A slideshow in dutch (Internet Explorer only).

1 Development and application of methods for detection of stress in Mediterranean fish species in aquaculture

The overall objective of the project is to develop a non-invasive method for detecting general and specific stressor-related stress effects in fish reared in aquaculture. Within the general research framework the project aims at two interrelated objectives:

• To detect, analyze and quantify stress effects, under both laboratory and fish farm conditions, in two Mediterranean marine fish species, gilthead seabream (Sparus auratus) and European sea bass (Dicentrarchus labrax), which are of major economic importance for European aquaculture, using as parameters changes in the skin and gill epithelia, and the mucus layer covering the skin: (a) changes in tissue morphology, (b) marker enzymatic activities in skin and skin mucus, (c) concentrations of the stress hormone cortisol in blood serum and skin mucus and (d) immunological parameters. The stressors to be applied (low dissolved oxygen levels, high ammonia levels, and handling stress) are relevant to aquaculture.

• To select subsequently the analytical methods and stress parameters that will prove to be reliable and easy to perform, using a sampling procedure from which the sampled fish can easily recover. The selected methods will be combined as bio-indicators through the establishment of a new stress-index, the "Fish Stress Condition Index" (FISCI), that will enable the characterization of stress condition in cultured fish and will reflect both fish health and water quality. This will provide fish farmers and managers of nature reserves with a protocol to check the stress condition of their fish.

The participants will study systematically the effect of low dissolved oxygen levels, high ammonia levels, and handling stress, which are three aquaculture relevant stressors, on structure and ultrastructure of the skin and gills of gilthead sea bream and European sea bass, reared under both summer and winter temperature and light regime conditions. The analysis will be both qualitative and quantitative, in order to identify general tissue responses to the stressors and specific responses to each stressor separately.

The combination of histopathological and biochemical methods to be applied on skin and gill biopsies and mucus samples is meant to lead to the development of the FISCI index. The FISCI will be used as an (almost) non-invasive method that will not require killing the fish. This method may enable the detection of fish stress status and will enable in many cases, through the stressor specific responses, the determination of the detrimental factor in the water.

The main experiments will be performed in Athens, by researchers from the National Center for Marine Research (Athens), the Department of Animal Physiology, Radboud University Nijmegen, and the Department of Biology, University of Jerusalem.

2 The immuno-neuroendocrine interactions in fish strains selected for stress response or immune capacity

Improvement of genetic disease resistance will reduce fish mortalities caused by diseases, and thus improve fish welfare. However, because of the complicated interaction between the immune system and the neuroendocrine system, it is expected that the stress response of fish will affect disease resistance, even in fish strains genetically selected for improved fish health. In this project we will investigate how this selection has changed the interaction between the immune system and the brain-pituitary-interrenal (BPI) axis. Interleukin-like factors produced by the immune system, and cortisol, the end product of the BPI-axis, are two of the most important mediating factors involved in this interaction. Interleukin-like factors have been reported to stimulate the BPI-axis. Cortisol, at least at the high levels typical for severe stress in fish, has suppressive actions on the immune system, which is explained as moderating mechanisms to prevent overreaction of the immune system. The central hypothesis of this project is that selection for either stress response or immune competence will disturb the interaction between both systems, with negative effects for fish health and welfare. First, the effects of the standard stressor (crowding) on the activity of the BPI axis (ACTH, MSH, b-END and cortisol production) will be compared for different strains selected for either stress response or immune competence (the same strains as used in the other projects). Second, the concentration/response relationship between IL-1 and this axis, and between cortisol and different immune parameters will be investigated in these strains in order to elucidate the physiological mechanisms involved.

Part of the experiments will be performed at the University of Cadiz (Spain) and at the research center for aquaculture Cupimar near Cadiz.

