A novel oxyconforming response in the freshwater fish Galaxias maculatus.

How fish oxygen consumption is modulated by external PO(2) has long been a matter of interest, yet is an experimentally complicated question to answer. In this study closed and semi-closed respirometry were used to evaluate the oxygen consumption rate of the scaleless galaxiid fish, inanga (Galaxias maculatus) as a function of decreasing external PO(2). Both respirometry techniques showed that as environmental oxygen levels declined, oxygen consumption rates also decreased. At no point did inanga regulate oxygen consumption. This is strong evidence that inanga is an oxyconformer. Partitioned respirometry experiments showed that skin plays an important role in oxygen uptake in this fish species, and cutaneous oxygen uptake may have an important role in shaping the oxygen consumption response to hypoxia.

Leap of faith: voluntary emersion behaviour and physiological adaptations to aerial exposure in a non-aestivating freshwater fish in response to aquatic hypoxia.

Lowland stream fauna in areas of intensive agriculture are increasingly under threat from anthropogenic activities leading to eutrophication and subsequent hypoxia. Survival of hypoxic episodes depends upon a combination of behavioural and physiological adaptations. Responses of inanga (Galaxias maculatus: Galaxiidae) to aquatic hypoxia were investigated in the laboratory. Contrary to expectation inanga did not display behaviour that might reduce energy expenditure during oxygen limitation, with swimming activity slightly, but significantly elevated relative to normoxia. Instead, as dissolved oxygen concentrations decreased, the fish moved higher in the water column, increased their swimming speed and exhibited aquatic surface respiration. Physiological changes such as enhanced opercular frequency were also noted. As hypoxia deepened inanga started to leap out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to hypoxic fish. Thus inanga appear better adapted to escape hypoxia (a behavioural adaptation) rather than tolerate it (physiological adaptation). The emersion strategy used for inanga in response to severe hypoxia is in agreement with their ability to take up more oxygen from the air than from hypoxic water and therefore may justify the potentially increased risks of desiccation and predation associated with leaving the water.

Effects of rested harvesting on muscle metabolite concentrations and K-values in Chinook salmon (Oncorhynchus tshawytscha) fillets during storage at 15 degrees C.

Improvement of harvesting procedures in aquaculture may also improve the quality and storage properties of the fish. The use of an anesthetic allows fish to be harvested with reduced stress and exhaustion, which affect fillet properties. We report here on the effects of rested harvesting on the postharvest metabolic profiles and K-values in Chinook salmon (Oncorhynchus tshawytscha) fillets stored near to the fish's acclimation temperature at 15 degrees C for 36 h. Fresh rested fillets were obtained by anesthesia with AQUI-S. They had high cut surface pHs (7.63) and high concentrations of adenosine triphosphate (ATP) and creatine phosphate (3.75 and 8.73 micromol g(-1) respectively), which depleted over 12 h. In contrast, fresh exhausted fillets had low cut surface pHs (6.66) and ATP and creatine phosphate were depleted. Adenosine diphosphate (ADP) and beta-nicotinamide adenine dinucleotide (NAD(+)) concentrations also remained significantly higher during the first 12 h of storage in rested fillets. In fresh rested fillets inosine monophosphate (IMP) concentrations reached maximum after 12 h storage (4.78 micromol g(-1)), whereas maximum IMP concentrations occurred immediately postharvest in the exhausted fillets (6.42 micromol g(-1)). After 36 h storage, K-values in exhausted fillets reached 52.11% compared to 19.27% in rested fillets. Rested harvesting of Chinook salmon improved the fillets' metabolic potential postharvest, extending metabolite depletion times, changing IMP concentrations and reducing K-values.

Oxygen-dependence of metabolic rate in the muscles of craniates.

