Acute Stress in Lesser-Spotted Catshark (Scyliorhinus canicula Linnaeus, 1758) Promotes Amino Acid Catabolism and Osmoregulatory Imbalances.

Acute-stress situations in vertebrates induce a series of physiological responses to cope with the event. While common secondary stress responses include increased catabolism and osmoregulatory imbalances, specific processes depend on the taxa. In this sense, these processes are still largely unknown in ancient vertebrates such as marine elasmobranchs. Thus, we challenged the lesser spotted catshark (Scyliorhinus canicula) to 18 min of air exposure, and monitored their recovery after 0, 5, and 24 h. This study describes amino acid turnover in the liver, white muscle, gills, and rectal gland, and plasma parameters related to energy metabolism and osmoregulatory imbalances. Catsharks rely on white muscle amino acid catabolism to face the energy demand imposed by the stressor, producing NH4+. While some plasma ions (K+, Cl- and Ca2+) increased in concentration after 18 min of air exposure, returning to basal values after 5 h of recovery, Na+ increased after just 5 h of recovery, coinciding with a decrease in plasma NH4+. These changes were accompanied by increased activity of a branchial amiloride-sensitive ATPase. Therefore, we hypothesize that this enzyme may be a Na+/H+ exchanger (NHE) related to NH4+ excretion. The action of an omeprazole-sensitive ATPase, putatively associated to a H+/K+-ATPase (HKA), is also affected by these allostatic processes. Some complementary experiments were carried out to delve a little deeper into the possible branchial enzymes sensitive to amiloride, including in vivo and ex vivo approaches, and partial sequencing of a nhe1 in the gills. This study describes the possible presence of an HKA enzyme in the rectal gland, as well as a NHE in the gills, highlighting the importance of understanding the relationship between acute stress and osmoregulation in elasmobranchs.

The isosmotic point as critical salinity limit for growth and osmoregulation, but not survival, in the wolf eel Anarrhichthys ocellatus.

Members of wolf fish family Anarhichadidae have emerged as potential cold-water marine aquaculture species. This study examined growth performance and osmoregulation in juvenile wolf eel (Anarrhichthys ocellatus) held in a series of dilute salinities (30, 14, 9, and 6 ‰) over an 8-week trial. At the conclusion of the growth study, fish were sampled for analysis of gill and intestine enzyme activity, plasma ion content, and muscle moisture. Growth rate remained positive in all salinities throughout the 8-week trial. Specific growth rate was maintained above 3.0% mass day-1 at salinities of 30 and 14 ‰, but was significantly reduced at 9 (2.9% mass day-1) and 6 ‰ (2.0% mass day-1). Muscle water content increased with increasing salinity dilution (77.9% water in 30 ‰; 79.8% water in 6 ‰), and plasma osmolality (~ 320 mOsm kg-1) was maintained in salinities as dilute as 9 ‰ but was significantly lower (~ 280 mOsm kg-1) in the most dilute salinity of 6 ‰. Segmental linear regression analyses revealed that the calculated isosmotic point for wolf eel of ~ 10.6 ‰ was a critical limit for maintaining growth performance and osmoregulatory homeostasis. It is an important finding that fish considered to be a typical marine stenohaline organism could maintain ion and water balance as low as the isosmotic point, and exhibit survival and positive growth rates in salinities as dilute as 6 ‰. This work delivers a fundamental step in the empirical examination of this emerging aquaculture species and provides a model for evaluating osmoregulatory performance of marine stenohaline fishes in low-salinity aquaculture.

Interaction of osmoregulatory and acid-base compensation in white sturgeon (Acipenser transmontanus) during exposure to aquatic hypercarbia and elevated salinity.

Migratory fishes encounter a variety of environmental conditions, including changes in salinity, temperature and dissolved gases, and it is important to understand how these fishes are able to acclimate to multiple environmental stressors. The gill is the primary site of both acid-base balance and ion regulation in fishes. Many ion transport mechanisms involved with acid-base compensation are also required for the regulation of plasma Na(+) and Cl(+), the predominant extracellular ions, potentially resulting in a strong interaction between ionoregulation and acid-base regulation. The present study examined the physiological interaction of elevated dissolved CO2 (an acid-base disturbance) on osmoregulation during seawater acclimation (an ionoregulatory disturbance) in juvenile white sturgeon (Acipenser transmontanus). Blood pH (pHe), plasma [HCO3 (-)], [Na(+)], [Cl(-)] and osmolality, white muscle water content, and gill Na(+)/K(+)-ATPase (NKA) and Na(+)/K(+)/2Cl(-) co-transporter (NKCC) abundance were examined over a 10 day seawater (SW) acclimation period under normocarbia (NCSW) or during prior and continued exposure to hypercarbia (HCSW), and compared with a normocarbic freshwater (NCFW) control. Hypercarbia induced a severe extracellular acidosis (from pH 7.65 to pH 7.2) in HCSW sturgeon, and these fish had a 2-fold greater rise in plasma osmolarity over NCSW by day 2 of SW exposure. Interestingly, pHe recovery in HCSW was associated more prominently with an elevation in plasma Na(+) prior to osmotic recovery and more prominently with a reduction in plasma Cl(-) following osmotic recovery, indicating a biphasic response as the requirements of osmoregulation transitioned from ion-uptake to ion-excretion throughout SW acclimation. These results imply a prioritization of osmoregulatory recovery over acid-base recovery in this period of combined exposure to acid-base and ionoregulatory disturbances.