3 Calcium metabolism in fish and ParaThyroid Hormone related Protein (PTHrP)

Despite improved methodologies for farming of marine fish species, egg and larval viability is still in most cases no more than 20-30%, as a consequence of high mortalities and/or a high incidence of larvae with skeletal deformities (dystrophies). Often dystrophies are not immediately apparent leading to wasteful use of food, energy, space and human resources until dystrophic fish are graded and removed. Skeletal deformities result from abnormal calcification of cartilaginous tissue and hypercalcaemic factors are likely involved. Therefore, the elucidation of the processes controlling ossification in fish larvae is of extreme importance to devise culture methodologies minimizing abnormal development. Calcium is further important in a range of other physiological processes of practical importance to aquaculture, such as in reproduction when high concentrations of calcium are required for vitellogenesis and growth; the latter requires bone remodeling and ossification. Surprisingly little information is available about how calcium requirements vary in teleost fish during their life cycle or the physiological mechanisms employed to raise plasma calcium levels. This project aims to determine calcium homeostasis and studies calcium availability during critical stages of the life cycle of the teleost fish Sparus aurata, an important marine aquaculture species in Southern European countries.

Maintenance of calcium ion levels between narrow limits is vital for normal function of the cardiovascular, neural and muscular systems and thus for larval and skeletal development, as well as for body growth and gonad growth and maturation, particularly egg production. Endocrine systems of the brain, pituitary and gonads play key roles in these events and integrate with calcium-regulating hormones. In fish the mechanisms controlling calcium homeostasis and their interaction with other hormones have long been the focus of research in Nijmegen. Hypocalcaemic factors, such as stanniocalcin and calcitonin, have been identified and characterized, but until now only prolactin has been recognized as a slow acting hypercalcaemic hormone. We have now cloned and identified the sea bream gene for a potential hypercalcaemic factor, parathyroid hormone-related protein (PTHrP). Initial experiments have shown that teleost PTHrP does elevate internal calcium. This discovery indicates that raising circulating calcium levels is an important physiological function but the mechanisms by which this is achieved are currently unknown.

To determine experimentally the roles of PTHrP in whole animal calcium homeostasis, tissues sensitive to changes in calcium availability, including gut, kidney and gills will be identified by in vitro and in vivo techniques. This will be carried out at stages in which calcium requirements are high, namely during skeletal development, reproduction and growth. A multidisciplinary approach will be used including molecular techniques to produce recombinant PTHrP for in vivo treatments and production of specific antisera. The latter will be used to establish specific assays for PTHrP in tissues and plasma, for its receptors and for quenching of PTHrP actions.

The overall achievement of our research objectives will be to provide new information about calcium physiology in sea bream hitherto unavailable. This will allow the identification of causal factors in abnormal bone development in larvae, the mechanism by which calcium is incorporated into vitellogenin, the general requirements of calcium for normally growing fish and the function of PTHrP in these processes.

4 Regulation of food intake in the common carp (Cyprinus carpio)

The obesity pandemic taking place in the entire Western world nowadays, enhances the interest in the genetic and molecular basis of this problem. Energy balance is made up of two components, energy intake and energy expenditure. Whereas the latter is mainly comprised of metabolic rate and physical activity, the former consists of food intake and digestion.

Energy homeostasis is under multiple endocrine and neural control that involves both central and peripheral hormones and neuropeptides. Disorders of energy balance (e.g. obesitas and anorexia nervosa) are caused by subtle dysregulations of these regulatory mechanisms.

The hypothalamic arcuate nucleus is a main site of central regulation in mammals, where two distinct subpopulations of neurons co-express either neuropeptide Y (NPY) and agouti related protein (AgRP) or proopiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART); the former set of peptides increase food intake, the latter decrease food intake and obviously affect energy metabolism.

The two hypothalamic sets of neuropeptides are under peripheral control. Hormones like insulin, ghrelin and leptin stimulate or inhibit the two neuron-populations in the arcuate nucleus. Knowledge on these peripheral hormones is scant outside mammals.

Comparative analysis of these peripheral mechanisms may shed new light on the function and evolution of the mechanisms at the basis of energy homeostasis. The recent identification of leptin in several fish species including common carp provides new insights and opportunities to enhance our understanding of the regulation of food intake.

Despite low amino acid identity, the unique gene structure, the conservation of both cysteines that form leptin's single disulphide bridge, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and carp leptins. In carp, the liver is a major (but not the only) site of leptin production whereas in mammals, the major site of leptin production is subcutaneous fat.

Our goal is to gain more insight in the evolution of leptin by comparative research on teleost fish, which are the oldest vertebrates on the planet.

Part of the experiments will be performed in collaboration with the Salk institute in La Jolla (USA).

For information (and possibilities for an interneeship) please enquire with Prof. Dr. Gert Flik (G.Flik@science.ru.nl).