Hon Jung Liew

Modulation of Rh glycoproteins, ammonia excretion and Na+ fluxes in three freshwater teleosts when exposed chronically to high environmental ammonia

SUMMARY We investigated relationships among branchial unidirectional Na+ fluxes, ammonia excretion, urea excretion, plasma ammonia, plasma cortisol, and gill transporter expression and function in three freshwater fish differing in their sensitivity to high environmental ammonia (HEA). The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia-sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were exposed chronically (12–168 h) to 1 mmol l−1 ammonia (as NH4HCO3; pH 7.9). During HEA exposure, carp and goldfish elevated ammonia excretion (JAmm) and Na+ influx rates () while trout experienced higher plasma ammonia (TAmm) and were only able to restore control rates of JAmm and . All three species exhibited increases in Na+ efflux rate (). At the molecular level, there was evidence for activation of a ‘Na+/NH4+ exchange metabolon’ probably in response to elevated plasma cortisol and TAmm, though surprisingly, some compensatory responses preceded molecular responses in all three species. Expression of Rhbg, Rhcg (Rhcg-a and Rhcg-b), H+-ATPase (V-type, B-subunit) and Na+/K+-ATPase (NKA) mRNA was upregulated in goldfish, Rhcg-a and NKA in carp, and Rhcg2, NHE-2 (Na+/H+ exchanger) and H+-ATPase in trout. Branchial H+-ATPase activity was elevated in goldfish and trout, and NKA activity in goldfish and carp, but NKA did not appear to function preferentially as a Na+/NH4+-ATPase in any species. Goldfish alone increased urea excretion rate during HEA, in concert with elevated urea transporter mRNA expression in gills. Overall, goldfish showed more effective compensatory responses towards HEA than carp, while trout were least effective.

Differential responses in ammonia excretion, sodium fluxes and gill permeability explain different sensitivities to acute high environmental ammonia in three freshwater teleosts.

We examined the acute physiological responses to high environmental ammonia (HEA), particularly the linkages between branchial ammonia fluxes and unidirectional Na(+) fluxes, as well as urea excretion, cortisol, and indicators of gill permeability in three freshwater teleosts differing in their sensitivities to ammonia; the highly sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less sensitive cyprinid Cyprinus carpio (common carp) and the highly resistant cyprinid Carassius auratus (goldfish). Fish were acutely exposed to two sub-lethal ammonia concentrations (as NH(4)HCO(3)) at pH 7.9: 1 mM for a period of 12 h, identical for all species, and 5 mM for the cyprinids and 1.4 mM for the trout for 3 h. Elevation of plasma cortisol at both levels of HEA was apparent in all species. At 1 mM, ammonia excretion (J(amm)) was inhibited to a greater extent in trout than cyprinids and concurrently a significantly higher plasma ammonia level was evident in trout. However J(amm) was reversed in all species at 5 or 1.4 mM. Goldfish showed a significant increase in urea excretion rate (J(urea)) during HEA exposure. In carp and trout, neither level of HEA elevated J(urea) but urea production was increased as evidenced by a considerable elevation of plasma urea. At 1mM HEA, Na(+) imbalance became progressively more severe in trout and carp due to a stimulation of unidirectional Na(+) efflux (J(out)(Na)) without a concomitant increase in unidirectional Na(+) influx (J(in)(Na)). Additionally, a transient reduction of J(in)(Na) was evident in trout. Goldfish showed an opposite trend for J(out)(Na) with reduced efflux rates and a positive Na(+) balance during the first few hours of HEA. However, after 12 h of exposure, both J(in)(Na) and J(out)(Na) were also increased in both carp and goldfish, whereas only J(out)(Na) was increased in trout, leading to a net Na(+) loss. Na(+) homeostasis was entirely disrupted in all three species when subjected to the 5 or 1.4 mM ammonia for 3 h: J(in)(Na) was significantly inhibited while considerable activation of J(out)(Na) was observed. Diffusive water efflux rates and net K(+) loss rates across the gills were enhanced during HEA only in trout, indicating an increment in gill transcellular permeability. Transepithelial potential was increased in all the species during ammonia exposure, but to the least extent in goldfish. Overall, for several different physiological systems, trout were most disturbed, and goldfish were least disturbed by HEA, helping to explain the differential ammonia tolerance of the three species.

