The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus)

In order to replace the diffusive loss of water to the surrounding environment, seawater (SW)-acclimated euryhaline fishes have gastrointestinal tracts with higher ion/water flux in concert with greater permeability, and contrast that to freshwater (FW)-acclimated fish. To understand the cellular basis for these differences, we examined cell proliferation and apoptosis in the anterior intestine of mudskipper transferred from one-third SW to FW or to SW for 1 and 7 days, and those kept out of water for 1 day. The intestinal apoptosis (indicated by DNA laddering) increased during seawater acclimation. TUNEL staining detected numerous apoptotic cells over the epithelium of SW-acclimated fish. Cell proliferation ([³H]thymidine incorporation) in the FW fish was greater than those in SW 7 days after transfer. Labeling with a Proliferating cell nuclear antigen (PCNA) antibody indicated that proliferating cells were greater in number and randomly distributed in the epithelium of FW fish, whereas in SW fish they were almost entirely in the troughs of the intestinal folds. There were no changes in cell turnover in fish kept out of water. During acclimation to different salinities, modification of the cell turnover and abundance may play an important role in regulating the permeability (and transport capacity) of the gastrointestinal tract of fish.     

精氨酸代谢途径抗酸关键基因对乳酸乳球菌Lactococcus lactis NZ9000胁迫抗性的影响

【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗性。【结果】与对照菌株对比,重组菌株在环境胁迫下表现了较高的生长性能、存活率和发酵性能。生理学分析发现,酸胁迫环境下,重组菌株细胞有较高的胞内NH4+、ATP含量和H+-ATPase活性,并显著提高了精氨酸脱亚胺酶(ADI)途径中的氨基酸浓度。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调。【结论】在L.lactis NZ9000中过量表达ASS或ASL可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,为微生物,尤其是工业微生物提高胁迫抗性提供了新思路。     

Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Alterations of gene expression profiles induced by sulfur dioxide in rat lungs

Sulfur dioxide (SO2) is a ubiquitous air pollutant presents in low concentrations in urban air and in higher concentrations in working environment.Few data are avail-able on the effects of being exposed to this pollutant on the molecular mechanism,although some biochemical changes in lipid metabolism,intermediary metabolism and oxidative stress have been detected.The present investigation aimed at analyzing the gene expression profiles of the lungs of Wistar rats short-term (20 ppm,6 h/day,for seven days) and long.term (5 ppm,1 h/day,for 30 days) exposed to SO2 by Affymetrix GeneChip (RAE230A) analysis.It was found that 31 genes,containing 18 known genes and 13 novel genes were up-regulated,and 31 genes,containing 20 known genes and 11 novel genes,were down-regulated in rats short-term exposed to SO2 compared with control rats.While there were 176 genes,containing 82 known genes and 94 novel genes were up-regulated,and 85 genes,containing 46 known genes and 39 novel genes,were down-regulated in rats long-term exposed to SO2 compared with control rats.It is suggested that:(1) SO2 exerts its effects by different mechanisms in vivo at high-dose short-term inhalation and at low-dose long-term inhalation;(2) a notable feature of the gene expression profile was the decreased expression of genes related to oxidative phosphorylation in lungs of rats short-term exposed to SO2,which shows high-dose short-term exposed to SO2 may cause the deterioration of mitochondrial functions;(3)discriminating genes in lungs of rats long-term exposed to SO2 included those involved in fatty acid metabolism,immune,inflammatory,oxidative stress,oncogene,tumor suppresser and extracellular matrix.The mechanism of low-dose long-term exposed to SO2 is more complex.     

硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响

【目的】利用转录组测序研究硫酸锌添加提高絮凝酿酒酵母SPSC01乙酸胁迫耐性的分子机理。【方法】在10.0 g/L乙酸胁迫条件下,添加0.03 g/L硫酸锌,取对数期酿酒酵母细胞,与不添加硫酸锌的对照组细胞进行比较转录组分析。【结果】添加硫酸锌的实验组与对照组相比较,50个基因转录水平上调,162个基因转录水平下调,这些转录水平变化明显的基因涉及糖代谢、甲硫氨酸合成、维生素合成等多条代谢途径,此外,转录水平变化的基因还包括抗氧化酶基因等关键胁迫响应基因。【结论】硫酸锌添加可改变酿酒酵母全局基因转录水平,提高抗氧化酶及其他胁迫耐性相关基因的表达,影响细胞氧化还原平衡和能量代谢,通过对多基因转录的调控提高酿酒酵母乙酸耐受性。     

The ultrastructural characterization of mitochondria‐rich cells as a response to variations in salinity in two types of teleostean pseudobranch: milkfish (Chanos chanos) and Mozambique tilapia (Oreochromis mossambicus)