Interaction of osmoregulatory and acid-base compensation in white sturgeon (Acipenser transmontanus) during exposure to aquatic hypercarbia and elevated salinity.

Migratory fishes encounter a variety of environmental conditions, including changes in salinity, temperature, and dissolved gases, and it is important to understand how these fishes are able to acclimate to multiple environmental stressors. The gill is the primary site of both acid-base balance and ion regulation in fishes. Many ion transport mechanisms involved with acid-base compensation are also required for the regulation of plasma Na+ and Cl+, the predominant extracellular ions, potentially resulting in a strong interaction between iono- and acid-base regulation. The present study examined the physiological interaction of elevated dissolved CO2 (an acid-base disturbance) on osmoregulation during seawater acclimation (an ionoregulatory disturbance) in juvenile white sturgeon (Acipenser transmontanus). Blood pH (pHe), plasma [HCO3-], [Na+], [Cl-], and osmolality, white muscle water content, and gill Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC) abundance were examined over a 10-day seawater (SW) acclimation period under normocarbia (NCSW) or during prior and continued exposure to hypercarbia (HCSW), and compared to a normocarbic freshwater (NCFW) control. Hypercarbia induced a severe extracellular acidosis (from pH 7.65 to pH 7.2) in HCSW sturgeon, and these fish had a 2-fold greater rise in plasma osmolarity over NCSW by day 2 of SW exposure. Interestingly, pHe recovery in HCSW was associated more prominently with an elevation in plasma Na+ prior to osmotic recovery and more prominently with a reduction in plasma Cl- following osmotic recovery, indicating a biphasic response as the requirements of osmoregulation transitioned from ion-uptake to ion-excretion throughout SW acclimation. These results imply a prioritization of osmoregulatory recovery over acid-base recovery in this period of combined exposure to acid-base and ionoregulatory disturbances.

Differential expression of Na+, K(+)-ATPase α-1 isoforms during seawater acclimation in the amphidromous galaxiid fish Galaxias maculatus.

Inanga (Galaxias maculatus) is an amphidromous fish with a well-known capacity to withstand a wide range of environmental salinities. To investigate the molecular mechanisms facilitating acclimation of inanga to seawater, several isoforms of the Na(+), K(+)-ATPase ion transporter were identified. This included three α-1 (a, b and c), an α-2 and two α-3 (a and b) isoforms. Phylogenetic analysis showed that the inanga α-1a and α-1b formed a clade with the α-1a and α-1b isoforms of rainbow trout, while another clade contained the α-1c isoforms of these species. The expression of all the α-1 isoforms was modulated after seawater exposure (28‰). In gills, the expression of the α-1a isoform was progressively down-regulated after seawater exposure, while the expression of the α-1b isoform was up-regulated. The α-1c isoform behaved similarly to the α-1a, although changes were less dramatic. Physiological indicators of salinity acclimation matched the time frame of the changes observed at the molecular level. A 24-h osmotic shock period was highlighted by small increases in plasma osmolality, plasma Na(+) and a decrease in muscle tissue water content. Thereafter, these values returned close to their pre-exposure (freshwater) values. Na(+), K(+)-ATPase activity showed a decreasing trend over the first 72 h following seawater exposure, but activity increased after 240 h. Our results indicate that inanga is an excellent osmoregulator, an ability that is conferred by the rapid activation of physiological and molecular responses to salinity change.

Changes in gill H+-ATPase and Na+/K+-ATPase expression and activity during freshwater acclimation of Atlantic salmon (Salmo salar).

Few studies have examined changes in salmon gill ion transporter expression during the transition from seawater to freshwater, a pivotal moment in the salmonid life cycle. Seawater-acclimated Atlantic salmon were transferred to freshwater and blood and gill tissue were sampled over 30 days of acclimation. Salmon held in seawater had stable plasma osmolality and sodium and chloride levels throughout the experiment. Following freshwater exposure, plasma sodium and chloride levels and total osmolality decreased significantly before returning towards control levels over time. Gill H(+)-ATPase activity increased by more than 45% 14 days after exposure to freshwater, whereas H(+)-ATPase mRNA levels were not affected by the salinity change. Within 4 days of freshwater exposure, gill Na(+)/K(+)-ATPase activity increased ∼43% over control levels, remaining significantly higher until the 30 day sampling group when it declined back to control levels. This increase in activity was associated with a more than 7-fold increase in Na(+)/K(+)-ATPase isoform α1a mRNA level and a ∼60% decrease in Na(+)/K(+)-ATPase isoform ß1b mRNA level. The mRNA levels of Na(+)/K(+)-ATPase isoforms α1c and α3 did not change as a result of freshwater exposure. The time courses for mRNA expression of the small membrane protein FXYD 11 and the ß1-subunit were very similar, with levels increasing significantly 7 days following freshwater exposure before subsiding back to control levels at 30 days. Taken together, these data suggest an important role for Na(+)/K(+)-ATPase in freshwater acclimation in Atlantic salmon.

