The present study aimed to compare effects of increasing chronic stress load on the stress response of European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) to identify neuroendocrine functions that regulate this response. Fish were left undisturbed (controls) or exposed to three levels of chronic stress for 3 weeks and then subjected to an acute stress test (ACT). Chronic stress impeded growth and decreased feed consumption in seabass, not in seabream. In seabass basal cortisol levels are high and increase with stress load; the response to a subsequent ACT decreases with increasing (earlier) load. Basal cortisol levels in seabream increase with the stress load, whereas the ACT induced a similar response in all groups. In seabass and seabream plasma α-MSH levels and brain stem serotonergic activity and turnover were similar and not affected by chronic stress. Species-specific molecular neuro-regional differences were seen. In-situ hybridization analysis of the early immediate gene cfos in the preoptic area showed ACT-activation in seabream; in seabass the expression level was not affected by ACT and seems constitutively high. In seabream, expression levels of telencephalic crf, crfbp, gr1, and mr were downregulated; the seabass hypothalamic preoptic area showed increased expression of crf and gr1, and decreased expression of mr, and this increased the gr1/mr ratio considerably. We substantiate species-specific physiological differences to stress coping between seabream and seabass at an endocrine and neuroendocrine molecular level. Seabass appear less resilient to stress, which we conclude from high basal activities of stress-related parameters and poor, or absent, responses to ACT. This comparative study reveals important aquaculture, husbandry, and welfare implications for the rearing of these species.

Recently, we established an inhibitory avoidance paradigm in Tupfel Long-Fin (TL) zebrafish. Here, we compared task performance of TL fish and fish from the AB strain; another widely used strain and shown to differ genetically and behaviourally from TL fish. Whole-body cortisol and telencephalic gene expression related to stress, anxiety and fear were measured before and 2 h post-task. Inhibitory avoidance was assessed in a 3-day paradigm: fish learn to avoid swimming from a white to a black compartment where a 3V-shock is given: day 1 (first shock), day 2 (second shock) and day 3 (no shock, sampling). Tupfel Long-Fin fish rapidly learned to avoid the black compartment and showed an increase in avoidance-related spatial behaviour in the white compartment across days. In contrast, AB fish showed no inhibitory avoidance learning. AB fish had higher basal cortisol levels and expression levels of stress-axis related genes than TL fish. Tupfel Long-Fin fish showed post-task learning-related changes in cortisol and gene expression levels, but these responses were not seen in AB fish. We conclude that AB fish show higher cortisol levels and no inhibitory avoidance than TL fish. The differential learning responses of these Danio strains may unmask genetically defined risks for stress-related disorders.

Partial replacement of fish ingredients with vegetable ingredients has elevated levels of polycyclic aromatic hydrocarbons (PAHs) in Atlantic salmon reared on these feeds. PAH uptake in the intestinal tract is postulated to occur in association with lipid absorption and could well be affected by fatty acid composition. We therefore investigated the effects of a fish oil and vegetable oil fatty acid, eicosapentaenoic acid (EPA; 20:5n-3) and oleic acid (18:1n-9) respectively, on the uptake of benzo[a]pyrene (BaP) and phenanthrene (PHE) across the intestinal brush border membrane in the salmonid species rainbow trout (Oncorhynchus mykiss). BaP and PHE were solubilized in mixed micelles composed of either EPA or oleic acid and administrated to isolated brush border membrane vesicles (BBMV) derived from the pyloric caeca, proximal intestine and distal intestine. In the absence of free fatty acids (FFA) trans-membrane uptake of BaP and PHE was 2-7 times lower than the fraction associated to or in the membrane. In the presence of FFA, trans-membrane BaP uptake had decreased by 80 and 40% at the highest EPA and oleic acid concentration, respectively, whereas PHE uptake was virtually unaffected. In the presence of BaP, but not PHE, trans-membrane EPA uptake in BBMV had decreased. This study obtained evidence for PAH-dependent interactions with FFA uptake. We conclude that intestinal BaP uptake is reduced by luminal FFA contents whereas PHE uptake is not. A large fraction of the administrated BaP and PHE remains associated with the cellular membrane of enterocytes and may interfere with uptake of nutrients.

Temporal patterns of whole-body α-MSH concentrations and of transcripts of melanocortin receptors during early development as well as the endocrine response (α-MSH, cortisol, MCR mRNAs) to stress at the end of the larval period were characterized in Dicentrarchus labrax. Immunohistochemistry showed α-MSH positive cells in the pituitary pars intermedia in all stages examined. As development proceeds, α-MSH content gradually increases; mRNA levels of mc2r and mc4r remain low until first feeding where peak values are observed. Mc1r expression was constant during development, pomc mRNA levels remain low until the stage of flexion after which a significant increase is observed. At the stage of the formation of all fins, whole-body cortisol and α-MSH concentrations responded with peak values at 2 h post stress. Additionally, the stress challenge resulted in elevated transcript levels of pomc, mc2r and mc4r but not in mc1r, with a pattern characterized by peak values at 1 h post stress and a strong correlation with whole body α-MSH concentrations was found. Our data provide for the first time a view on the importance of the α-MSH stress response in early development of European sea bass, an additional and relatively poorly understood signal involved in the stress response in teleosts.

The inclusion of vegetable oils in aquafeeds introduces contaminating polycyclic aromatic hydrocarbons (PAHs) in salmonids. Since lipophilic PAHs solubilize in micelles composed of lipids, bile salts and fatty acids, dietary lipid composition can alter intestinal transepithelial PAH transfer. We studied the uptake of two PAHs, viz. benzo[a]pyrene (BaP) and phenanthrene (PHE), in rainbow trout (Oncorhynchus mykiss) intestine. We also investigated the effects of two fatty acids, viz. fish oil-derived eicosapentaenoic acid (EPA, 20:5n-3) and vegetable oil-derived oleic acid (18:1n-9) on intestinal uptake. Radiolabeled PAHs were solubilized in micelles composed of tritiated EPA and oleic acid, respectively, and administrated to intestinal segments mounted in Ussing chambers. In the absence of micelles, PHE accumulation was two times higher than BaP in the mucosal and serosal layers of proximal and distal intestine. Administration of PHE in micelles composed of oleic acid resulted in a 50% lower accumulation of PHE in the mucosal layers of the proximal intestine compared to EPA-composed micelles. Accumulation of EPA and oleic acid in the proximal intestinal mucosa correlated negatively with the transepithelial transfer of these fatty acids across the proximal intestinal epithelium. Transepithelial PHE transfer across the proximal intestine was reduced by 30% in co-exposure with EPA-composed micelles compared to 80% with oleic acid micelles. BaP was not transferred across the intestine. We conclude that the lipid composition of an aquafeed is an important determinant of PAH bioavailability. Therefore, lipid composition should be an important consideration in choosing vegetable oils as alternatives for fish oil in aquafeeds.