We present evidence that oxygen consumption (VO2) is oxygen partial pressure (PO2)dependent in striated muscles and PO2-independent in the vasculature in representatives of three craniate taxa: two teleost fish, a hagfish and a rat. Blood vessel VO2 displayed varying degrees of independence in a PO2 range of 15-95 mmHg, while VO2 by striated muscle tissue slices from all species related linearly to PO2 between 0 and 125 mmHg, despite VO2 rates varying greatly between species and muscle type. In salmon red muscle, lactate concentrations fell in slices incubated at a PO2 of either 30 or 100 mmHg, suggesting aerobic rather than anaerobic metabolism. Consistent with this finding, potential energy, a proxy of ATP turnover, was PO2-dependent. Our data suggest that the reduction in VO2 with falling PO2 results in a decrease in ATP demand, suggesting that the hypoxic signal is sensed and cellular changes effected. Viability and diffusion limitation of the preparations were investigated using salmon cardiac and skeletal muscles. Following the initial PO2 depletion, reoxygenation of the Ringer bathing salmon cardiac muscle resulted in VO2S that was unchanged from the first run. VO2 increased in all muscles uncoupled with p-trifluoromethoxylphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP). Mitochondrial succinate dehydrogenase activity, quantified by reduction of 3-(4,5-dimethylthiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, was constant over the course of the experiment. These three findings indicate that the tissues remained viable over time and ruled out diffusion-limitation as a constraint on VO2.

Oxygen consumption, ventilation frequency and cytochrome c oxidase activity in blue cod (Parapercis colias) exposed to hydrogen sulphide or isoeugenol.

The effects of hydrogen sulphide (H(2)S) and isoeugenol exposure on activity, oxygen consumption (VO(2)), ventilation frequency (Vf) and cytochrome c oxidase activity in a teleost fish are reported. In H(2)S (200 microM Na(2)S) exposed animals VO(2) and Vf decreased significantly (both to 40% of resting) after 30 min, concurrent with a loss of equilibrium and narcosis. Post-flushing, VO(2) increased to resting values, but Vf remained depressed (P<0.05) until 30 min of recovery. Subsequently, equilibrium and mobility were regained accompanied by increases in VO(2) (66%) and Vf (15%) between 60-70 min of recovery. Isoeugenol (0.011 g L(-1)) exposed fish reached stage 4-5 of anaesthesia accompanied by decreases (P<0.05) in VO(2) (64%) and Vf (38%) by 35 min. Post-flushing, VO(2) and Vf recovered to resting values, followed by a rise (P<0.05) in VO(2) (45%) and Vf (25%). Overall, VO(2) in relation to the resting rate was reduced in isoeugenol treated animals. Conversely, VO(2) was increased (P<0.05) relative to the resting rate in H(2)S exposed fish. 20 and 200 microM Na(2)S reduced cytochrome c oxidase activity (P<0.05) in skeletal muscle and gill lamellae by between 69 and 97%, while isoeugenol had no effect in any tissue.

Lipid oxidation is inhibited by isoeugenol exposure in chinook Salmon (Oncorhynchus Tshawytscha) fillets during storage at 15 degrees C.

The method of harvest for farmed fish and the postharvest tissue metabolism can have a significant effect on the quality and storage stability of the resulting fillets. We have examined the effects of rested harvesting and isoeugenol exposure on tissue oxidation and the loss of tissue antioxidants in fillets of chinook salmon (Oncorhyncus tshawytscha) during storage at a normal metabolic temperature of 15 degrees C. Isoeugenol is a lipid soluble phenolic antioxidant used as an anesthetic in the aquaculture industry (AQUI-S). Fillets from salmon harvested in rested and exhausted physiological states with and without isoeugenol were prepared and stored in air at 15 degrees C for 96 h. Exposure to isoeugenol resulted in significantly decreased late-stage lipid peroxidation (TBARS) levels in the fillets during storage regardless of the harvest method. Protein carbonyl concentrations increased 73% in the fillets during storage (from 406 to 703 nmol/g wet weight) and were not affected by the harvest method. Fillet vitamin C concentrations decreased 92% (from 49 to 4 nmol/g wet weight) but were also not affected by the harvest method. Although significant late-stage lipid oxidation was observed with exhausted harvesting, no significant vitamin E loss was observed in any of the fillets during storage. Our results show that rested harvesting of chinook salmon does not affect their oxidative stability immediately postharvest and that isoeugenol can function as an antioxidant in fish fillets as it prevented late-stage lipid oxidation.