Fasting goldfish, Carassius auratus, and common carp, Cyprinus carpio, use different metabolic strategies when swimming

Fish need to balance their energy use between digestion and other activities, and different metabolic compromises can be pursued. We examined the effects of fasting (7 days) on metabolic strategies in goldfish and common carp at different swimming levels. Fasting had no significant effect on swimming performance (U(crit)) of either species. Feeding and swimming profoundly elevated total ammonia (T(amm)) excretion in both species. In fed goldfish, this resulted in increased ammonia quotients (AQ), and additionally plasma and tissue ammonia levels increased with swimming reflecting the importance of protein contribution for aerobic metabolism. In carp, AQ did not change since oxygen consumption (MO(2)) and T(amm) excretion followed the same trend. Plasma ammonia did not increase with swimming suggesting a balance between production and excretion rate except for fasted carp at U(crit). While both species relied on anaerobic metabolism during exhaustive swimming, carp also showed increased lactate levels during routine swimming. Fasting almost completely depleted glycogen stores in carp, but not in goldfish. Both species used liver protein for basal metabolism during fasting and muscle lipid during swimming. In goldfish, feeding metabolism was sacrificed to support swimming metabolism with similar MO(2) and U(crit) between fasted and fed fish, whereas in common carp feeding increased MO(2) at U(crit) to sustain feeding and swimming independently.

Cortisol emphasizes the metabolic strategies employed by common carp, **Cyprinus carpio** at different feeding and swimming regimes

The objective of this study was to investigate the interaction between feeding, exercise and cortisol on metabolic strategies of common carp over a 168h post-implant period. Feeding provided readily available energy and clearly increased muscle and liver protein and glycogen stores. Swimming, feeding and cortisol all induced aerobic metabolism by increasing oxygen consumption, and stimulated protein metabolism as demonstrated by the increased ammonia and urea excretion and ammonia quotient. Hypercortisol stimulated ammonia self-detoxifying mechanisms by enhancing ammonia and urea excretion, especially during severe exercise. At high swimming level, higher branchial clearance rates in cortisol treated fish succeeded in eliminating the elevation of endogenous ammonia, resulting in reduced plasma Tamm levels compared to control and sham implanted fish. Carp easily induced anaerobic metabolism, both during routine and active swimming, with elevated lactate levels as a consequence. Both feeding and cortisol treatment increased this dependence on anaerobic metabolism. Hypercortisol induced both glycogenesis and gluconeogenesis resulting in hyperglycemia and muscle and liver glycogen deposition, most likely as a protective mechanism for prolonged stress situations and primarily fuelled by protein mobilization.

Cortisol affects metabolic and ionoregulatory responses to a different extent depending on feeding ration in common carp, Cyprinus carpio

Interacting effects of feeding and stress on corticoid responses in fish were investigated in common carp fed 3.0% or 0.5% body mass (BM) which received no implant, a sham or a cortisol implant (250 mg/kg BM) throughout a 168 hour post-implant period (168 h-PI). At 12h-PI, cortisol implants elevated plasma cortisol, glucose and lactate. Plasma osmolality and ions remained stable, but cortisol increased gill and kidney Na(+)/K(+) ATPase (NKA) and H(+) ATPase activities. Gill NKA activities were higher at 3%-BM, whereas kidney H(+) ATPase activity was greater at 0.5%-BM. Cortisol induced liver protein mobilization and repartitioned liver and muscle glycogen. At 3%-BM, this did not increase plasma ammonia, reflecting improved excretion efficiency concomitant with upregulation of Rhesus glycoprotein Rhcg-1 in gill. Responses in glucocorticoid receptors (GR1/GR2) and mineralocorticoid receptor (MR) to cortisol elevation were most prominent in kidney with increased expression of all receptors at 24 h-PI at 0.5%-BM, but only GR2 and MR at 0.5%-BM. In the liver, upregulation of all receptors occurred at 24 h-PI at 3%-BM, whilst only GR2 and MR were upregulated at 0.5%-BM. In the gill, there was a limited upregulation: GR2 and MR at 72 h-PI and GR1 at 168 h-PI at 3%-BM but only GR2 at 72 h-PI at 0.5%-BM. Thus cortisol elevation led to similar expression patterns of cortisol receptors in both feeding regimes, while feeding affected the type of receptor that was induced. Induction of corticoid receptors occurred simultaneously with increases in Rhcg-1 mRNA expression (gill) but well after NKA and H(+) ATPase activities increased (gill/kidney).

An in vitro study of urea, water, ion and CO2/HCO3− transport in the gastrointestinal tract of the dogfish shark (Squalus acanthias): the influence of feeding

