Individual variation in the stress response in fish

Sjoerd E. Wendelaar Bonga

Department of Animal Physiology, University of Nijmegen, Toernooiveld 1, Nijmegen, The Netherlands
(wendelaar@sci.kun.nl)

The physiological response of fishes to a stressor is essentially adaptive and results in adjustments of body functions with enhanced probability of survival. When exposed to severe challenges the stress response may show maladaptive features such as severe structural damage to the gills and increased susceptibility to diseases which have been linked to high circulating catecholamine and cortisol levels during acute and chronic stress. An important finding in several species is the presence of individual differences in the release of cortisol in response to stressors (high and low responders) related to behavioural differences in the stress response. We have studied such differences in males of the African cichlid teleost Oreochromis mossambicus.
In this species high and low cortisol responses appear to be related to social hierarchy, with the high responders having a low hierarchical position. The hypothesis was tested that in a group with a hierarchical structure stressors, such as acute exposure to air, net confinement of chronic cadmium exposure, will have a differential impact on different group members, dependent on their hierarchical position. Substantial differences were observed between dominant and subordinate males. The latter showed a higher rise in plasma cortisol and a more severe drop in plasma osmolarity and sodium levels than the first. Plasma glucose and lactate levels were also higher, as well as gill damage such as epithelial lifting. The subordinate males further showed a higher increase in cell turnover of the branchial pavement and chloride cells, and skin pavement cells, as appeared from enhanced rates of apoptosis, necrosis and mitosis of these cells. Osmoregulatory activity was also higher, as reflected by highly stimulated branchial NaK ATPase activity.
These results indicate that at least part of the variability of the response of a group of tilapia to stressors is related to individual differences in rank. This phenomenon seems independent of the type of stressor and was also observed in carp (Cyprinus carpio). For this species a program was started to produce clones of strains with a high or a low cortisol response to stressors.

Physiological responses of fish to toxins occurring in freshwaters and in the marine environment

FB Eddy¹, MJ Gubbins², JH Best¹, GA Codd¹, RM Stagg² and Susan Gallacher²

¹Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, UK and
²FRS Marine Laboratories, PO Box 101, Victoria Road, Aberdeen AB11 9DB, Scotland, UK.

Substances toxic to fish may enter the aquatic environment from industrial and agricultural activities and from blooms of micro-organisms including dinoflagellates and cyanobacteria. Paralytic shellfish poisoning toxins (PSPs) are produced by several species of marine dinoflagellates and the best known is saxitoxin (SXT) and its derivatives. During blooms these toxins accumulate in shellfish and in vertebrate consumers their toxic action is to block sodium channels leading to paralysis. It has been suggested that such blooms may be increasing prevalence due to eutrophication by anthropogenic activity, and PSPs have often been implicated as the causative agent in bloom related fish kills. Little is known of the metabolism of PSPs in fish and studies on marine salmon aimed to investigate the role of xenobiotic metabolising enzymes including induction of the Phase II enzyme, glutathione S-transferase (GST).
Cyanobacterial (blue green algae) produce a variety of toxins in fresh water including neurotoxins and endotoxins and a group of cyclic peptides, the microcystins which are hepatotoxins. Ingestion by mammals leads to symptoms including vomiting and diarrhoea, followed by damage to liver parenchyma which may be fatal. Fish kills sometimes occur during blooms of cyanobacteria though it is difficult to attribute mortality to a specific cause. Mortality could result from deterioration of water quality often characterised by lowered oxygen levels, increased ammonia and pH levels, and toxins which may be released during senescence of the bloom. A variety of other substances are present during blooms and there is recent interest in lipopolysaccharides (LPS) produced by the cyanobacteria and bacteria present in blooms. Our studies aimed to investigate the effects of LPS on circulatory and osmoregulatory physiology of juvenile salmonids. Of particular interest is the possibility t! hat LPS may act via the cytokine system, stimulate the inducible form of nitric oxide synthase (iNOS) leading to increased levels of nitric oxide (NO) whose effects include vasodilation. Effects of LPS and NO on circulation and osmoregulation in juvenile salmonids are discussed.

References
MJ Gubbins, FB Eddy, S Gallacher and RM Stagg (2000). Paralytic shellfish poisoning toxins induce xenobiotic metabolising enzymes in Atlantic salmon (Salmo salar). Marine Environmental Research 50, 469-483.
JH Best, FB Eddy and GA Codd (2001). Effects of purified microcystin-LR and cell extracts of Microcystis strains PCC 7813 and CYA-43 on cardiac function in brown trout (Salmo trutta) alevins. Fish Physiology and Biochemistry. In press.

Morphologic respiratory and osmoregulatory adaptations in Oreochromis alcalicus grahami, a cichlid fish that subsists in hot alkaline Lake Magadi of Kenya

J.N. Maina

Department of Anatomical Sciences, The University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa.

O. a. grahami, a small cichlid fish, is found in the highly exacting and dynamic shallow peripheral lagoons of the volcanic Lake Magadi of Kemya. The temperature may be as high as 46°C, pH 10; -CO3-2 and -HCO3-1 ions concentration ~ 200 mmol/L; Na+ ions 340 mmol/L; Cl- ions 100 mmol/L, osmolarity ~560 mOsm/kg; total CO2 level 180 mmol/L; conductivity 1.6x105 µmho/cm and the O2 levels shift from virtual anoxia at night (~0.3 kPa) to supersaturation (~53 kPa) during the day. The lagoons are undoubtedly some of the most severe environments inhabited by a vertebrate.
The morphology and morphometry of the gills of O. a. grahami have been investigated and compared with those of O. niloticus, a closely related freshwater fish. Adaptively, the gills of O. a. grahami have a relatively more extensive respiratory surface area, a thinner water-blood barrier and a significantly greater number and more specialized chloride cells. Among fish, the diffusing capacity of the gills of O. a. grahami for O2 is only exceeded by those of the highly energetic tuna. The characteristic behaviour of skimming the surface of water with the mouth open allows the gills to be passively ventilated with well-aerated water and the air-bladder to be filled with air, a strategy that maximizes O2 acquisition at night when the water is virtually anoxic. By adopting multiple respiratory and osmoregulatory adaptive strategies, O. a. grahami has been able to thrive in an extremely severe habi! tat.