Cardiac chymase converts rat proAngiotensin-12 (PA12) to angiotensin II: effects of PA12 upon cardiac haemodynamics.

AIMS: The aim of this study was to observe the direct physiological and biochemical cardiac effects in response to a newly identified putative component of the renin-angiotensin system, proangiotensin-12 (PA12); and investigate whether PA12 can serve as a substrate for Angiotensin II (AngII) generation. METHODS AND RESULTS: The direct cardiac actions of PA12 and its role as a substrate for chymase-dependent AngII generation were investigated in Sprague-Dawley rats using an isolated heart model of cardiac ischaemia-reperfusion injury. PA12 potently constricted coronary arteries with no significant effect on left-ventricular contractility. PA12 impaired recovery from global ischaemia, maintaining coronary constriction and markedly increasing release of creatine kinase and troponin I (TnI), indicating greater myocardial injury. Analysis of perfusate collected after transcardiac passage revealed a marked increase in AngII production from hearts infused with PA12. Cardiac AngII production was not blocked by angiotensin-converting enzyme inhibitors, whereas inhibition of chymase with chymostatin significantly reduced AngII production and attenuated PA12-induced vasoconstriction and myocardial damage following ischaemia. Furthermore, Angiotensin II type 1 receptor (AT(1)R) blockade abolished PA12 activity. In vitro, PA12 was efficiently and precisely converted to AngII as assessed on reverse phase-high performance liquid chromatography coupled to tandem mass spectrometry. This conversion was blocked by chymostatin. CONCLUSION: PA12 may act as a circulating substrate for cardiac chymase-mediated AngII production, in contrast to ACE-mediated AngII production from AngI.

Oxygen consumption and blood flow distribution in perfused skeletal muscle of chinook salmon.

An isolated, perfused salmon tail preparation showed oxyconformance at low oxygen delivery rates. Addition of pig red blood cells to the perfusing solution at a haematocrit of 5 or 10% allowed the tail tissues to oxyregulate. Below ca. 60 ml O(2) kg(-1) h(-1) of oxygen delivery (DO(2)), VO(2) was delivery dependent. Above this value additional oxygen delivery did not increase VO(2) of resting muscle above ca. 35 ml O(2) kg(-1) h(-1). Following electrical stimulation, VO(2) increased to ca. 65 ml O(2) kg(-1) h(-1), with a critical DO(2) of ca. 150 ml O(2) kg(-1) h(-1). Dorsal aortic pressure fell to 69% of the pre-stimulation value after 5 min of stimulation and to 54% after 10 min. Microspheres were used to determine blood flow distribution (BFD) to red (RM) and white muscle (WM) within the perfused myotome. Mass specific BFD ratio at rest was found to be 4.03 +/- 0.49 (RM:WM). After 5 min of electrical stimulation the ratio did not change. Perfusion with saline containing the tetrazolium salt 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) revealed significantly more mitochondrial activity in RM. Formazan production from MTT was directly proportional to time of perfusion in both red and WM. The mitochondrial activity ratio (RM:WM) did not change over 90 min of perfusion.

Oxygen dependency of hydrogen sulfide-mediated vasoconstriction in cyclostome aortas.

