Effects of the cyanobacterial neurotoxin beta-N-methylamino-L-alanine on the early-life stage development of zebrafish (Danio rerio).

beta-N-Methylamino-L-alanine (BMAA), a neurotoxic amino acid, is produced by members of all known groups of cyanobacteria. In the presence of added carbonate, BMAA generates an analogue of glutamate which has been associated with motor neuron (MN) diseases via a mechanism of motor neurone specific excitotoxicity. The toxicity of BMAA has been established in various mammalian test models, but the widespread aquatic production of BMAA raises questions of BMAA toxicity to aquatic organisms. Zebrafish (Danio rerio) embryos were exposed to varying concentrations of BMAA (5-50,000 microgl(-1)) with and without added carbonate. BMAA exposure induced a range of neuro-muscular and developmental abnormalities in D. rerio, which can be directly related to disruptions to glutamatergic signalling pathways. When exposed to BMAA plus added carbonate, the incidence of pericardial oedema increased by up to 21% in test subjects, correlating with a reduction in heart rate. Increased incidence of abnormal spinal axis formation was seen in all D. rerio larvae exposed to BMAA concentrations of >or=50microgl(-1), with a further 10% increase from >or=500 microgl(-1) BMAA when carbonate species were present. A dose-dependent increase in clonus-like convulsions was observable in embryos exposed to >or=5 microgl(-1) BMAA+/-added carbonate. This is the first study on the neuro-muscular and developmental effects of BMAA exposure on aquatic vertebrates. The present findings, plus the potentially widespread production of BMAA in aquatic cyanobacteria, indicate a need for information of exposure levels, duration and toxic outcomes in aquatic biota.

Role of nitric oxide in larval and juvenile fish.

Fish are known to express the three isoforms of nitric oxide synthase (NOS), the constitutive forms endothelial or eNOS, neuronal or nNOS and the inducible form iNOS. Most studies in fish have focussed on possible roles for NO in cardiovascular physiology although there has been recent attention on the role of nNOS in embryonic development. However compared to mammalian studies there have been relatively few studies on effects of nitric oxide (NO) on fish. Studies on heart and blood vessel preparations from various fish species appear to show results specific to the species or to the particular preparation. Possible roles of NO in the in vivo biology of adult fish or larval fish have received little attention. This article reviews effects of nitric oxide on cardiovascular physiology in fish with special emphasis on larval fish. It introduces some experimental work on possible signaling pathways in larval fish and introduces the possibility that NO could be an important environmental influence for some aquatic organisms. In higher vertebrates LPS (lipopolysaccharide) is known to activate the cytokine signaling system and stimulate increased expression of iNOS and increased production of NO, but this remains less investigated in fish. The effects of LPS on cardiovascular and osmoregulatory physiology of larval and juvenile salmonids are discussed and a possible role of NO in stress-induced drinking is suggested.

Effects of Microcystis cells, cell extracts and lipopolysaccharide on drinking and liver function in rainbow trout Oncorhynchus mykiss Walbaum.

Liver mass (hepatosomatic index, HSI) increased by approximately 18% and water content in the gut by approximately 13 ml kg(-1) in freshwater rainbow trout exposed for 24 h to intact cells of a microcystin-producing cyanobacterium (Microcystis PCC 7813) together with administration of heterotrophic bacterial LPS. Exposure to broken (ultrasonicated) cyanobacterial cells together with administration of bacterial LPS increased HSI by approximately 50% and water content in the gut by almost 30 ml kg(-1). Exposure to broken or unbroken Microcystis cells without administration of bacterial LPS resulted in increased water content of the gut (by approximately 13 ml kg(-1)) with insignificant changes in HIS. Drinking rate increased with increasing dosage of bacterial LPS alone. The increased volume of water in the gut potentially increases the opportunity for uptake of waterborne toxins, including microcystins, and increased liver mass is a symptom consistent with the toxic effects of microcystins. It is concluded that exposure of fish to the cell contents of cyanobacteria (e.g. Microcystis PCC 7813) promotes osmoregulatory imbalance resulting from stimulation of the drinking response, increased volume of fluid in the gut and inability to remove excess water.

Effects of nitric oxide synthase inhibitors and a substrate, L-arginine, on the cardiac function of juvenile salmonid fish.

