Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.).

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.

Branchial nitrogen cycle symbionts can remove ammonia in fish gills.

Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.

Voluntary timing of food intake increases weight gain and reduces basal plasma cortisol levels in common carp (Cyprinus carpio L.).

We investigated the effect of timing of food intake on growth in common carp (Cyprinus carpio L.). Juvenile carp were demand-fed for 22 days using a computerized pendulum feeder that registered meal requests. Controls were pair-fed at 10:00 h, both groups were kept at 12L:12D (lights on at 06:30 h). Demand-fed fish displayed highest food intake at 22:00 h, and the lowest at 10:00. After 22 days, demand-fed fish had grown by 20% of their initial body weight, compared to 4% of the pair-fed control. Plasma cortisol levels in demand-fed fish were remarkably low and stable, whereas in the control group levels had increased 60-fold at 10:00 h compared to 22:00 h. Hepatic mRNA expression of leptin-a1 and leptin-a2 also differed markedly between groups and time points, with leptin-a2 expression being lowest in the demand-fed group at the time point of lowest food intake. We conclude that timing of food intake is an important determinant of endocrine status, growth and welfare.

Comparative thyroidology: thyroid gland location and iodothyronine dynamics in Mozambique tilapia (Oreochromis mossambicus Peters) and common carp (Cyprinus carpio L.).

In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several species thyroid follicles are found in, for example, heart, head kidney and kidney. Such heterotopic thyroid follicles are active, and considered to work in concert with the subpharyngeal thyroid. In Mozambique tilapia (Oreochromis mossambicus) thyroid activity is, indeed, restricted to the subpharyngeal region; in common carp (Cyprinus carpio) the functional endocrine thyroid is associated with renal tissues. The subpharyngeal follicles of carp comprise only 10% of the total thyroid tissue, and these follicles neither accumulate iodide nor synthesize or secrete thyroid hormones to a significant degree. Although the shape and size of carp subpharyngeal and renal follicles vary, the epithelial cell height of the thyrocytes and thyroxine immunoreactivity do not differ, which suggests that the activity of the carp subpharyngeal thyroid follicles is dormant. Differences in thyroid physiology between the two fish species were further assessed at the level of peripheral thyroid hormone metabolism. Carp clears plasma of thyroid hormones faster than tilapia does. Furthermore, a significant amount of conjugated thyroid hormones was observed in the plasma of tilapia, which was preceded by the occurrence of thyroid hormone conjugates in the subpharyngeal region and coincides with the appearance of conjugates in the surrounding water. Apparently, plasma thyroid hormone conjugates in tilapia originate from the thyroid gland and function in the excretion of thyroid hormones. Our data illustrate the variability in teleostean thyroidology, an important notion for those studying thyroid physiology.

Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation.

Leptin is a key factor in the regulation of food intake and is an important factor in the pathophysiology of obesity. However, more than a decade after the discovery of leptin in mouse, information regarding leptin in any nonmammalian species is still scant. We report the identification of duplicate leptin genes in common carp (Cyprinus carpio). The unique gene structure, the conservation of both cysteines that form leptin's single disulfide bridge, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and carp leptins, despite low amino acid identity. The liver is a major yet not the only site of leptin expression. However, neither 6 d nor 6 wk of fasting nor subsequent refeeding affected hepatic leptin expression, although the carp predictably shifted from carbohydrate to lipid metabolism. Animals that were fed to satiation grew twice as fast as controls; however, they did not show increased leptin expression at the termination of the study. Hepatic leptin expression did, however, display an acute and transient postprandial increase that follows the postprandial plasma glucose peak. In summary, leptin mRNA expression in carp changes acutely after food intake, but involvement of leptin in the long-term regulation of food intake and energy metabolism was not evident from fasting for days or weeks or long-term feeding to satiation. These are the first data on the regulation of leptin expression in any nonmammalian species.

Stress response to waterborne Cu during early life stages of carp, Cyprinus carpio.

Following in vitro fertilization, eggs/embryos and larvae of the common carp (Cyprinus carpio) were exposed to 0 (control), 0.3 or 0.8 micromol.l(-1) Cu in artificially prepared fresh water for 168 h. The total amounts of Cu, Na, Ca, adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and cortisol were measured in homogenates of eggs (up to 60 h post fertilization, hpf), isolated embryos (between 60 hpf and hatching) and free-swimming larvae. Only in embryos of eggs exposed to 0.8 micromol.l(-1) Cu a significant accumulation of Cu was observed as well as a concurrent increase in the incidence of spinal cord deformation and larval mortality. Further, when exposed to 0.8 micromol.l(-1) Cu, the whole-body Ca and Na contents were lower at 48 and 72 hpf compared to the controls and those exposed to 0.3 micromol.l(-1) Cu. Conversely, in larvae (>72 hpf) exposed to 0.3 micromol.l(-1) Cu, the Ca content was elevated from 96 hpf onwards. At 48 hpf and onwards, the whole-body ACTH and cortisol contents of the embryos exposed to 0.8 micromol.l(-1) Cu were higher than those in either controls or those exposed to 0.3 micromol.l(-1) Cu. By 96 hpf, ACTH and cortisol contents of the group exposed to 0.3 micromol.l(-1) Cu also surpassed those in controls. The alpha-MSH content in both Cu exposed groups was lower than in controls from 48 hpf onwards. It thus appears that ACTH cells and MSH cells in early life stages of carp exposed to waterborn Cu respond differently; we conclude that in prehatch carp pituitary corticotropes and interrenal cortisol producing cells respond to the chemical stressor Cu and that the resulting hormonal changes provide a sensitive diagnosis for stress as well as toxicity tests.