Hydrogen sulfide (H(2)S) has been proposed to mediate hypoxic vasoconstriction (HVC), however, other studies suggest the vasoconstrictory effect indirectly results from an oxidation product of H(2)S. Here we examined the relationship between H(2)S and O(2) in isolated hagfish and lamprey vessels that exhibit profound hypoxic vasoconstriction. In myographic studies, H(2)S (Na(2)S) dose-dependently constricted dorsal aortas (DA) and efferent branchial arteries (EBA) but did not affect ventral aortas or afferent branchial arteries; effects similar to those produced by hypoxia. Sensitivity of H(2)S-mediated contraction in hagfish and lamprey DA was enhanced by hypoxia. HVC in hagfish DA was enhanced by the H(2)S precursor cysteine and inhibited by amino-oxyacetate, an inhibitor of the H(2)S-synthesizing enzyme, cystathionine beta-synthase. HVC was unaffected by propargyl glycine, an inhibitor of cystathionine lambda-lyase. Oxygen consumption (M(O(2))) of hagfish DA was constant between 15 and 115 mmHg P(O(2)) (1 mmHg=0.133 kPa), decreased when P(O(2)) <15 mmHg, and increased after P(O(2)) exceeded 115 mmHg. 10 micromol l(-1) H(2)S increased and > or =100 micromol l(-1) H(2)S decreased M(O(2)). Consistent with the effects on HVC, cysteine increased and amino-oxyacetate decreased M(O(2)). These results show that H(2)S is a monophasic vasoconstrictor of specific cyclostome vessels and because hagfish lack vascular NO, and vascular sensitivity to H(2)S was enhanced at low P(O(2)), it is unlikely that H(2)S contractions are mediated by either H(2)S-NO interaction or an oxidation product of H(2)S. These experiments also provide additional support for the hypothesis that the metabolism of H(2)S is involved in oxygen sensing/signal transduction in vertebrate vascular smooth muscle.

Different sensitivities of arteries and veins to vasoactive drugs in a hagfish, Eptatretus cirrhatus.

We used myography on five different arteries and three veins of the hagfish, Eptatretus cirrhatus, to test the response of vessels to vasoactive drugs. Concentration-response curves were generated for carbachol, endothelin-1, arginine vasotocin and the adrenergic agonists, phenylephrine and isoprenaline. pEC50 values indicated that veins were more sensitive to endothelin-1 than were arteries, but the arteries were more sensitive to the cholinergic agonist, carbachol. Segmental arteries did not react to arginine vasotocin, but all other vessels did, and on a molar basis it was the most potent agonist tested. That ventral and dorsal aortas were more sensitive to arginine vasotocin than smaller vessels might indicate that this neurohypophysial peptide has the potential to exert a profound influence on branchial vascular resistance and cardiac output in hagfishes. The results also demonstrate the potential for a variety of endogenous peptides to contribute to central venous tone.

Effects of potential mediators of an intestinal brake mechanism on gut motility in Chinook salmon (Oncorhynchus tshawytscha).

Potential humoral factors controlling an intestinal brake mechanism in Chinook salmon were characterised in terms of their effect on frequency and amplitude of spontaneous contractions in gastrointestinal (GI) rings. Concentration-response curves of gut contractility were produced for cholecystokinin-8 (CCK-8), gastrin-1, glucagon-like peptide-1 (GLP-1) and 5-hydroxytryptamine (5-HT) using gut rings from cardiac stomach (CS), pyloric stomach (PY), pyloric sphincter (Psp) and intestine (Int). Calculated log10 molar (M) EC50 values for CCK-8 (n=7) were: CS -8.15+/-0.90, PY -7.88+/-0.48, Psp -8.98+/-0.68, Int -8.93+/-0.64. Log10 M EC50 values calculated for gastrin 1 (n=7) were: CS -12.45+/-0.66, PY -12.55+/-0.63, Psp -9.35+/-0.78, Int -12.69+/-1.12. Log10 M EC50 values calculated for 5-HT (n=6) were: CS -4.78+/-1.05 and Psp -6.18+/-1.14. GLP -1 (n=4) produced no response in any of the tissues examined. Spontaneous contractions, measured as spikes per minute and the peak force generated were also measured for each hormone-tissue combination. The Psp generated the greatest mass-specific force, with stomach rings generating the least force. Dilutions of serum from fish diagnosed with gastric dilation air sacculitis (GDAS +ve) increased gut contractility compared to controls (GDAS -ve).