Control of cardiac function was investigated juvenile brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) using inhibitors of nitric oxide synthase (NOS), (L-NAME, NG-nitro-L-arginine and L-NMMA, NG-monomethyl-L-arginine) and a substrate of NOS (L-arginine). Salmonid alevins are excellent models for such studies since they are transparent, the beating heart is easily observed, diffusing distances are small, and they respond within a few seconds to exogenously administered chemicals. The response to inhibitors of NOS (L-NAME or L-NMMA) was tachycardia interpreted as vasoconstriction through lowered capacity for synthesis of NO. This could be reversed by addition of L-arginine and the subsequent bradycardia was explained as a vasodilation resulting from increased synthesis of NO. Blood flow into the heart is mainly via the vitelline vein and changes of flow resulting from constriction or dilation of this vessel may be probably major determinants of heart rate. The results provide evidence for the presence NOS in juvenile fish and indicate a physiological role for NO in cardiovascular control.

Sodium-dependent copper uptake across epithelia: a review of rationale with experimental evidence from gill and intestine.

The paper reviews the evidence for apparent sodium-dependent copper (Cu) uptake across epithelia such as frog skin, fish gills and vertebrate intestine. Potential interactions between Na(+) and Cu during transfer through epithelial cells is rationalized into the major steps of solute transfer: (i) adsorption on to the apical/mucosal membrane, (ii) import in to the cell (iii) intracellular trafficking, and (iv) export from the cell to the blood. Interactions between Na(+) and Cu transport are most likely during steps (i) and (ii). These ions have similar mobilities (lambda) in solution (lambda, Na(+), 50.1; Cu(2+), 53.6 cm(2) Int. ohms(-1) equiv(-1)); consequently, Cu(2+) may compete equally with Na(+) for diffusion to membrane surfaces. We present new data on the Na(+) binding characteristics of the gill surface (gill microenvironment) of rainbow trout. The binding characteristics of Na(+) and Cu(2+) to the external surface of trout gills are similar with saturation of ligands at nanomolar concentrations of solutes. At the mucosal/apical membrane of several epithelia (fish gills, frog skin, vertebrate intestine), there is evidence for both a Cu-specific channel (CTR1 homologues) and Cu leak through epithelial Na(+) channels (ENaC). Cu(2+) slows the amiloride-sensitive short circuit current (I(sc)) in frog skin, suggesting Cu(2+) binding to the amiloride-binding site of ENaC. We present examples of data from the isolated perfused catfish intestine showing that Cu uptake across the whole intestine was reduced by 50% in the presence of 2 mM luminal amiloride, with 75% of the overall inhibition attributed to an amiloride-sensitive region in the middle intestine. Removal of luminal Na(+) produced more variable results, but also reduced Cu uptake in catfish intestine. These data together support Cu(2+) modulation of ENaC, but not competitive entry of Cu(2+) through ENaC. However, in situations where external Na(+) is only a few millimoles (fish gills, frogs in freshwater), Cu(2+) leak through ENaC is possible. CTR1 is a likely route of Cu(2+) entry when external Na(+) is higher (e.g. intestinal epithelia). Interactions between Na(+) and Cu ions during intracellular trafficking or export from the cell are unlikely. However, effects of intracellular chloride on the Cu-ATPase or ENaC indicate that Na(+) might indirectly alter Cu flux. Conversely, Cu ions inhibit basolateral Na(+)K(+)-ATPase and may increase [Na(+)](i).

Effects of enteric bacterial and cyanobacterial lipopolysaccharides, and of microcystin-LR, on glutathione S-transferase activities in zebra fish (Danio rerio).

