Role of the melanocortin system in zebrafish skin physiology.

The agouti-signaling protein (ASIP) acts as both a competitive antagonist and inverse agonist of melanocortin receptors which regulate dorsal-ventral pigmentation patterns in fish. However, the potential role of ASIP in the regulation of additional physiological pathways in the skin is unknown. The skin plays a crucial role in the immune function, acting as a physical limitation against infestation and also as a chemical barrier due to its ability to synthesize and secrete mucus and many immune effector proteins. In this study, the putative role of ASIP in regulating the immune system of skin has been explored using a transgenic zebrafish model overexpressing the asip1 gene (ASIPzf). Initially, the structural changes in skin induced by asip1 overexpression were studied, revealing that the ventral skin of ASIPzf was thinner than that of wild type (WT) animals. A moderate hypertrophy of mucous cells was also found in ASIPzf. Histochemical studies showed that transgenic animals appear to compensate for the lower number of cell layers by modifying the mucus composition and increasing lectin affinity and mucin content in order to maintain or improve protection against microorganism adhesion. ASIPzf also exhibit higher protein concentration under crowding conditions suggesting an increased mucus production under stressful conditions. Exposure to bacterial lipopolysaccharide (LPS) showed that ASIPzf exhibit a faster pro-inflammatory response and increased mucin expression yet severe skin injures and a slight increase in mortality was observed. Electrophysiological measurements show that the ASIP1 genotype exhibits reduced epithelial resistance, an indicator of reduced tissue integrity and barrier function. Overall, not only are ASIP1 animals more prone to infiltration and subsequent infections due to reduced skin epithelial integrity, but also display an increased inflammatory response that can lead to increased skin sensitivity to external infections.

Ocean acidification compromises energy management in Sparus aurata (Pisces: Teleostei).

The effects of ocean acidification mediated by an increase in water pCO2 levels on marine organisms are currently under debate. Elevated CO2 concentrations in the seawater induce several physiological responses in teleost fish, including acid-base imbalances and osmoregulatory changes. However, the consequences of CO2 levels enhancement on energy metabolism are mostly unknown. Here we show that 5 weeks of exposure to hypercapnia (950 and 1800 µatm CO2) altered intermediary metabolism of gilthead seabream (Sparus aurata) compared to fish acclimated to current ocean values (440 µatm CO2). We found that seabream compromises its physiological acid-base balance with increasing water CO2 levels and the subsequent acidification. Intestinal regions (anterior, mid, and rectum) engaged in maintaining this balance are thus altered, as seen for Na+/K+-ATPase and the vacuolar-type H+-ATPase activities. Moreover, liver and muscle counteracted these effects by increasing catabolic routes e.g., glycogenolysis, glycolysis, amino acid turnover, and lipid catabolism, and plasma energy metabolites were altered. Our results demonstrate how a relatively short period of 5 weeks of water hypercapnia is likely to disrupt the acid-base balance, osmoregulatory capacity and intermediary metabolism in S. aurata. However, long-term studies are necessary to fully understand the consequences of ocean acidification on growth and other energy-demanding activities, such as reproduction.

High rates of intestinal bicarbonate secretion in seawater tilapia (Oreochromis mossambicus).

Osmoregulation in fish is a complex process that requires the orchestrated cooperation of many tissues. In fish facing hyperosmotic environments, the intestinal absorption of some monovalent ions and the secretion of bicarbonate are key processes to favor water absorption. In the present study, we showed that bicarbonate levels in the intestinal fluid are several fold higher in seawater than in freshwater acclimated tilapia (Oreochromis mossambicus). In addition, we analyzed gene expression of the main molecular mechanisms involved in HCO3- movements i.e. slc26a6, slc26a3, slc4a4 and v-type H-ATPase sub C in the intestine of tilapia acclimated to both seawater and freshwater. Our results show an anterior/posterior functional regionalization of the intestine in tilapia in terms of expression patterns, which is affected by environmental salinity mostly in the anterior and mid intestine. Analysis of bicarbonate secretion using pH-Stat in tissues mounted in Ussing chambers reveals high rates of bicarbonate secretion in tilapia acclimated to seawater from anterior intestine to rectum ranging between ~900 and ~1700nmolHCO3-cm-2h-1. However, a relationship between the expression of slc26a6, slc26a3, slc4a4 and the rate of bicarbonate secretion seems to be compromised in the rectum. In this region, the low expression of the bicarbonate transporters could not explain the high bicarbonate secretion rates here described. However, we postulate that the elevated v-type H-ATPase mRNA expression in the rectum could be involved in this process.

Prolactin regulates luminal bicarbonate secretion in the intestine of the sea bream (Sparus aurata L.).

The pituitary hormone prolactin is a pleiotropic endocrine factor that plays a major role in the regulation of ion balance in fish, with demonstrated actions mainly in the gills and kidney. The role of prolactin in intestinal ion transport remains little studied. In marine fish, which have high drinking rates, epithelial bicarbonate secretion in the intestine produces luminal carbonate aggregates believed to play a key role in water and ion homeostasis. The present study was designed to establish the putative role of prolactin in the regulation of intestinal bicarbonate secretion in a marine fish. Basolateral addition of prolactin to the anterior intestine of sea bream mounted in Ussing chambers caused a rapid (<20 min) decrease of bicarbonate secretion measured by pH-stat. A clear inhibitory dose-response curve was obtained, with a maximal inhibition of 60-65% of basal bicarbonate secretion. The threshold concentration of prolactin for a significant effect on bicarbonate secretion was 10 ng ml(-1), which is comparable with putative plasma levels in seawater fish. The effect of prolactin on apical bicarbonate secretion was independent of the generation route for bicarbonate, as shown in a preparation devoid of basolateral HCO(3)(-)/CO(2) buffer. Specific inhibitors of JAK2 (AG-490, 50 µmol l(-1)), PI3K (LY-294002, 75 µmol l(-1)) or MEK (U-012610, 10 µmol l(-1)) caused a 50-70% reduction in the effect of prolactin on bicarbonate secretion, and demonstrated the involvement of prolactin receptors. In addition to rapid effects, prolactin has actions at the genomic level. Incubation of intestinal explants of anterior intestine of the sea bream in vitro for 3 h demonstrated a specific effect of prolactin on the expression of the Slc4a4A Na(+)-HCO(3)(-) co-transporter, but not on the Slc26a6A or Slc26a3B Cl(-)/HCO(3)(-) exchanger. We propose a new role for prolactin in the regulation of bicarbonate secretion, an essential function for ion/water homeostasis in the intestine of marine fish.