Hyperosmotic Environment Research Articles

Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium model alter in response to acute seawater exposure

Gill epithelium permeability and qualitative/quantitative aspects of gill claudin (cldn) tight junction (TJ) protein transcriptomics were examined with a primary cultured model gill epithelium developed using euryhaline puffer fish (Tetraodon nigroviridis) gills. The model was prepared using seawater-acclimated fish gills and was cultured on permeable cell culture filter supports. The model is composed of 1–2 confluent layers of gill pavement cells (PVCs), with the outer layer exhibiting prominent apical surface microridges and TJs between adjacent cells. During development of electrophysiological characteristics, the model exhibits a sigmoidal increase in transpithelial resistance (TER) and plateaus around 30kΩcm2. At this point paracellular movement of [3H]polyethylene glycol (PEG) 4000 was low at ~1.75cms−1×10−7. When exposed to apical seawater (SW) epithelia exhibit a marked decrease in TER while PEG flux remained unchanged for at least 6h. In association with this, transcript encoding cldn TJ proteins cldn3c, -23b, -27a, -27c, -32a and -33b increased during the first 6h while cldn11a decreased. This suggests that these proteins are involved in maintaining barrier properties between gill PVCs of SW fishes. Gill cldn mRNA abundance also altered 6 and 12h following abrupt SW exposure of puffer fish, but in a manner that differed qualitatively and quantitatively from the cultured model. This most likely reflects the cellular heterogeneity of whole tissue and/or the contribution of the endocrine system in intact fish. The current study provides insight into the physiological and transcriptomic response of euryhaline fish gill cells to a hyperosmotic environment.

Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

BackgroundMarine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9) comparing the lineages of cetaceans and terrestrial mammals.ResultsOur results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182), whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45), AQP4 (74), AQP7 (342, 343, 356) was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater.ConclusionsOur results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

Effects on the metabolism, growth, digestive capacity and osmoregulation of juvenile of Sub-Antarctic Notothenioid fish Eleginops maclovinus acclimated at different salinities

In this study we assessed the influence of three different environmental salinities (5, 15 and 31 psu during 90 days) on growth, osmoregulation, energy metabolism and digestive capacity in juveniles of the Notothenioid fish Eleginops maclovinus. At the end of experimental time samples of plasma, liver, gill, intestine, kidney, skeletal muscle, stomach and pyloric caeca were obtained. Growth, weight gain, hepatosomatic index and specific growth rate increased at 15 and 31 psu and were lower at 5 psu salinity. Gill Na(+), K(+)-ATPase (NKA) activity presented a "U-shaped" relationship respect to salinity, with its minimum rates at 15 psu, while this activity correlated negatively with salinity at both anterior and posterior intestinal portions. No significant changes in NKA activity were observed in kidney or mid intestine. Large changes in plasma, metabolite levels and enzymatic activities related to energy metabolism in liver, gill, intestine, kidney and muscle were generally found in the groups exposed to 5 and 31 psu compared to the 15 psu group. Only the pepsin activity (digestive enzymes) assessed enhanced with environmental salinity, while pyloric caeca trypsin/chymotrypsin ratio decreased. This study suggests that juvenile of E. maclovinus presents greater growth near its iso-osmotic point (15 psu) and hyperosmotic environment (31 psu). Acclimation to low salinity increased the osmoregulatory expenditure as seen by the gill and anterior intestine results, while at high salinity, branchial osmoregulatory activity was also enhanced. This requires the mobilization of lipid stores and amino acids, thereby holding the growth of fish back. The subsequent reallocation of energy sources was not sufficient to maintain the growth rate of fish exposed to 5 psu. Thus, E. maclovinus juveniles present better growth efficiencies in salinities above the iso-osmotic point and hyperosmotic environment of this species, showing their best performance at 15 psu as seen by the main osmoregulatory and energy metabolism enzymatic activities.