Cyanobacteria (blue-green algae) can produce a variety of toxins including hepatotoxins e.g. microcystins, and endotoxins such as lipopolysaccharides (LPS). The combined effects of such toxins on fish are little known. This study examines the activities of microsomal (m) and soluble (s) glutathione S-transferases (GST) from embryos of the zebra fish, Danio rerio at the prim six embryo stage, which had been exposed since fertilisation to LPS from different sources. A further aim was to see how activity was affected by co-exposure to LPS and microcystin-LR (MC-LR). LPS were obtained from Salmonella typhimurium, Escherichia coli, a laboratory culture of Microcystis CYA 43 and natural cyanobacterial blooms of Microcystis and Gloeotrichia. Following in vivo exposure of embryos to each of the LPS preparations, mGST activity was significantly reduced (from 0.50 to between 0.06 and 0.32 nanokatals per milligram (nkat mg(-1)) protein). sGST activity in vivo was significantly reduced (from 1.05 to between 0.19 and 0.22 nkat mg(-1) protein) after exposure of embryos to each of the cyanobacterial LPS preparations, but not in response to S. typhimurium or E. coli LPS. Activities of both m- and sGSTs were reduced after co-exposure to MC-LR and cyanobacterial LPS, but only mGST activity was reduced in the S. typhimurium and E. coli LPS-treated embryos. In vitro preparations of GST from adult and prim six embryo D. rerio showed no significant changes in enzyme activity in response to the LPS preparations with the exception of Gloeotrichia bloom LPS, where mGST was reduced in adult and embryo preparations. The present study represents the first investigations into the effects of cyanobacterial LPS on the phase-II microcystin detoxication mechanism. LPS preparations, whether from axenic cyanobacteria or cyanobacterial blooms, are potentially capable of significantly reducing activity of both the s- and mGSTs, so reducing the capacity of D. rerio to detoxicate microcystins. The results presented here have wide ranging implications for both animal and human health.

Paralytic shellfish poisoning toxins induce xenobiotic metabolising enzymes in Atlantic salmon (Salmo salar).

Paralytic shellfish poisoning (PSP) toxins have been implicated as the causative agent of a number of fish kills. Exposure experiments indicate that fish are susceptible to PSPs by intraperitoneal (i.p.) and oral administration, while sampling of fish affected by toxic blooms reveals that these toxins can be accumulated. In spite of the potential impact to marine fisheries, little research has been conducted on the potential metabolism and detoxification of PSPs in marine fishes. Previous work by this group has shown that the xenobiotic metabolising enzyme (XME) cytochrome P-450 (CYP1A) is induced in Atlantic salmon (Salmo salar) following i.p. exposure to saxitoxin (STX). Salmon injected i.p. with sub-lethal doses of STX show a four- to eight-fold induction of hepatic CYP1A (as shown by ethoxyresorufin-O-deethylase activity) over controls after 96 h. Results presented here show that the phase II XME glutathione S-transferase (GST) is also induced in salmon following PSP exposure. Post smolts were exposed to three injections of PSPs (2 micrograms STXeq/kg) over 21 days. Injection of both STX and PSPs extracted from a toxic strain of dinoflagellate (Alexandrium fundyense, CCMP 1719) resulted in induction of hepatic GST, as measured by activity for 1-chloro 2,4-dinitrobenzene. Such inductions indicate a potential role for XMEs in PSP metabolism. Possible roles for other enzymes are also discussed.

Ionic regulation and nitrogenous excretion in rainbow trout exposed to buffered and unbuffered freshwater of pH 10.5.

Rainbow trout exposed to unbuffered water of pH 10.5 initially showed significant increases in blood pH, plasma cortisol and glucose, partial pressure of NH3 (PNH3), NH4+, and HCO3- values as well as loss of plasma Cl-, reduced partial pressure of CO2 (PCO2), and inhibition of total ammonia excretion rate. After the first day, fish resisted further change, and new levels were established (for blood pH and plasma PCO2 and PNH3 levels) or imbalances corrected, either partially (for total ammonia excretion) or completely (for plasma Cl-, HCO3-, cortisol, and glucose values). During the 7-d exposure, 80% of fish in unbuffered water survived, but in buffered water (0.75 mmol L(-1) glycine-buffered KOH at pH 10.5), survival was only 50% after 3 d, and ion regulatory failure was evident. Fish in buffered and unbuffered alkaline waters had similar total ammonia excretion rates, which suggests that glycine-buffered KOH was not sufficient to significantly reduce gill boundary layer acidification. After 7 d in unbuffered alkaline water, 30% of total ammonia excretion was linked with an amiloride-sensitive (0.1 mmol L(-1)) Na+ uptake mechanism. Treatment of alkaline-exposed trout with waterborne acetazolamide (1.5 mmol L(-1)) indicated that gill boundary layer H+ production, through hydration of CO2, had a role in excretion of total ammonia. Exposure to 4-acetamino-4'-isothiocyantostilbene-2,2'-disulphonic acid (SITS; 0.1 mmol L(-1)) following 24-h exposure to unbuffered alkaline water resulted in increased plasma HCO3- and lowered plasma Cl- concentrations, indicating the role of branchial Cl-/HCO3- exchange in regaining Cl- lost and eliminating the HCO3- accumulated during exposure to alkaline water.

