Effects of temperature on acid-base regulation, gill ventilation and air breathing in the clown knifefish, Chitala ornata.

Chitala ornata is a facultative air-breathing fish, which at low temperatures shows an arterial PCO2  (PaCO2 ) level only slightly elevated above that of water breathers. By holding fish with in-dwelling catheters at temperatures from 25 to 36°C and measuring blood gasses, we show that this animal follows the ubiquitous poikilotherm pattern of reducing arterial pH with increasing temperature. Surprisingly, the temperature increase caused an elevation of PaCO2  from 5 to 12 mmHg while the plasma bicarbonate concentration remained constant at around 8 mmol l-1 The temperature increase also gave rise to a larger fractional increase in air breathing than in gill ventilation frequency. These findings suggest that air breathing, and hence the partitioning of gas exchange, is to some extent regulated by acid-base status in air-breathing fish and that these bimodal breathers will be increasingly likely to adopt respiratory pH control as temperature rises, providing an interesting avenue for future research.

Angiotensin II dependent cardiac remodeling in the eel Anguilla anguilla involves the NOS/NO system.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System (RAS), plays an important role in controlling mammalian cardiac morpho-functional remodelling. In the eel Anguilla anguilla, one month administration of AngII improves cardiac performance and influences the expression and localization of molecules which regulate cell growth. To deeper investigate the morpho-functional chronic influences of AngII on the eel heart and the molecular mechanisms involved, freshwater eels (A. anguilla) were intraperitoneally injected for 2 months with AngII (1 nmol g BW-1). Then the isolated hearts were subjected to morphological and western blotting analyses, and nitrite measurements. If compared to control animals, the ventricle of AngII-treated hearts showed an increase in compacta thickness, vascularization, muscle mass and fibrosis. Structural changes were paralleled by a higher expression of AT2 receptor and a negative modulation of the ERK1-2 pathway, together with a decrease in nitrite concentration, indicative of a reduced Nitric Oxide Synthase (NOS)-dependent NO production. Moreover, immunolocalization revealed, particularly on the endocardial endothelium (EE) of AngII-treated hearts, a significant reduction of phosphorylated NOS detected by peNOS antibody accompanied by an increased expression of the eNOS disabling protein NOSTRIN, and a decreased expression of the positive regulators of NOS activity, pAkt and Hsp90. On the whole, results suggest that, in the eel, AngII modulates cardiac morpho-functional plasticity by influencing the molecular mechanisms that control NOS activity and the ERK1-2 pathway.

Impact and tissue metabolism of nitrite at two acclimation temperatures in striped catfish (Pangasianodon hypophthalmus).

Elevated concentrations of nitrite develop occasionally in various aquatic habitats and aquaculture facilities, providing a potential danger for freshwater fish that take up nitrite via the gill chloride uptake mechanism. We studied the uptake, effects and metabolism of nitrite in blood, heart and skeletal muscle at two temperatures in striped catfish Pangasianodon hypophthalmus, a facultative air-breathing fish that is heavily cultivated in Southeast Asia. Exposure to 0.8 mM ambient nitrite increased blood [nitrite] and [methaemoglobin] (metHb) to high values at day 1, but values subsequently decreased towards controls at day 7. Blood [nitrite] and metHb content were unexpectedly higher at 27 °C (∼1.2 mM; 69% at day 1) than at 33 °C (∼0.9 mM; 55%), reflecting a lower nitrite uptake at the highest temperature, possibly via an increased reliance on air-breathing relative to water-breathing with temperature increase. A large fraction of the nitrite taken up was effectively eliminated by being detoxified to nitrate. Further, erythrocyte metHb reductase activity was increased during nitrite exposure, efficiently reducing metHb to functional haemoglobin. The uptake of nitrite into white skeletal musculature (main part of the fish) was much lower than into heart tissue. While heart [nitrite] was close to blood plasma levels, muscle [nitrite] peaked at ∼0.2 mM at day 1 and subsequently declined to ∼0.05 mM at day 7, which is below levels reported in various commercial cured meat products. Nitrite was partly metabolized to iron-nitrosyl, S-nitroso and N-nitroso compounds. The increase in nitros(yl)ated compounds was marginal in skeletal muscle and more pronounced in heart tissue.

The effects of elevated environmental CO2 on nitrite uptake in the air-breathing clown knifefish, Chitala ornata.

Nitrite and carbon dioxide are common environmental contaminants in the intensive aquaculture ponds used to farm clown knifefish (Chitala ornata) in the Mekong delta, Vietnam. Here we tested the hypothesis that hypercapnia reduces nitrite uptake across the gills, because pH regulation will reduce chloride uptake and hence nitrite uptake as the two ions compete for the same transport route via the branchial HCO3-/Cl- exchanger. Fish fitted with arterial catheters were exposed to normocapnic/normoxic water (control), nitrite (1 mM), hypercapnia (21 mmHg CO2), or combined hypercapnia (acclimated hypercapnia) and nitrite for 96 h. Blood was sampled to measure acid-base status, haemoglobin derivatives and plasma ions. Plasma nitrite increased for 48 h, but levels stayed below the exposure concentration, and subsequently decreased as a result of nitrite detoxification to nitrate. The total uptake of nitrite (evaluated as [NO2-] + [NO3-]) was significantly decreased in hypercapnia, in accordance with the hypothesis. Methemoglobin and nitrosylhemoglobin levels were similarly lower during hypercapnic compared to normocapnic nitrite exposure. The respiratory acidosis induced by hypercapnia was half-compensated by bicarbonate accumulation in 96 h, which was mainly chloride-mediated (i.e. reduced Cl- influx via the branchial HCO3-/Cl- exchanger). Plasma osmolality and main ions (Na+, Cl-) were significantly decreased by hypercapnia and by nitrite exposure, consistent with inhibition of active transport. We conclude that hypercapnia induces a long-lasting, and mainly chloride-mediated acid-base regulation that reduces the uptake of nitrite across the gills.

Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to up-regulation of methaemoglobin reductase activity.

The clown knifefish is a facultative air breather, which is widely farmed in freshwater ponds in Vietnam. Here we report a very high nitrite tolerance (96h LC50 of 7.82mM) in this species and examine the effects of 1mM (LC5) and 2.5mM (LC10) ambient nitrite on haemoglobin (Hb) derivatives, electrolyte levels, acid-base status, and total body water content during 7days of exposure. Furthermore, we tested the hypothesis that erythrocyte methaemoglobin (metHb) reductase activity is upregulated by nitrite exposure. Plasma nitrite levels increased for 2-3days but stayed below environmental levels and fell towards control values during the last half of the exposure period. Plasma nitrate, in contrast, rose continuously, reflecting detoxification of nitrite to nitrate. MetHb generated from the reaction between nitrite and erythrocyte Hb reached 38% at day 2, but then decreased to 17% by the end of experiment. The first order rate constant for metHb reduction by erythrocyte metHb reductase increased from 0.01 in controls to 0.046min-1 after 6days of nitrite exposure, showing up-regulation of this enzyme. While such upregulation has been suggested in nitrite-exposed fish species, this study provides the first experimental evidence.