Short-term response of different Saccharomyces cerevisiae strains to hyperosmotic stress caused by inoculation in grape must: RT-qPCR study and metabolite analysis

During the winemaking process, glycerol synthesis represents the first adaption response of Saccharomyces cerevisiae to osmotic stress after inoculation in grape must. We have implemented an RT-qPCR (Reverse Transcription-quantitative PCR) methodology with a preventive evaluation of candidate reference genes, to study six target genes related to glycerol synthesis (GPD1, GPD2, GPP2 and GPP1) and flux (STL1 and FPS1), and three ALD genes coding for aldehyde dehydrogenase involved in redox equilibrium via acetate production. The mRNA level in three strains, characterized by different metabolite production, was monitored in the first 120 min from inoculation into natural grape must. Expression analysis shows a transient response of genes GPD1, GPD2, GPP2, GPP1 and STL1 with differences among strains in term of mRNA abundance, while FPS1 was expressed constitutively. The transient response and different expression intensity among strains, in relation to the intracellular glycerol accumulation pattern, prove the negative feedback control via the HOG (High Osmolarity Glycerol) signalling pathway in S. cerevisiae wine strains under winery conditions. Among the ALD genes, only ALD6 was moderately induced in the hyperosmotic environment but not in all strains tested, while ALD3 and ALD4 were drastically glucose repressed. The intensity of transcription of ALD6 and ALD3 seems to be related to different acetate production found among the strains.

Na⁺/K⁺-ATPase α1 mRNA expression in the gill and rectal gland of the Atlantic stingray, Dasyatis sabina, following acclimation to increased salinity.

BackgroundThe salt-secreting rectal gland plays a major role in elasmobranch osmoregulation, facilitating ion balance in hyperosmotic environments in a manner analogous to the teleost gill. Several studies have examined the central role of the sodium pump Na+/K+-ATPase in osmoregulatory tissues of euryhaline elasmobranch species, including regulation of Na+/K+-ATPase activity and abundance in response to salinity acclimation. However, while the transcriptional regulation of Na+/K+-ATPase in the teleost gill has been well documented the potential for mRNA regulation to facilitate rectal gland plasticity during salinity acclimation in elasmobranchs has not been examined. Therefore, in this study we acclimated Atlantic stingrays, Dasyatis sabina (Lesueur) from 11 to 34 ppt salinity over 3 days, and examined changes in plasma components as well as gill and rectal gland Na+/K+-ATPase α1 (atp1a1) mRNA expression.ResultsAcclimation to increased salinity did not affect hematocrit but resulted in significant increases in plasma osmolality, chloride and urea. Rectal gland atp1a1 mRNA expression was higher in 34 ppt-acclimated D. sabina vs. controls. There was no significant change in gill atp1a1 mRNA expression, however mRNA expression of this gene in the gill and rectal gland were negatively correlated.ConclusionsThis study demonstrates regulation of atp1a1 in the elasmobranch salt-secreting gland in response to salinity acclimation and a negative relationship between rectal gland and gill atp1a1 expression. These results support the hypothesis that the gill and rectal gland play opposing roles in ion balance with the gill potentially facilitating ion uptake in hypoosmotic environments. Future studies should further examine this possibility as well as potential differences in the regulation of Na+/K+-ATPase gene expression between euryhaline and stenohaline elasmobranch species.

Genetic similarity of the Hainan medaka populations collected from hyper- and hypo-osmotic environments in northern Vietnam

Ricefishes of the genus Oryzias, including Japanese medaka (O. latipes), are known as excellent model organisms for studies in various fields of science. Some species of the genus inhabit brackish water, and such species are recognized to be useful to investigate physiological phenomena in seawater. However, only a limited number of species have been recorded from brackish waters. In addition, there is no information about the genetic relationship among populations inhabiting sites with different salinities. Here we report the discovery of Oryzias fish in two locations near Haiphong, northern Vietnam, a brackish mangrove planting area and a freshwater pond. A phylogenetic analysis using mitochondrial 12S and 16S ribosomal RNA (rRNA) gene sequences indicated that the fish from the two localities are the same species, Hainan medaka, O. curvinotus. Population genetic analysis using the mitochondrial 12S and 16S rRNA gene sequences revealed a close genetic relationship between the two populations. These results suggest that O. curvinotus is adaptable to both hyperosmotic and hypoosmotic environments. Due to its osmotic adaptability and ease of rearing in the laboratory, this species is expected to become a model for marine environmental and toxicological studies, as well as for studies of osmotic adaptation mechanisms.