References
Coe, M.J. (1966). The biology of Tilapia grahami (Boulenger) in Lake Magadi. Acta Tropica 23:146-177.
Maina J.N., Kisia, S.M., Wood, C.M., Narahara, A.B., Bergman, H.L., Laurent, P. and P.J. Walsh (1996). A comparative allometric study of the morphometry of the gills of an alkalinity adapted cichlid fish, Oreochromis alcalicus grahami of Lake Magadi, Kenya. Int. J. Salt Lake Res. 5: 131-156.

Antifreeze proteins from the Antarctic fish Dissostichus mawsoni studied by differential scanning calorimetry and nanoliter osmometry.

Hans Ramløv

Dept. of Life Sciences and Chemistry, Build. 18.2, P.O. Box 260, Roskilde University, DK-4000 Roskilde, Denmark

The polar oceans are either in the winter (Arctic) or year round (Antarctic at high latitudes) at about -1.9°C, the freezing point of seawater. During these periods the seas are covered with ice. Typical marine teleosts have a melting point of -0.5 to -0.9°C thus they are in the cold periods supercooled by 1°C or more. Any contact with the ice by the fish would therefore lead to freezing. The Antarctic notothenioid Dissostichus mawsoni lives at 300-500 m where the temperature is -1.9°C. At least eight size classes of antifreeze glyco proteins (AFGP’s) are found in the blood of D. mawsoni ranging from 2.6 kDa to 34 kDa.
Antifreeze proteins inhibit the growth of ice crystals by interaction with specific crystal faces on the ice crystals thus giving rise to a separation of the melting point and the freezing point. Antifreeze proteins show the expected colligative effect on the melting point but depress the freezing point 200 – 300 times more than expected on a colligative basis. This is called “thermal hysteresis” or “the antifreeze effect” (Cheng & DeVries 1991).
It has been observed that AFGP 1-5 are more efficient in inhibiting ice growth than AFGP 6-8. Also a small initial ice growth is observed when the temperature is decreased in an AFGP 1-5 solution containing a minute polycrystalline ice crystal. In the present investigation solutions of AFGP 1-5, 1-8 and 7 & 8 was studied by differential scanning calorimetry (DSC). Two exotherms were found in the AFGP 1-5 and 1-8 solutions whereas in the 7&8 solution only a “disturbance” of the ice growth was observed.

References
Cheng, C.C. & DeVries, A.L. (1991) The role of antifreeze glycopeptides and peptides in the freezing avoidance of cold-water fish. Pp. 1- 14 In “Life under extreme conditions” (G. di Prisco, ed.), Springer-Verlag, Berlin Heidelberg.

Effects of copper toxicity to larvae of Oreochromis mossambicus

Felister M. Urasa¹ and Sjoerd E. Wendelaar Bonga²

¹Department of Zoology, University Dar es Salaam, P.O. Box 35064, Dar es Salaam, Tanzania
(urasa@udsm.ac.tz)
²Department of Animal Physiology, University of Nijmegen, Toernooiveld 1, Nijmegen, The Netherlands
(wendelaar@sci.kun.nl)

Although copper (Cu) is of physiological importance, it is known to become toxic when concentrations are higher than normal. In fish, branchial ion transport, in particular sodium, is disturbed by high concentrations of copper, mainly by inhibition of NaK-ATPase activity in the chloride cells. In this study early post hatch larvae of Mozambique tilapia (Oreochromis mossambicus) were exposed to Cu concentrations of 0.3 µmol.l^-1 and 0.8 µmol.l^-1 at 7.8 and 270 for 14 days. Growth of the larvae (body length and weight) was monitored at intervals of two or three days. The ultrastructure of the chloride cells was studied. Whole body concentrations of Cu, Ca, Mg, Na, K and cortisol were determined.
The larvae exposed to copper concentrations of 0.3 µmol.l^-1 and 0.8 µmol.l^-1 showed a lower increase in length and weight than the control larvae. Structural damage such as uplifting of the epithelium of the gill filaments was observed. Necrosis of some chloride cells was indicated by the destruction of the cytoplasm and the presence of vacuoles in larvae exposed to 0.8 µ mol.l^-1. Phagosomes were present in some chloride cells. In the intact chloride cells the tubular system was more developed than in the control larvae. Also apoptosis, reflected by densification of cells nuclei and mitochondria, was observed. The whole body Cu concentration increased with the duration of exposure. Larval whole body calcium and sodium content decreased. Cortisol levels were high directly after the start of Cu exposure and remained elevated. The results show that the osmoregulatory system of the young life stages of Oreochromis mossambicus is sensitive to copper, and that these fish show a stress response.

References
Hellawell, J.M., 1988. Toxic substances in rivers and streams. Environ. Pollut. 50: 61-85.
Stouthart, A.J.H.X., J.L.M.Haans, R.A.C. Lock and S.E.Wendelaar Bonga, 1996. Ambient water pH determines toxicity of copper to embryonic and larval stages of the common carp. Environ.Toxicol.Chem.15: 376-383
Wilson, R.W. and E.W.Taylor. 1993. The physiological responses of freshwater rainbow trout, Oncorhynchus mykiss, during acutely lethal copper exposure. J. Comp.Physiol. 163B: 38-47