality of cDNA was evaluated by qualitative amplification of the RPL6 gene. qPCRs were performed and the obtained results were examined using the Ct method to evaluate the relative expression of each gene in schooling and complete yolk sac absorption phases compared to hatching phase. Statistical analysis of the data was performed using the 2-way ANOVA with temperatures and developmental stages as main factors of the SAS (version 8.1, Cary Inc., NC). The data were presented as least squared means ± SEM. Differences were considered significant at P < 0.05 and P < 0.01. Results and Discussion No differences were found regarding larval body weight (Fig.1f), during the schooling stage among temperatures; at the yolk-sac absorption stage, larvae maintained at 16°C presented a significantly higher body weight than larvae subjected to 19°C (P<0.05), but did not differ from those reared at 22°C. Heat-shock Proteins 70, 90a and 90b, were more expressed at 16°C than at 19° and 22°C (P<0,01 for HSP70 and 90a and P<0,05 for HSP90b) during the schooling stage (Fig.1a-c); at the complete yolk sac absorption phase. There were no significant differences in the expression of all HSP’s. At the schooling stage Myog (Fig.1d) showed a higher expression at 16°C than at 19°C (P<0.05) and 22°C (P<0.01); no differences were found in myog expression in larvae reared at 19° or 22°C. At the end of the trial, this situation was inverted: larvae reared at 22°C showed a higher expression of this gene than larvae reared at 16°C (P<0.05) and 19°C (P<0.01). During the schooling stage IGF1 was significantly more expressed at the lowest temperature (Fig.1e; P<0,05) when compared to the highest temperature; at the yolk-sac absorption phase there was no difference between 16° and 22°C, but these were significantly higher than 19°C (P<0,05) (Fig.1e). At the schooling stage there was a higher expression of all the three HSPs in larvae reared at 16°C.The expression of these proteins in fish is not only related with heat-shock, but also with cold-shock, transport, metals exposure, viruses, etc (Iwama et al, 1998). This high expression seemed not to cause a negative influence on growth, taking into account that larvae reared at 16°C showed the higher levels of expression of both myog and IGF1 but also an increased weight at the end of the trial. Larvae reared at 22°C seemed to be less affected by stress but showed lower expression of both myog and IGF1 at the schooling stage; from this stage on, larvae reared at 22°C showed a sharp increase in myog and IGF1, which led to an increased weight at the end of the trial. It would seem like commercial hatchery could adopt either 16° or 22°C for this species for these stages of development. However, further studies would be necessary to deepen the effects of the high expression of HSPs observed on larvae reared at 16°C.