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Contains fulltext : 199551.pdf (Publisher’s version ) (Open Access)

How seed-eating animals influence the germination of endozoochorous plant species is of great relevance to our understanding of long-distance seed dispersal. To evaluate the effect of gut passage on the germination speed of aquatic and riparian plants, we fed seeds of 19 species to common carp ( Cyprinus carpio ) and Mozambique tilapia ( Oreochromis mossambicus ). Carp has a pharyngeal ‘mill’, compensating for lack of a stomach and teeth, while tilapia has tiny teeth and an acid stomach. Potamogeton species, Alisma plantago-aquatica and Sagittaria sagittifolia showed a continuous germination with relatively low, and constant germination rates throughout the germination period. In contrast, the seeds of Myriophyllum spicatum and Nymphoides peltata and those of the riparian species exhibited discontinuous germination, i.e. almost all seeds germinated within a few days. Compared to control seeds, we found on average a negative effect of gut passage on the germination speed for carp, but no effect for tilapia. However, several plant species deviated from this general pattern. For example, gut passage in carp enhanced germination speed of viable seeds relative to controls for Potamogeton alpinus , but reduced it for Filipendula ulmaria . Similarly, tilapia enhanced germination speed for P . alpinus , but reduced it for P. pusillus . We conclude that carp’s efficient mastication affects the germination speed of plant species after gut passage, whereas stomach-passage in tilapia generally does not influence the germination speed when compared to control seeds. However, inherent variation across species in germination speed proved to be greater than effects of fish gut passage.

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.

Successive exposure to aquaculture-related stressors may compromise the allostatic capacity of African catfish and lead to allostatic overload and poor welfare. Therefore, we tested the effect of (i) feeding during the light or dark phase, (ii) density (51 fish per 140 L versus 51 fish per 43 L) and (iii) altered available resting space/shelter on stress handling were studied in African catfish (Clarias gariepinus). Available resting space/shelter was manipulated by providing PVC-tubes in the tanks. Growth, baseline stress load (cortisol and osmolality), metabolism (glucose, lactate and non-esterified fatty acids) and aggression (measured by skin scar incidence) were assessed. Upon completion of the experimental period, we determined the adaptive capacity of the fish by the response to air exposure. We show that night-feeding enhances growth and lowers feed conversion ratio compared to day-feeding. No effects of density were found for baseline stress hormones, basal metabolism or aggression. Low density seems to stimulate aggressive behaviour after air exposure. Unexpectedly, providing PVC-tubes under high-density conditions increased aggression, raised baseline cortisol levels and decreased basal metabolism as well as growth; also the endocrine response to air exposure was stronger. We argue that these effects relate to a reduction in available space, i.e. a further increase in density, or to an increased competition for shelter. Overall, our data suggest that night-feeding optimizes growth and that care should be taken in using objects as shelter/enrichment for African catfish.

For aquatic and riparian plants, the important role of fish in seed dispersal is increasingly recognised. While the propensity of seeds to disperse is known to be a function of morphological, physical and chemical traits of the seed, in the case of fish-mediated seed dispersal (ichthyochory), it is largely unknown how seed traits modulate the potential for seed ingestion and their subsequent survival through the gut. Furthermore, which seed traits are important may vary among fish species. To evaluate the role of both seed and fish traits in ichthyochory, we fed seeds of 19 aquatic and riparian plant species to fish with differing feeding mechanisms. Cyprinus carpio (common carp) has a pharyngeal ‘mill’, which it uses physically to crush hard food, while Oreochromis mossambicus (Mozambique tilapia) has only tiny oral and pharyngeal teeth and instead relies more on chemical digestion. A number of seed traits, including hardness, size and shape, were important determinants of the potential of seeds for ichthyochory. Certain traits (e.g. seed dimensions) were more important during ingestion, whereas other traits were more important for seed survival and subsequent germination (e.g. seed hardness, mucilaginous coat). Compared to controls, germination of retrieved seeds in carp was lower in 10 and higher in two plant species, whereas for tilapia, it was lower in seven and higher in three species. Overlap between these plant species was low, indicating clear difference between the fish studied in their potential for seed dispersal. Carp increased in size during the experiment and concomitant decreases in seed survival and retrieval were found, suggesting that body size and the correlated bite force is an important fish trait in ichthyochory. Overall, seed hardness, size and shape appear crucial for the survival of seeds passing through the guts of carp and tilapia. Beyond this general pattern, a greater complexity of trait-performance relationships appeared: different seed traits are involved during each of the stages of ichthyochory. Moreover, the importance of seed traits differed between carp and tilapia, with some traits having interactive and contrasting effects in both fish species. Aquatic plants with floating seeds adapted to hydrochorous dispersal were less likely to be dispersed by tilapia than plants with non-floating seeds, suggesting a dispersal trade-off between ichthyochory and hydrochory. Thus, depending on their seed characteristics, fish may offer an additional dispersal route to aquatic and riparian plants.