Cardiovascular responses in vivo to angiotensin II and the peptide antagonist saralasin in rainbow trout Oncorhynchus mykiss.

The effects of [Asn1,Val5]-angiotensin II (AngII) and [Sar1,Val5, Ala8]-angiotensin II (saralasin) on dorsal aortic blood pressure, pulse pressure and heart rate were examined in rainbow trout in vivo. AngII when administered as a single dose of 25 microg kg-1 induced a biphasic response in blood pressure, with a significant hypertensive response during the initial 10 min, followed by a significant hypotension of 70-75 % compared with the initial blood pressure after 50 min and continuing until approximately 80 min post-injection. The co-administration of AngII (25 microg kg-1) and saralasin (50 microg kg-1) resulted in the same hypertensive response during the initial phase, but abolished the hypotensive effect of AngII. Heart rate was significantly increased in response to AngII, but the administration of AngII and saralasin together attenuated the increase by approximately 44 %. Stimulation of the endogenous renin-angiotensin system using a vasodilator, sodium nitroprusside, significantly increased drinking rate in rainbow trout fry, a response inhibited by saralasin, indicating a role for AngII-induced hypotension in drinking. For the first time, a decrease in blood pressure in response to AngII in vivo has been demonstrated in fish, and this is discussed in relation to homeostasis of blood pressure and a possible role in the control of drinking.

Drinking in Atlantic salmon presmolts and smolts in response to growth hormone and salinity.

Drinking rate in freshwater Atlantic salmon presmolts (about 0.1 ml/kg/h) was unaffected by daily injections of ovine GH (50 micrograms/fish) for a week but upon transfer to sea water an immediate and full drinking response was developed compared to saline treated fish (3.34 +/- 0.16 vs. 2.23 +/- 0.27 ml/kg/h). Smolting did not affect drinking rates in freshwater but after 7 days in sea water, salmon smolts imbibed 3.88 +/- 0.25 ml/ kg/h, significantly higher than the rate for saline injected presmolts (p < 0.05, one-way ANOVA), but not significantly different from oGH treated presmolts. Smolting and oGH treatment were without effect on plasma Na+ levels in freshwater fish and 7 days after transfer to sea water both groups showed a better regulation of plasma Na+ levels compared to saline treated presmolts. Atlantic salmon smolts showed higher levels of plasma Cl- than presmolts in freshwater, and after 7 days in sea water, both oGH presmolts and smolts showed significantly lower levels of plasma Cl- than saline injected presmolts. GH treatment in freshwater presmolts improved hypoosmoregulatory capacity following transfer to seawater and these results are discussed in relation to the physiology of smolting, and control of drinking.

Effect of manipulation of the renin-angiotensin system in control of drinking in juvenile Atlantic salmon (Salmo salar L) in fresh water and after transfer to sea water.

Drinking in Atlantic salmon (Salmo salar) juveniles was investigated in fresh water and following transfer to sea water. There was a significant effect of fish size on drinking, and smolts (20-30 g) imbibed about ten times less water than alevins of 0.2-0.3 g. Freshwater smolts drank at a rate of 0.15 +/- 0.03 ml.kg-1.h-1 and administration of doses of 10 or 20 mg.kg-1 of papaverine (stimulator of the renin-angiotensin system RAS) or [Asn1, Val5]-Angiotensin II (0.4 mumol.kg-1) resulted in significant increases in drinking, while administration of the angiotensin converting enzyme inhibitor, enalapril (50 mg.kg-1) had no effect on drinking. Transfer of Atlantic salmon smolts to 1/3, 2/3 and full strength sea water resulted in significant increases in drinking to 1.06 +/- 0.12, 1.24 +/- 0.0.16 and 3.89 +/- 0.28 ml.kg-1.h-1, respectively. In sea water, stimulation of the endogenous RAS by administration of papaverine (20 mg.kg-1) resulted in a 20% increase in drinking, while administration of enalapril to doses of 50 and 200 mg.kg-1 lowered drinking to 1.99 +/- 0.48 and 0.32 +/- 0.06 ml.kg-1.h-1, respectively. All treatments were without effect on blood plasma levels of Na+ and Cl- in fresh water, while in sea water smolts both stimulation and inhibition of drinking resulted in hemoconcentration of Na+ and Cl-. The role of the renin angiotensin system in control of drinking and hydromineral balance in Atlantic salmon is discussed.