Enhanced D-arabitol production by Zygosaccharomyces rouxii JM-C46: isolation of strains and process of repeated-batch fermentation.

A new strain producing high yield of D-arabitol was isolated from hyperosmotic environments and the ITS rDNA sequencing analysis revealed it as Zygosaccharomyces rouxii. In addition, using a pH control and repeated-batch fermentation strategy in a 5-L reactor, the maximum yield and the highest volumetric productivity of D-arabitol were 93.48 ± 2.79 g/L and 1.143 g/L h, respectively. Volumetric productivity was successfully improved from 0.86 to 1.143 g/L h, which was increased by 32.9 % after 72 h of fermentation. Z. rouxii JM-C46 has potential to be used for D-arabitol and xylitol production from glucose via D-arabitol route.

Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana

The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in osmotic conditions disrupt the equilibrium of the plasma membrane: hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root meristem cells. Acute hyperosmotic treatments (ionic and nonionic) enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presumably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin-mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

Morphofunctional remodelling of the osmoregulatory system in starred sturgeon Acipenser stellatus (Acipenseridae) during transition from hyperosmotic to hypoosmotic regulation

Morphological changes in the complex of functionally linked organs responsible for maintaining the osmotic homeostasis in immature euryhaline acipenserid species—starred sturgeon Acipenser stellatus—in the process of adaptation to hyperosmotic environmental salinity of 12.5–14.6‰ (403–472 mOsm/L) have been studied with the following characteristics: dynamics of blood serum osmolarity, morphofunctional changes in the neuroendocrine structures (preoptic and tuberal nuclei in the hypothalamus, adrenocorticotropic cells of hypophysis), peripheral endocrine glands (thyroid and interrenal), effector organs (chloride cells of gills and kidney), dynamics of concentrations of cortisol and thyroid hormones (T3 and T4) in the blood serum, activity of Na+/K+ ATPase in homogenates of gills, kidney, and directly in chloride cells. Transition from hyperosmotic (in fresh water) to hypoosmotic (in hyperosmotic environment) regulation is determined by the essential morphofunctional remodelling of the osmoregulatory organs under study. During this period (72 h) serum osmolarity increases. The neuroendocrine and endocrine systems respond to changes in the environmental salinity in a short time interval (1–24 h) thus stimulating morphofunctional changes in the effector organs and their transition to hypoosmotic regulation, which restores serum osmolarity close to the initial level.

Functional presence of Na+‐K+‐2Cl‐ cotransporter in rabbit lacrimal gland ductal epithelial cells

AbstractPurpose Capability of fluid secretion of rabbit lacrimal gland duct epithelial cells was recently demonstrated by the authors using isolated duct segment model developed earlier. Inhibition of basolateral Na+‐K+‐2Cl‐ cotransporter (NKCC1) with bumetanide resulted in a complete stoppage of fluid secretion. Aim of the present work was to determine the activity and the regulation of NKCC1.Methods Experiments were performed on isolated rabbit lacrimal gland interlobular duct segments. Intracellular fluorophotometry with ammonium pulse technique was used to measure cotransporter activity. The rate of bumetanide sensitive cytosolic acidification after addition of NH4‐ can be used to quantify the activity of the cotransporter. Role of various potentially stimulatory effects were investigated.Results Unstimulated activity of NKCC1 was determined. Elevated intracellular cAMP level evoked by forskolin stimulation resulted in marked increase in NKCC1 activity. Beside increased intracellular cAMP levels, low cytosolic Cl‐ concentration and cell shrinkage evoked by hyperosmotic environment were significant determinants of increased cotransporter activity.Conclusion Our earlier results suggested the dominant role of Cl‐ dependent mechanisms in lacrimal gland ductal fluid secretion. The presented experimental work proves the substantial role of NKCC1 in basolateral Cl‐ uptake. Intracellular regulation of Cl‐ handling and luminal Cl‐ secretion needs further investigation.