The pseudobranchs of two euryhaline teleost species, the milkfish (Chanos chanos ) and the Mozambique tilapia (Oreochromis mossambicus ), were studied after acclimization to different salinities using optical and electron microscopy. The milkfish pseudobranch was the lamellae‐free type, with separate lamellae along the filaments containing two groups of mitochondria (Mt)‐rich cells: chloride cells (CCs) and pseudobranch type cells (PSCs). Conversely, the tilapia pseudobranch was the embedded type, covered with connective tissues and with only one group of Mt‐rich PSCs. Chloride cells were identified according to the apical openings and branched tubular networks around randomly distributed and diversely shaped Mt. Pseudobranchs type cells, however, were characterized according to the orderly arrangement of parallel tubules around closely packed Mt; both the tubules and the Mt were distributed in the vascular side of the cell, but were absent from the apical region. Compared with those of seawater (SW)‐acclimated milkfish, the pseudobranchial lamellae of freshwater (FW) specimens were longer on average, and the Mt of the CCs had fewer cristae, were less electron‐dense, and were often vacuolated. The Mt in the PSCs of FW‐acclimated milkfish and tilapia were larger and more electron‐dense than those of their SW‐acclimated counterparts; in addition, more tubules were found to aggregately surround the Mt and basolateral membranes in the PSCs of fish from the hypo‐osmotic environment. Conversely, the PSCs of tilapia were periodic acid‐Schiff (PAS)‐positive, and Mt in PSCs were concentrated with more parallel arrays of the tubule system than those of milkfish. Therefore, salinity‐dependent changes in the ultrastructures of PSCs suggest their potential role in energy metabolism of both lamellae‐free and embedded pseudobranchs, whereas the PAS‐positive staining characteristics suggest a role in releasing or storaging polysaccharides in the embedded pseudobranch. J. Morphol. 278:390–402, 2017. © 2017 Wiley Periodicals, Inc.     

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

Transcriptomic differences between euryhaline and stenohaline malaria vector sibling species in response to salinity stress

Evolution of osmoregulatory systems is a key factor in the transition of species between fresh‐ and saltwater habitats. Anopheles coluzzii and Anopheles merus are stenohaline and euryhaline malaria vector mosquitoes belonging to a larger group of sibling species, the Anopheles gambiae complex, which radiated in Africa within the last 2 million years. Comparative ecological genomics of these vector species can provide insight into the mechanisms that permitted the rapid radiation of this species complex into habitats of contrasting salinity. Here, we use RNA‐Seq to investigate gene expression differences between An. coluzzii and An. merus after briefly exposing both young and old larval instars of each species to either saltwater (SW) or freshwater (FW). Our study aims to identify candidate genes and pathways responsible for the greater SW tolerance of An. merus. Our results are congruent with the ability of gene induction to mediate salinity tolerance, with both species showing increasing amounts of differential gene expression between SW and FW as salt concentrations increase. Besides ion transporters such as AgAE2 that may serve as effectors for osmoregulation, we also find mitogen‐activated protein kinases that may serve in a phosphorylation signalling pathway responding to salinity, and report potential cross‐talk between the mosquito immune response and osmoregulation. This study provides a key step towards applying the growing molecular knowledge of these malaria vectors to improve understanding of their ecological tolerances and habitat occupancy.     

Salinity-dependent expression of the branchial Na+/K +/2Cl (-) cotransporter and Na+/K (+)-ATPase in the sailfin molly correlates with hypoosmoregulatory endurance

In the branchial mitochondrion-rich (MR) cells of euryhaline teleosts, the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) is an important membrane protein that maintains the internal Cl⁻ concentration, and the branchial Na⁺/K⁺-ATPase (NKA) is crucial for providing the driving force for many other ion-transporting systems. Hence this study used the sailfin molly (Poecilia latipinna), an introduced aquarium fish in Taiwan, to reveal that the potential roles of NKCC and NKA in sailfin molly were correlated to fish survival rates upon salinity challenge. Higher levels of branchial NKCC were found in seawater (SW)-acclimated sailfin molly compared to freshwater (FW)-acclimated individuals. Transfer of the sailfin molly from SW to FW revealed that the expression of the NKCC and NKA proteins in the gills was retained over 7 days in order to maintain hypoosmoregulatory endurance. Meanwhile, their survival rates after transfer to SW varied with the duration of FW-exposure and decreased significantly when the SW-acclimated individuals were acclimated to FW for 21 days. Double immunofluorescence staining showed that in SW-acclimated sailfin molly, NKCC signals were expressed on the basolateral membrane of MR cells, whereas in FW-acclimated molly, they were expressed on the apical membrane. This study illustrated the correlation between the gradual reductions in expression of branchial NKCC and NKA (i.e., the hypoosmoregulatory endurance) and decreasing survival rates after hyperosmotic challenge in sailfin molly.     

Survival and behavioral characteristics of amphidromous goby larvae of Sicyopterus japonicus (Tanaka, 1909) during their downstream migration

To understand the ecology and environmental tolerances of newly hatched larvae of the amphidromous fish Sicyopterus japonicus during their downstream migration, the salinity tolerance of eggs, 0-15 day old larvae, and adults, and the temperature tolerance, specific gravity and phototaxis of hatched larvae were examined. Tolerances of adults were measured as survival after a 24 h challenge in freshwater (FW), brackish water (1/3 SW) and seawater (SW). The survival rate of adult S. japonicus was 100% in FW and 1/3 SW, while none survived in SW. Hatching success of eggs (30 eggs each) was significantly higher in FW (mean: 73%) and 1/3 SW (73%) than in SW (19%). Tolerance of newly hatched larvae to salinity and temperature was investigated in different combinations of salinities (FW, 1/3 SW and SW) and temperatures (18, 23 and 28 °C). Larval survival was significantly different in each salinity and temperature. Survival rate was significantly higher in 1/3 SW than in FW and higher in SW than in FW at 23 °C and 28 °C. At the latter part of the experiment, there was no survival in FW and at 28 °C. Survival was higher in lower temperatures, but larval development did not occur in FW. Specific gravity of newly hatched larvae was 1.036 at 28 °C and 1.034 at 23 °C. When exposed to a light source on one side of an aquarium, larval distribution was not affected. Our results indicated larval S. japonicus are more adapted to brackish water and seawater than freshwater, while the adults and eggs are more adapted to freshwater and brackish water than seawater. This is consistent with their amphidromous life history with growth and spawning occurring in freshwater and the larval stage utilizing marine habitats.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.