Cardiovascular changes and catecholamine release following anaesthesia in Chinook salmon (Oncorhynchus tshawytscha) and snapper (Pagrus auratus).

We investigated recovery from anaesthesia in Chinook salmon (Oncorhynchus tshawytscha) with and without surgery. Fish either underwent light sedation on exposure to 60 ppm AQUI-S or surgical depth anaesthesia with 120 ppm AQUI-S. Surgical depth anaesthesia experiments were replicated using New Zealand snapper (Pagrus auratus). During light sedation, there was no evidence of catecholamine release in salmon despite changes in heart rate and blood pressure. Following surgical anaesthesia both salmon and snapper released high concentrations of catecholamines into the circulation. Plasma half-life of adrenaline in salmon was 9.3+/-0.7 min (n = 7) and in snapper was 4.4+/-3.3 min (n = 7). There was no further release of catecholamines, despite attempts by both species to escape their enclosures. Though clearance of the catecholamines was rapid, the cardiovascular effects of anaesthesia were prolonged. Dorsal aortic blood pressure (P(DA)) and heart rate (HR) were high following anaesthesia, falling by 60 min in the 60 ppm exposed salmon but remaining high in the 120 ppm group. Following anaesthesia ventral aorta blood pressure (P(VA)) in snapper was positively correlated with HR, as was P(DA) and haematocrit in salmon. Recovery of cardiovascular control processes is prolonged in recovery from anaesthesia if the fish become hypoxic.

Cardiovascular responses of Chinook salmon (Oncorhynchus tshawytscha) during rapid anaesthetic induction and recovery.

The effects of three anaesthetics on induction and recovery were compared in Chinook salmon (Oncorhynchus tshawytscha). Heart rate (HR), cardiac output (Q), dorsal aortic pressure (DAP) and stroke volume (SV) were measured in minimally disturbed salmon during 5 min anaesthetic inductions with approximately equi-potent concentrations of MS222 (100 ppm), metomidate (6-10 ppm) and AQUI-S (60 ppm). MS222 induction caused a steady decline in DAP only, while metomidate induction did not affect any cardiovascular variable. AQUI-S caused a biphasic response, and within 2 min had depressed HR, Q, DAP and SV by between 20 and 50%. In the final 3 min HR returned to pre-anaesthesia levels, and Q and SV climbed to greater than pre-anaesthesia levels. Blood samples taken pre- and post-anaesthesia showed all inductions caused hypoxaemia (oxygen partial pressure of dorsal aortic blood (PaO2): MS222 47 mmHg, metomidate 35 mmHg, AQUI-S 21 mmHg). Haematocrit and plasma adrenaline and noradrenaline levels increased slightly in AQUI-S treated fish only. Recovery was monitored for 6 h post-anaesthesia, and was similar for each anaesthetic. All cardiovascular variables had returned to control levels within 5 min with the exception of DAP, which was initially slightly elevated (up to 20%) but returned to control values within 30 min. Anaesthesia is usually preceded by handling. Netting prior to anaesthesia caused significant increases in HR, Q and SV, which masked any anaesthetic dependent effects. Recovery from anaesthesia combined with surgery was also generally anaesthetic independent and recovery was prolonged, compared to anaesthesia alone. These data suggest limiting fish handling/manipulation is more important in minimising cardiovascular disturbance than the choice of anaesthetic.

STATISTICAL COVARIANCE AS A MEASURE OF PHYLOGENETIC RELATIONSHIP.

Abstract- The method of parsimony in phylogenetic inference is often taken to mean two things: (1) that one should favor the genealogical hypothesis that minimizes the required number of homoplasies (matchings of independently evolved derived character states), and (2) that symplesiomorphies (matchings of primitive character states) have little or no evidential value for phylogenetic relationship. This paper shows both theses to be false by undermining recent likelihood arguments for them and by providing a more secure likelihood proof of a new method, which is incompatible with both (1) and (2).