Influence of colonization by arbuscular mycorrhizal fungi on three strawberry cultivars under salty conditions

Plant adaptation to hyperosmotic environments is generally associated with reduced growth and ultimately yield loss, making farming difficult. The potential of mycorrhizal symbioses to alleviate salt stress has been documented and benefits to plant revealed to be specific and dependent to both plant cultivars and fungal strains. A factorial greenhouse experiment was performed to determine the effects of three arbuscular mycorrhizal fungi (AMF) species (Funneliformis caledonius, F. mosseae and Rhizophagus irregularis) on three ‘day-neutral’ strawberry (Fragaria × ananassa Duch.) cultivars (‘Albion’, ‘Charlotte’ and ‘Seascape’), and a mixture of R. irregularis and F. mosseae on ‘Seascape’, under four salt conditions (0–200 mM NaCl). The overall results showed that plant biomass decreased with increasing salinity. The cultivars responded differently to both AMF and salinity, and ‘Seascape’ was more tolerant to salinity than the other cultivars. AMF enhanced plant growth and improved salt tolerance by increasing the proportion of medium (0.5<ɸ≤1.5 mm) and coarse (ɸ>1.5 mm) diameter roots. The mixture of two AMF species increased root and shoot mass to a higher degree than each species alone at low salinity (0–50 mM) but reduced fruit quality. At higher levels (100–200 mM), R. irregularis alleviated salt stress and improved fruit quality to a higher degree than the other AMF species. Our results support the use of bio-inoculants in saline horticultural areas. Because cultivars respond differently to fungal inoculants, and inoculants prefer specific environmental conditions, fungal inoculants need to be screened on a cultivar- and condition-specific basis.

Environmental salinity and osmoregulatory processes in cultured flatfish

The aim of this study was to carry out a comparative analysis of the osmoregulatory properties and associated energy metabolism of euryhaline flatfish species that are cultured in the world. Culture of flatfish (pleuronectiformes) requires stage- and species-dependent osmotic conditions for rearing. Additionally, geographic origin of broodstock animals is another factor to be taken into account for the culture of pleuronectiformes. Larval and juvenile stages of many flatfish species are cultured in large nurseries situated in estuaries and shallow marine habitats, where the environmental salinity is close to the iso-osmotic point of their internal milieu. This fact implicates an advantage in terms of energy savings for osmoregulatory purposes. Thus, this ‘saved’ energy can be derived to other physiological processes, such as somatic growth. However, this scientific presumption does not always results in an optimal growth for many flatfish species. Indeed, iso-osmotic culture conditions can evoke a higher allostatic load than that in the usual hyper-osmotic environment where flatfish species live wildly. Optimization of flatfish culture thus requires adjustments of the osmotic culture conditions to the specific osmoregulatory and metabolic demands that eventually determine the allostatic load and consequently condition growth rates. In this sense, the geographical location of aquaculture farms and the osmoregulatory-based selection of the species to be cultivated in a particular area become critical factors to be considered for optimal flatfish culture.

Pituitary control of branchial NCC, NKCC and Na+, K+-ATPase α-subunit gene expression in Nile tilapia, Oreochromis niloticus