Should I stay or should I go? The Ectodysplasin locus is associated with behavioural differences in threespine stickleback.

Adaptive divergence may be facilitated if morphological and behavioural traits associated with local adaptation share the same genetic basis. It is therefore important to determine whether genes underlying adaptive morphological traits are associated with variation in behaviour in natural populations. Positive selection on low-armour alleles at the Ectodysplasin (Eda) locus in threespine stickleback has led to the repeated evolution of reduced armour, following freshwater colonization by fully armoured marine sticklebacks. This adaptive divergence in armour between marine and freshwater populations would be facilitated if the low allele conferred a behavioural preference for freshwater environments. We experimentally tested whether the low allele is associated with preference for freshwater by measuring the preference of each Eda genotype for freshwater versus saltwater after acclimation to either salinity. We found no association between the Eda low allele and preference for freshwater. Instead, the low allele was significantly associated with a reduced preference for the acclimation environment. This behaviour may facilitate the colonization of freshwater habitats from the sea, but could also hinder local adaptation by promoting migration of low alleles between marine and freshwater environments.

Sodium pump localization in epithelia.

In epithelial cells, the sodium pump, in coordination with several other ion transporting proteins and channels, acts to regulate directional water and ion flux across the epithelial barrier. This function is dependant on the polarized localization of the sodium pump to a single plasma membrane domain. In most epithelial cell types the sodium pump is found in an exclusively basolateral position. Despite the clear importance of maintaining a polarized distribution of the sodium pump, surprisingly little is known about the specific mechanisms responsible for the targeting and trafficking of the sodium pump to the basolateral surface. We briefly discuss our current understanding of factors which may act to regulate the cellular distribution of the sodium pump, including the potential role of the sodium pump beta-subunit. Several previous, studies have suggested that the expression of the beta2 isoform (instead of beta1) may cause the apical localization of the sodium pump. This appeared to be confirmed by Wilson et al. Am J Pathol, 156: 253-268, 2000 who found that MDCK cells stably transfected with the beta2 subunit express the sodium pump at the apical surface. However, careful examination by Laughery et al.,Am J Physiol, 292: F1718-F1725, 2007, showed that the apical targeting of the pump was caused by the presence of butyrate in the cell growth media and was not due to the presence of the beta2 isoform. These findings are discussed below, along with potential explanations as to how butyrate may influence the polarity of the sodium pump in epithelial cells.

Temperature effects on trimethylamine oxide accumulation and the relationship between plasma concentration and tissue levels in smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) were maintained in a long term acclimation study to elucidate temperature effects on the accumulation of trimethylamine oxide (TMAO) and to determine if the activity of trimethylamine oxidase (TMAoxi) plays a role in modulating the seasonally variable levels of TMAO. In the same experiment, the TMAO content was determined for several tissues at varying plasma TMAO concentrations. TMAO accumulation begins at 5-7 degrees C, well above that which might be normally associated with an antifreeze response. The plasma concentration reached a plateau of 20 mM as temperatures reached 0 degrees C. Plasma TMAO concentration drops to pre-accumulation levels, less than 5 mM, when fish are held at elevated temperature (8-11 degrees C) and increases when fish are chilled below ambient seawater temperatures. However, despite temperatures near or below 0 degrees C, plasma TMAO decreases after the winter season. Changes in TMAoxi activity do not correlate with TMAO levels, suggesting that the activity of this enzyme does not play a key role in regulating TMAO concentrations in smelt. For the first time in any teleost fish, tissue TMAO contents in liver, kidney, brain, and intestine were found to strongly correlate with plasma TMAO concentrations. For these tissues, the intracellular and extracellular concentration of TMAO appears to be approximately equal. Conversely, the heart and white muscle accumulate TMAO, and in the case of white muscle, intracellular concentration is maintained at a constant level of approximately 35 mmol/kg, despite fluctuating plasma concentrations over a range from 0 to over 25 mM.

Na+/K+-ATPase alpha-isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer.

We identified five Na+/K+-ATPase alpha-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate alpha1 isoforms (designated alpha1a, alpha1b and alpha1c), one isoform was closely related to alpha2 isoforms (designated alpha2) and the fifth was closely related to alpha3 isoforms (designated alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase alpha-isoforms were expressed in trout gills (alpha1a, alpha1b, alpha1c and alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase alpha1a and alpha1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPase alpha1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase alpha1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase alpha1a- and alpha1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.