The nitrite threshold concentration in rearing water of African catfish (Clarias gariepinus) was assessed. African catfish with an initial mean (SD) weight of 219.7 (57.8) g were exposed to an increasing range of water nitrite from 6 (Control) to 928 µM nitrite for 28 days. Mean (SD) plasma nitrite concentrations increased from 5.0 (3.6) to 32.5 (12.6) µM at 928 µM ambient nitrite. The increase in nitrite was accompanied by gradual increase in plasma nitrate from 41.6 (28.4) µM to 420.2 (106.4) µM. Haematocrit, haemoglobin, methemoglobin, plasma concentrations of cortisol, glucose, lactate, osmolality, gill morphology and branchial Na+/K+-ATPase activity were not affected. Feed intake, final weight, SGR, FCR and mortality were not affected. We advise not to exceed a water nitrite concentration of 43 µM (0.6 mg L-1 NO2--N) to prevent the risk of reduced growth and feed intake in African catfish aquaculture.

Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus-pituitary-interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE(®), may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S(®). Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.

The threshold concentration for NH 3 in rearing water of African catfish ( Clarias gariepinus ) was assessed. African catfish with an initial mean (SD) weight of 141.0 (24) g were exposed to five different T amm [sum of NH 3 and NH 4 + ] concentrations: 0.37 (Control), 1.06, 2.12, 5.16 and 19.7 mM, which concurs with NH 3 concentrations of 4 (Control), 14, 38, 176 and 1084 μM. Plasma concentrations of NH 4 + , cortisol, glucose and lactate, plasma osmolality, gill morphology, branchial Na + /K + -ATPase activity, feed intake and specific growth rate were monitored. No effect of water NH 3 on plasma NH 4 + concentrations was detected. Feed intake and specific growth rate were severely affected at exposure to water NH 3 concentrations above 90 μM (calculated EC 10 values: 89 and 122 μM). No major disturbances in physiological blood parameters were observed at these NH 3 concentrations, but gill morphology (a remarkably sensitive stress indicator) deteriorated significantly. Based on the lower limit of the 95% confidence interval for EC 10 , we advise for African catfish not to exceed a water NH 3 concentration of 24 μM (0.34 mg NH 3 -N/L). This finding is relevant for design and management of African catfish production systems.

The effect of polychlorinated biphenyls (PCBs) on the energy consumption of fasting silver European eel (Anguilla anguilla L.) was studied over a 27-day period during which the animals were at rest or were swimming 800 km in Blazka swim tunnels. Three-year-old female hatchery eels (silver stage) between 73 and 80 cm long weighing around 1 kg were dosed intraperitoneally with PCBs at a nominal dosage of 10x the consumption standard as a mixture representative for planar (7 microg PCB126/kg eel), non-planar (5 mg PCB153/kg eel) and metabolizable PCBs (50 microg PCB77/kg eel) found in wild eel, or only with the vehicle (corn oil, 10 ml/kg eel). Four major observations were made: (1) PCB-exposed animals lose less weight compared to their unexposed controls; (2) PCB-concentrations on a lipid basis are 2.8-14 times higher in swimming compared to resting animals; (3) the standard metabolic rate is significantly lower in the PCB-exposed animals than in unexposed controls. In addition, PCB-exposure significantly reduces oxygen consumption during swimming, and starting at 400 km (18 days) this effect increases with time; (4) the relative spleen and liver weight significantly increased in the PCB-swim animals but not in the PCB-rest animals. The swimming animals lost about 75% more weight compared to resting animals and had about 50% lower plasma fat content. Hematocrit, haemoglobin, plasma pH, ion levels (sodium and potassium), and plasma lactate were not affected by PCB-exposure or swimming. Apparently, the current levels of PCBs and other dioxin-like compounds may seriously impair the reproduction of the European eel.

The cyclooxygenase (COX) pathway converts arachidonic acid (ArA) into prostaglandins (PGs), which interact with the stress response in mammals and possibly in fish as well. Acetylsalicylic acid (ASA) is a COX inhibitor and was used to characterize the effects of PGs on the release of several hormones and the stress response of tilapia ( Oreochromis mossambicus). Plasma PGE2 was significantly reduced at 100 mg ASA/kg body wt, and both basal PGE2 and cortisol levels correlated negatively with plasma salicylate. Basal plasma 3,5,3′-triiodothyronine (T3) was reduced by ASA treatment, whereas prolactin (PRL)188 increased at 100 mg ASA/kg body wt. ASA depressed the cortisol response to the mild stress of 5 min of net confinement. As expected, glucose and lactate were elevated in the stressed control fish, but the responses were blunted by ASA treatment. Gill Na+-K+-ATPase activity was not affected by ASA. Plasma osmolarity increased after confinement in all treatments, whereas sodium only increased at the high ASA dose. This is the first time ASA has been administered to fish in vivo, and the altered hormone release and the inhibition of the acute stress response indicated the involvement of PGs in these processes.