This study investigated endocrine control of branchial ionoregulatory function in Nile tilapia (Oreochromis niloticus) by prolactin (Prl188 and Prl177), growth hormone (Gh) and cortisol. Branchial expression of Na(+)/Cl(-) cotransporter (ncc) and Na(+)/K(+)/2Cl(-) cotransporter (nkcc) genes were employed as specific markers for freshwater- and seawater-type ionocytes, respectively. We further investigated whether Prl, Gh and cortisol direct expression of two Na(+), K(+)-ATPase (nka)-α1 subunit genes, denoted nka-α1a and nka-α1b. Tilapia transferred to fresh water following hypophysectomy failed to adequately activate gill ncc expression; ncc expression was subsequently restored by Prl replacement. Prl188 and Prl177 stimulated ncc expression in cultured gill filaments in a concentration-related manner, suggesting that ncc is regulated by Prl in a gill-autonomous fashion. Tilapia transferred to brackish water (23‰) following hypophysectomy exhibited a reduced capacity to up-regulate nka-α1b expression. However, Gh and cortisol failed to affect nka-α1b expression in vivo. Similarly, we found no clear effects of Gh or cortisol on nkcc expression both in vivo and in vitro. When considered with patterns previously described in euryhaline Mozambique tilapia (O. mossambicus), the current study suggests that ncc is a conserved target of Prl in tilapiine cichlids. In addition, we revealed contrasting dependencies upon the pituitary to direct nka-α1b expression in hyperosmotic environments between Nile and Mozambique tilapia.

Concentrations of osmotically related constituents in plasma and urine of finless porpoise (Neophocaena asiaeorientalis): implications for osmoregulatory strategies for marine mammals living in freshwater

Abstract Background Most cetaceans inhabit the hyperosmotic marine environment with only a few species living in freshwater habitats. The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is the only freshwater subspecies of the genus. Our aim was to study whether the osmoregulation mechanism of the Yangtze finless porpoise is different from the marine subspecies, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri). We assayed and compared the concentrations of the constituents involved in osmoregulation in the blood and urine in the Yangtze finless porpoise and the East Asian finless porpoise. We also compared the corresponding urine constituents of the porpoises with existing data on fin whales (Balaenoptera physalus) and bottlenose dolphins (Tursiops truncatus). Results The mean plasma osmolality of Yangtze finless porpoise was significantly lower than that of the marine subspecies (P < 0.01). Similarly, the urine osmolality of Yangtze finless porpoise was also significantly lower than that of its marine counterpart (P < 0.05). However, the urine sodium concentration of freshwater finless porpoise was significantly lower than that in the marine subspecies (P < 0.01), even though their serum sodium has no significant difference. Moreover, the freshwater porpoise has significantly lower urine urea concentration but much higher serum urea than in the marine finless porpoise (P < 0.05). Conclusions These results suggest that the freshwater finless porpoise does have different osmoregulatory mechanism from marine cetaceans. Conserving sodium by excreting urine with low ion levels may be an essential strategy to maintain the serum electrolyte balance for the freshwater subspecies that also appears to be more susceptible to hyponatremia.

Claudin-6, -10d, and -10e contribute to seawater acclimation in the euryhaline puffer fishTetraodon nigroviridis

Expression profiles of claudin-6, -10d and -10e in the euryhaline teleost fish Tetraodon nigroviridis revealed claudin-6 in brain, eye, gill and skin tissue, while claudin-10d and -10e were found in brain, gill and skin only. In fishes, the gill and skin are important tissue barriers that interface directly with surrounding water, but these organs generally function differently in osmoregulation. Therefore, roles for gill and skin claudin-6, -10d and -10e in the osmoregulatory strategies of T. nigroviridis were investigated. In the gill epithelium, claudin-6, -10d and -10e co-localized with Na(+)-K(+)-ATPase immunoreactive (NKA-ir) ionocytes, and differences in sub-cellular localization could be observed in hypoosmotic (freshwater, FW) versus hyperosmotic (seawater, SW) environments. Claudin-10d and -10e abundance increased in the gills of fish acclimated to SW versus FW, while claudin-6 abundance decreased in the gills of fish acclimated to SW. Taken together with our knowledge of claudin-6 and -10 function in other vertebrates, data support the idea that in SW-acclimated T. nigroviridis, these claudins are abundant in gill ionocytes, where they contribute to the formation of a Na(+) shunt and 'leaky' epithelium, both of which are characteristic of salt-secreting SW fish gills. Skin claudin-10d and -10e abundance also increased in fish acclimated to SW versus those in FW, but so did claudin-6. In skin, claudin-6 was found to co-localize with NKA-ir cells, but claudin-10d and -10e did not. This study provides direct evidence that the gill epithelium contains salinity-responsive tight junction proteins that are abundant primarily in ionocytes. These same proteins also appear to play a role in the osmoregulatory physiology of the epidermis.

