Effect of different salinities on gene expression and activity of digestive enzymes in the thick-lipped grey mullet (Chelon labrosus).

The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.

Daily rhythms in the somatotropic axis of Senegalese sole (Solea senegalensis): The time of day influences the response to GH administration.

Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase-activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28-0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture.

Starving/re-feeding processes induce metabolic modifications in thick-lipped grey mullet (Chelon labrosus, Risso 1827).

The effects of starvation and re-feeding on metabolites and tissue composition, GH/IGF-I axis, and digestive enzyme activities in juvenile thick-lipped grey mullet (Chelon labrosus) were evaluated. Fish were divided into three feeding groups (n=72, 82.00±4.09 g initial body mass). The control group was fed 1% of their body mass once a day throughout the experiment with commercial pellets. The other two groups were deprived of feed for 21 days (starved), or re-fed for 7 days after 14 days of food deprivation (re-fed). Full-length cDNAs from pituitary GH and hepatic IGF-I were cloned by screening a cDNA library or by PCR techniques. Furthermore, changes in their mRNA expressions were assessed by real time PCR in specimens maintained under the different feeding patterns. Results showed a negative growth in starved and re-feeding groups. Starvation induced a significant increase in plasma triglycerides as well as a decrease in liver glucose and glycogen. Re-feeding increased plasma glucose, lactate and protein, as well as liver glucose and glycogen. In addition, starvation significantly increased pituitary GH expression, while re-feeding down-regulated it. No significant changes were observed in hepatic IGF-I expression in any dietary treatment. Digestive enzyme activities were not significantly affected either by starvation or by re-feeding. The results of the present work suggest that juveniles of the thick-lipped grey mullet may easily adjust their metabolism under situations characterized by a short-term starvation and re-feeding.

Daily rhythms in the hypothalamus-pituitary-interrenal axis and acute stress responses in a teleost flatfish, Solea senegalensis.

The endocrine axis controlling the stress response displays daily rhythms in many factors such as adrenal sensitivity and cortisol secretion. These rhythms have mostly been described in mammals, whereas they are poorly understood in teleost fish, so that their impact on fish welfare in aquaculture remains unexplored. In the present research, the authors investigated the daily rhythms in the hypothalamus-pituitary-interrenal (HPI) axis in the flatfish Solea senegalensis, which has both scientific and commercial interest. In a first experiment, hypothalamic expression of corticotropin-releasing hormone (crh) and its binding protein (crhbp), both pituitary proopiomelanocortin A and B (pomca and pomcb) expression, as well as plasma cortisol, glucose, and lactate levels were analyzed throughout a 24-h cycle. All variables displayed daily rhythms (cosinor, p < .05), with acrophases varying depending on the factor analyzed: crh and cortisol peaked at the beginning of the dark phase (zeitgeber time [ZT] = 14.5 and 14.4 h, respectively), pomca and pomcb as well as glucose at the beginning of the light phase (ZT = 1.2, 2.4, and 3.4 h, respectively), and crhbp and lactate at the end of the dark phase (ZT = 22.3 and 23.0 h, respectively). In a second experiment, the influence of an acute stressor (30 s of air exposure), applied at two different time points (ZT 1 and ZT 13), was tested. The stress response differed depending on the time of day, showing higher cortisol values (96.2 ± 10.7 ng/mL) when the stressor was applied at ZT 1 than at ZT 13 (52.6 ± 11.1 ng/mL). This research describes for the first time the daily rhythms in endocrine factors of the HPI axis of the flatfish S. senegalensis, and the influence of daytime on the stress responses. A better knowledge of the chronobiology of fish provides a helpful tool for understanding the circadian physiology of the stress response, and for designing timely sound protocols to improve fish welfare in aquaculture.