Drinking rate in juvenile Atlantic salmon,Salmo salar L fry in response to a nitric oxide donor, sodium nitroprusside and an inhibitor of angiotensin converting enzyme, enalapril.

Drinking in freshwater juvenile salmon was investigated in response to vasodilation by sodium nitroprusside (SNP), a nitric oxide donor, which significantly increased blood vessel diameter in Atlantic salmon alevins. Atlantic salmon fry (1-3 g), as previously shown, drank at a significant rate in fresh water which doubled to about 1.2 ml kg(-1) h(-1) following injection of SNP (100 µmol kg(-1)), through dilation of body vasculature and activation of a vasoconstrictive mechanism, the endogenous renin angiotensin system (RAS). This response was 50% inhibited by injection of about 100 mg kg(-1) enalapril. Fry increased drinking in response to SNP administered in the water, though the concentration required for maximal response, 1.6 mmol l(-1), was much greater than for injected SNP; this response was also inhibited by enalapril injection. Possible involvement of the gill vasculature and branchial osmoreceptors or baroreceptors in control of the drinking response is discussed.

K+ balance in rainbow trout gill and liver tissue, cell suspensions and cultured cells

Both intact gill and liver tissue from rainbow trout accumulated K+, as determined by 86Rb+ uptake, a process largely inhibited by ouabain, indicating the presence of functional NaKATPase. Cell suspensions, produced by disaggregation of gill or liver tissues, accumulated very little K+ compared to tissues (Less than 10 percent). Disaggregation resulted in depolarisation of cells with loss of intracellular K+ and although NaKATPase, as measured by 86Rb+ uptake rate, remained functional and inhabitable by ouabain, the activity was insufficient to replace the rapid K+ loss. While attached, cultured gill and liver cells showed normal K+/Na+ ratios and NaKATPase activity, but release from the substratum resulted in depolarisation and rapid K+ loss as seen in cell suspensions. These results suggest that care is required in interpreting ionic regulatory and other results from cell suspensions and that further research should be directed towards systems where cells can maintain normal ionic balance.

K+ balance in rainbow trout gill and liver tissue, cell suspensions and cultured cells.

Both intact gill and liver tissue from rainbow trout accumulated K+, as determined by 86Rb+ uptake, a process largely inhibited by ouabain, indicating the presence of functional NaKATPase. Cell suspensions, produced by disaggregation of gill or liver tissues, accumulated very little K+ compared to tissues (less than 10%). Disaggregation resulted in depolarisation of cells with loss of intracellular K+ and although NaKATPase, as measured by 86Rb+ uptake rate, remained functional and inhibitable by ouabain, the activity was insufficient to replace the rapid K+ loss. While attached, cultured gill and liver cells showed normal K+/Na+ ratios and NaKATPase activity, but release from the substratum resulted in depolarisation and rapid K+ loss as seen in cell suspensions. These results suggest that care is required in interpreting ionic regulatory and other results from cell suspensions and that further research should be directed towards systems where cells can maintain normal ionic balance.

Renal, respiratory and ionic regulation in Atlantic salmon (Salmo salar L.) kelts following transfer from fresh water to seawater.