Highly efficient gene transfection by a hyperosmotic polymannitol based gene tranporter through regulation of caveolae and COX-2 induced endocytosis.

The regulation of cellular uptake to cross the cell membrane is one of the key strategies of importance for efficient gene transfection of non-viral vectors. Hyperosmotic activity of polyplexes may facilitate crossing of the membrane barrier by elevating the osmolarity of the extracellular matrix. In this study, we demonstrated that a polymannitol based gene transporter (PMGT) utilizes the hyperosmoticity contributed by the polymannitol backbone leading to accelerated cellular uptake and enhanced gene transfection. Mannitol dimethacrylate (MDM) monomer was synthesized by esterification of mannitol and methacryloyl chloride. The prepared MDM was then cross-linked with low molecular weight (LMW) branched polyethyleneimine (bPEI) by Michael addition reaction to produce PMGT. PMGT provided polyplex stability in serum, low cytotoxicity, and degradability due to the ester linkages present in the polymannitol backbone. Elevated transfection activity and efficiency, both in vitro and in vivo, were achieved by modulating the mode of cellular uptake due to the effect of the hyperosmotic properties of PMGT. Cyclooxygenase-2 (COX-2) inhibition by SC58236 revealed the up-regulation of this osmoprotectant molecule against the hyperosmotic activity of polymannitol, inducing rapid endocytosis of PMGT in order to re-balance the hyperosmotic environment. Various inhibition studies of endocytosis showed caveolae-mediated endocytosis to be the main route of cellular internalization to account for the enhanced transgene expression.

Osmotic damage as a predictor of motility loss during convective desiccation of bovine sperm.

Current state-of-the art technologies are lagging in the application of desiccation storage to mammalian cells using nonreducing sugars. For bovine sperm, motility is irreversibly lost before reaching a sufficiently low moisture content necessary for preservation. It is hypothesized that much of the damage during drying is related to the osmotic stress encountered due to increased osmolarity of the extracellular environment. To test this hypothesis, we subjected sperm to liquid hyperosmotic environments for varying time-periods and measured their motility. We then extracted parameters for two models for motility loss based on these experiments: a first-order rate injury model (Fast or Slow) and a multi-modal (MM) injury model. The MM injury model incorporated an additional function accounting for damage induced by a time-independent osmotic change. Based on these models, we predicted sperm motility loss measured from natural and forced convective desiccation experiments. The MM injury model was able to closely bracket motility loss for desiccation as an osmotic change event with time-independent and time-dependent components. While the mechanistic basis of osmotic damage requires further exploration, the model can serve as a bracketing tool for predicting motility loss during desiccation based on excipients designed to minimize osmotic damage.

Adaptive evolution of the osmoregulation-related genes in cetaceans during secondary aquatic adaptation

BackgroundOsmoregulation was a primary challenge for cetaceans during the evolutionary transition from a terrestrial to a mainly hyperosmotic environment. Several physiological mechanisms have been suggested to maintain the water and salt balance in cetaceans, but their genetic and evolutionary bases remain poorly explored. The current study investigated the genes involved in osmoregulation in cetaceans and compared them with their counterparts in terrestrial mammals to test whether adaptive evolution occurred during secondary aquatic adaptation.ResultsThe present study analyzed the molecular evolution of 11 osmoregulation-related genes in 11 cetacean species, which represented all of the major cetacean clades. The results demonstrated positive selection acting on angiotensin converting enzyme (ACE), angiotensinogen (AGT), SLC14A2, and aquaporin 2 (AQP2). This evidence for the positive selection of AQP2 and SLC14A2 suggests that the adaptive evolution of these genes has helped to enhance the capacity for water and urea transport, thereby leading to the concentration of urine, which is an efficient mechanism for maintaining the water balance. By contrast, a series of positively selected amino acid residues identified in the ACE and AGT (two key members of the renin-angiotensin-aldosterone system, RAAS) proteins of cetaceans suggests that RAAS might have been adapted to maintain the water and salt balance in response to a hyperosmotic environment. Radical amino acid changes in positively selected sites were distributed among most internal and terminal branches of the cetacean phylogeny, which suggests the pervasively adaptive evolution of osmoregulation since the origin of cetaceans and their subsequent diversification.ConclusionsThis is the first comprehensive analysis of the molecular evolution of osmoregulation-related genes in cetaceans in response to selection pressure from a generally hyperosmotic environment. Four genes, i.e., AQP2, SLC14A2, ACE, and AGT were subject to positive selection in cetaceans, which suggests that cetaceans may have adapted to maintain their water and salt balance. This also suggests that cetaceans may have evolved an effective and complex mechanism for osmoregulation.