The objective of this study was to assess the effects of transportation of marketable eel (0.15 kg) in the Netherlands with respect to welfare. Eels (Anguilla anguilla) were obtained from a commercial farm and acclimatized for 7 weeks at the laboratory. Fish were transported according to regular commercial procedures. The animals were placed in water-filled transport tanks on the trailer. Fish density increased from 72 kg m-3 (husbandry) to 206 kg m-3 (fasting) and was further increased to 270–290 kg m-3 during transport. Fish transport lasted 3 h after which the eels were returned to laboratory recirculation systems to measure parameters indicative of stress load, i.e. mortality, plasma cortisol, lactate and non-esterified fatty acids (NEFA) as well as gill morphology. Samples were taken at 0, 6, 24, 48 and 72 h after transport in transported fish and non-transported counterparts (controls). Transportation affected water quality within known tolerable limits. No mortality during or after transport was observed. After 6 h, plasma cortisol levels had returned to baseline. However, energy metabolism had increased suggesting that transportation of eels resulted in an increased energy demand that lasted for at least 72 h in the fasted animals. Thus, it is conceivable that exposure to adverse conditions, prior to stunning/killing, in a slaughterhouse may result in allostatic overload in eel.

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.

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.

Consumer awareness of the need to improve fish welfare is increasing. Electrostunning is a clean and potentially efficient procedure more and more used to provoke loss of consciousness prior to killing or slaughtering (reviewed by Van de Vis et al. in Aquac Res 34:211–220, 2003). Little is known how (powerful) electrical stimuli, which do not stun immediately, are perceived by fish. We investigated responses of hand-held Mozambique tilapia (Oreochromis mossambicus) to a standardized electric shock applied to the tailfin. The handling with the resulting unavoidable acute stress response was carefully controlled for. Fish responses were analyzed up to 24 h following the shock. Electric shock resulted in slightly higher levels in plasma cortisol, lactate, ionic levels, and osmolality, than handling alone. Plasma glucose had significantly increased 6 h after shock compared to handling, indicative of enhanced adrenergic activity. Mucus release from the gills, branchial Na+/K+ ATPase activity, and chloride cell migration and proliferation, parameters that will change with strong adrenergic activation, were not affected. Decreased swimming activity and delay in resumption of chafing behavior indicated a stronger and differential response toward the electric shock. Responses to handling lasted shorter compared to those to an electric shock. The differential and stronger responses to the electric shock suggest that fish perceived the shock potentially as painful.

Contains fulltext : 35012.pdf ( ) (Open Access) 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.

Contains fulltext : 35747.pdf (Publisher’s version ) (Open Access) 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.

Contains fulltext : 35554.pdf ( ) (Open Access) Juvenile gilthead sea bream were exposed to diluted seawater (2.5 per thousand salinity; DSW) for 3 h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30 days. Branchial Ca(2+) influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca(2+) and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca(2+) (R(2)=0.30, N=39, P

Contains fulltext : 35553.pdf ( ) (Open Access) In fish, vitellogenin is an important nutritional precursor protein produced solely in the liver and released into the blood where it binds calcium. In the gilthead sea bream (Sparus auratus) 17beta-Estradiol (E2) plays an important role in the synthesis of vitellogenin, but also the pituitary hormones prolactin (PRL) and growth hormone (GH) can stimulate vitellogenin induction in fish. Considering the emerging involvement of PTHrP in fish calcium metabolism and the importance of calcium regulation in reproduction, we investigated the possible role of PTHrP in vitellogenesis. E2-naive and E2-primed sea bream hepatocytes were used in an in vitro primary hepatocyte culture and stimulated with a recombinant sea bream PTHrP (sbPTHrP) to establish the contribution of sbPTHrP alone or in combination with E2 to the regulation of hepatic vitellogenin synthesis. Hepatocytes stimulated solely with sbPTHrP were not affected in their vitellogenesis. However, in hepatocytes stimulated with E2 in combination with sbPTHrP a higher vitellogenin production was seen than with E2 alone. It is concluded that sbPTHrP has a potentiating effect on estradiol stimulation of vitellogenin production by sea bream hepatocytes. The sea bream provides a unique model where vitellogenesis regulation can be studied on E2-naive liver cells, both in vivo and in vitro.