Atlantic salmon may return to the sea after spawning in fresh water. These fish, known as kelts, reportedly show a limited ability to hypoosmoregulate. However, this study shows that fresh-water-adapted kelts exposed to seawater demonstrate rapid adaptation (within 48 h) in osmoregulatory parameters to values characteristics of seawater-adapted salmonids. The urine flow rate falls from 1.2 to 0.2 ml.kg-1.h-1 within 24 h. Over the same period, urine osmolality increases from 48 mosmol.kg-1 to become isosmotic with the plasma, and Mg2+ secretion by the kidney tubules elevates the urine concentration from 0.5 to 100 mmol.l-1. As is characteristic for marine teleosts, kelts drink seawater and process the ingested water in the gut to replace body water lost by osmosis to the hyperosmotic medium. Seawater exposure causes a marked hypoxia, arterial oxygen tension falling by 43% within minutes and persisting for at least 4 days at this low level. This is associated with large changes in blood pH and acid-base balance. The physiological mechanisms involved in adaptation to a hyperosmotic external medium are discussed, and the osmoregulatory capacity of kelts is compared with that of salmon at other stages of the life cycle.

Intestinal water transport in juvenile Atlantic salmon (Salmo salar L.) during smolting and following transfer to seawater.

1. The rate of mucosal to serosal water movement was measured in vitro in non-everted midgut segments in Atlantic salmon (Salmo salar L.) during parr-smolt transformation (February-July) and following transfer of smolts to seawater in May. 2. The rate increased significantly during smolting from 5.61 microliters/cm2/hr in the parr (February) to 11.03 microliters/cm2/hr in smolts in May. 3. Measured at intervals over a period of 20 days in seawater, the rate of water transport was not significantly different from that found in the freshwater-adapted smolts (11.20 microliters/cm2/hr). 4. Intestinal water transport is sodium-linked and inhibited by ouabain but is not stimulated by cortisol.

Circulatory and ionoregulatory effects of atrial natriuretic peptide on rainbow trout (Salmo gairdneri Richardson) fed normal or high levels of dietary salt.

Rainbow trout fed a normal salt diet (1.3% NaCl) or a high salt diet (12% NaCl for at least 6 months) were chronically cannulated in the dorsal aorta and received 10 µg kg(-1) ANP (1-28 human, UBC-Bioproducts) infused over a 10 min period. This had an insignificant influence on sodium balance, blood electrolytes and branchial sodium fluxes. In fish given a normal diet, the blood pressure and heart rate were uninfluenced by ANP, but pulse pressure was reduced by on average 60% and in some cases was not evident at all. Blood pressure in the fish fed a high salt diet was significantly higher than in the control fish; this together with heart rate and pulse pressure was not affected by ANP administration.

Kidney function in response to salt feeding in rainbow trout (Salmo gairdneri Richardson).

1. The effect of high levels of dietary salt up to 12% NaCl on kidney function in freshwater rainbow trout was investigated. 2. Renal response to dietary NaCl load includes increases in urinary flow rate and glomerular filtration rate, together with a slight reduction in ionic reabsorption capacity. 3. The renal salt excretion rate, which was doubled to about 100 microM/kg/hr, in fish fed the high salt diet, is not entirely a consequence of a reduction in tubular ionic reabsorption but also of increased glomerular filtration. 4. The role of the endocrine system in control of renal salt excretion is discussed.

Transepithelial ion exchange in smolting atlantic salmon (Salmo Salar L.).

Whole-body (but predominantly gill) Na(+) exchange, gill Na(+)/K(+)/ATPase activity and seawater tolerance were examined in juvenile Atlantic salmon during the smolting period. Transepithelial net Na(+) gain decreased steadily from late February showing a net loss in April and early May, returning to approximate equilibrium in mid-May. This seasonal net loss of Na(+) to the environment occurred slightly after maximal gill epithelial Na(+)/K(+)/ATPase activity and preceded maximal seawater tolerance. The results are discussed in relation to changes in gill permeability and salt intake via the diet.

Some effects of adrenaline on anion transport and nitrite-induced methaemoglobin formation in the rainbow trout (Salmo gairdneri Richardson).

The effects of adrenaline on branchial anion transport and nitrite-induced methaemoglobinaemia have been investigated in rainbow trout. Nitrite uptake and efflux results suggest that adrenaline effects a net anion efflux principally by stimulation of the unidirectional branchial anion efflux. In oxygenated whole blood nitrite-induced methaemogloblin was significantly reduced in the presence of adrenaline. The physiological and environmental consequences of nitrite-induced stress are discussed.