Characterization of Pseudomonas aeruginosa growth on O-acylcarnitines and identification of a short-chain acylcarnitine hydrolase.

To survive in various environments, from host tissue to soil, opportunistic bacterial pathogens must be metabolically flexible and able to use a variety of nutrient sources. We are interested in Pseudomonas aeruginosa's catabolism of quaternary amine compounds that are prevalent in association with eukaryotes. Carnitine and acylcarnitines are abundant in animal tissues, particularly skeletal muscle, and are used to shuttle fatty acids in and out of the mitochondria, where they undergo β-oxidation. We previously identified the genes required for carnitine catabolism as the first four genes in the carnitine operon (caiX-cdhCAB; PA5388 to PA5385). However, the last gene in the operon, PA5384, was not required for carnitine catabolism. We were interested in determining the function of PA5384. Bioinformatic analyses along with the genomic location of PA5384 led us to hypothesize a role for PA5384 in acylcarnitine catabolism. Here, we have characterized PA5384 as an l-enantiomer-specific short-chain acylcarnitine hydrolase that is required for growth and hydrolysis of acetyl- and butyrylcarnitine to carnitine and the respective short-chain fatty acid. The liberated carnitine and its downstream catabolic product, glycine betaine, are subsequently available to function as osmoprotectants in hyperosmotic environments and induce transcription of the virulence factor phospholipase C, plcH. Furthermore, we confirmed that acylcarnitines with 2- to 16-carbon chain lengths, except for octanoylcarnitine (8 carbons), can be utilized by P. aeruginosa as sole carbon and nitrogen sources. These findings expand our knowledge of short-chain acylcarnitine catabolism and also point to remaining questions related to acylcarnitine transport and hydrolysis of medium- and long-chain acylcarnitines.

Expression of aquaporin-3 and − 8 mRNAs in the parr and smolt stages of sockeye salmon, Oncorhynchus nerka: Effects of cortisol treatment and seawater acclimation

This study aimed to examine the role of 2 aquaporin (AQP) isoforms (AQP3, and −8) in sockeye salmon (Oncorhynchus nerka) in response to a hyperosmotic challenge from freshwater to seawater (SW) during the parr and smoltification (smolt) stages. AQP3 mRNA was primarily detected in the osmoregulatory organs, such as gills, while AQP8 mRNA was primarily found in the intestine. These results suggested that AQP isoforms play a role in osmoregulation in specific osmoregulatory organs. Similarly, AQP3 mRNA expression in the gills (mean values:1.06±0.05 [parr] and 1.29±0.07 [smolt]) was significantly higher than AQP8 mRNA levels (parr: 0.04±0.003; smolt: 0.14±0.004), and in the intestine, AQP8 mRNA expression (parr: 0.89±0.007; smolt: 1.91±0.03) was significantly higher than AQP3 mRNA levels (parr: 0.24±0.006; smolt: 0.83±0.005); these expression patterns were similar in vivo and in vitro. Additionally, AQP mRNA levels were lower in cortisol treated than in control groups. Therefore, these results suggest that AQPs play important roles in the water absorption mechanisms associated with multiple AQP isoforms, and that cortisol enhances the hypo-osmoregulatory capacity of fish in SW, and also controls the expression of AQPs in a hyperosmotic environment.