SUMMARY In vitro gut sac preparations made from the cardiac stomach (stomach 1), pyloric stomach (stomach 2), intestine (spiral valve) and colon were used to examine the impact of feeding on transport processes in the gastrointestinal tract of the dogfish shark. Preparations were made from animals that were euthanized after 1–2 weeks of fasting, or at 24–48 h after voluntary feeding on a 3% ration of teleost fish (hake). Sacs were incubated under initially symmetrical conditions with dogfish saline on both surfaces. In comparison to an earlier in vivo study, the results confirmed that feeding caused increases in H+ secretion in both stomach sections, but an increase in Cl− secretion only in stomach 2. Na+ absorption, rather than Na+ secretion, occurred in both stomach sections after feeding. All sections of the tract absorbed water and the intestine strongly absorbed Na+ and Cl−, regardless of feeding condition. The results also confirmed that feeding increased water absorption in the intestine (but not in the colon), and had little influence on the handling of Ca2+ and Mg2+, which exhibited negligible absorption across the tract. However, K+ was secreted in the intestine in both fasted and fed preparations. Increased intestinal water absorption occurred despite net osmolyte secretion into the mucosal saline. The largest changes occurred in urea and CO2/HCO3− fluxes. In fasted preparations, urea was absorbed at a low rate in all sections except the intestine, where it was secreted. Instead of an increase in intestinal urea secretion predicted from in vivo data, feeding caused a marked switch to net urea absorption. This intestinal urea transport occurred at a rate comparable to urea reabsorption rates reported at gills and kidney, and was apparently active, establishing a large serosal-to-mucosal concentration gradient. Feeding also greatly increased intestinal CO2/HCO3− secretion; if interpreted as HCO3− transport, the rates were in the upper range of those reported in marine teleosts. Phloretin (0.25 mmol l−1, applied mucosally) completely blocked the increases in intestinal urea absorption and CO2/HCO3− secretion caused by feeding, but had no effect on Na+, Cl− or water absorption.

Kidney activity increases in copper exposed goldfish (Carassius auratus auratus).

In the present study, the effect of copper was examined in the common goldfish (Carassius auratus auratus). Fish were fasted and exposed to either a high (0.84μM), a low (0.34μM) or a control copper concentration (0.05μM) for 1 and 7days. Swimming performance was not affected by either fasting or copper exposure. Food deprivation alone had no effect on ionoregulation, but low plasma osmolality levels and plasma Na+ were noticed in fasted fish exposed to Cu for 7days. Both gill Na+/K+-ATPase and H+-ATPase activities were undisturbed, while both kidney ATPase activities were up-regulated when challenged with the high Cu levels. Up-regulated kidney ATPase activities likely acted as compensatory strategy to enhance Na+ reabsorption. However, this up-regulation was not sufficient to restore Na+ to control levels in the highest exposure group.

Feeding and swimming modulate iono-and-hormonal regulation differently in goldfish, Carassius auratus and common carp, Cyprinus carpio.

Feeding and swimming can influence ion balance in fish. Therefore we investigated their impact on ionoregulation and its hormonal control in goldfish and common carp. As expected due to the osmorespiratory compromise, exhaustive swimming induced increases in gill Na(+)/K(+) ATPase (NKA) activity in both species, resulting in stable levels of plasma ions. In contrast to our expectations, this only occurred in fed fish and feeding itself increased NKA activity, especially in carp. Fasting fish were able to maintain ion balance without increasing NKA activity, we propose that the increase in NKA activity is related to ammonia excretion rather than ion uptake per se. In goldfish, this increase in NKA activity coincided with a cortisol elevation whilst no significant change was found in carp. In goldfish, high conversion of plasma T4 to T3 was found in both fed and fasted fish resulting in low T4/T3 ratios, which increased slightly due to exhaustive swimming. In starved carp the conversion seemed much less efficient, and high T4/T3 ratios were observed. We propose that thyroid hormone regulation in carp was more related to its role in energy metabolism rather than ionoregulation. The present research showed that both species, whether fed or fasted, are able to sufficiently adapt their osmorepiratory strategy to minimise ions losses whilst maintaining gas exchange under exhaustive swimming.

Kinematics of mouthbrooding in Oreochromis niloticus (Cichlidae)

ABSTRACT Many species from several different families of fishes perform mouthbrooding, where one of the sexes protects and ventilates the eggs inside the mouth cavity. This ventilation behaviour differs from gill ventilation outside the brooding period, as the normal, small-amplitude suction-pump respiration cycles are alternated with actions including near-simultaneous closed-mouth protrusions and high-amplitude depressions of the hyoid. The latter is called churning, referring to its hypothetical function in moving around and repositioning the eggs by a presumed hydrodynamic effect of the marked shifts in volume along the mouth cavity. We tested the hypothesis that churning causes the eggs located posteriorly in the mouth cavity to move anteriorly away from the gill entrance. This would prevent or clear accumulations of brood at the branchial basket, which would otherwise hinder breathing by the parent. Dual-view videos of female Nile tilapias (Oreochromis niloticus) during mouthbrooding showed that churning involves a posterior-to-anterior wave of expansion and compression of the head volume. Flow visualisation with polyethylene microspheres revealed a significant inflow of water entering the gill slits at the zone above the pectoral fin base, followed by a predominantly ventral outflow passing the ventrolaterally flapping branchiostegal membranes. X-ray videos indicated that particularly the brood located close to the gills is moved anteriorly during churning. These data suggest that, in addition to mixing of the brood to aid its oxygenation, an important function of the anterior flow through the gills and buccal cavity during churning is to prevent clogging of the eggs near the gills. Highlighted Article: Mouthbrooding cichlids regularly use posterior-to-anterior waves of head expansion and significant inflow through the opercular slits to move the brood anteriorly away from the gills.