Regulation of PGC-1α of the Mitochondrial Energy Metabolism Pathway in the Gills of Indian Medaka (Oryzias dancena) under Hypothermal Stress.

Ectothermic fish exposure to hypothermal stress requires adjusting their metabolic molecular machinery, which was investigated using Indian medaka (Oryzias dancena; 10 weeks old, 2.5 ± 0.5 cm) cultured in fresh water (FW) and seawater (SW; 35‱) at room temperature (28 ± 1 °C). The fish were fed twice a day, once in the morning and once in the evening, and the photoperiod was 12 h:12 h light: dark. In this study, we applied two hypothermal treatments to reveal the mechanisms of energy metabolism via pgc-1α regulation in the gills of Indian medaka; cold-stress (18 °C) and cold-tolerance (extreme cold; 15 °C). The branchial ATP content was significantly higher in the cold-stress group, but not in the cold-tolerance group. In FW- and SW-acclimated medaka, the expression of genes related to mitochondrial energy metabolism, including pgc-1α, prc, Nrf2, tfam, and nd5, was analyzed to illustrate differential responses of mitochondrial energy metabolism to cold-stress and cold-tolerance environments. When exposed to cold-stress, the relative mRNA expression of pgc-1α, prc, and Nrf2 increased from 2 h, whereas that of tfam and nd5 increased significantly from 168 h. When exposed to a cold-tolerant environment, prc was significantly upregulated at 2 h post-cooling in the FW and SW groups, and pgc-1α was significantly upregulated at 2 and 12 h post-cooling in the FW group, while tfam and nd5 were downregulated in both FW and SW fish. Hierarchical clustering revealed gene interactions in the cold-stress group, which promoted diverse mitochondrial energy adaptations, causing an increase in ATP production. However, the cold-tolerant group demonstrated limitations in enhancing ATP levels through mitochondrial regulation via the PGC-1α energy metabolism pathway. These findings suggest that ectothermic fish may develop varying degrees of thermal tolerance over time in response to climate change. This study provides insights into the complex ways in which fish adjust their metabolism when exposed to cold stress, contributing to our knowledge of how they adapt.

Differential expression of two ATPases revealed by lipid raft isolation from gills of euryhaline teleosts with different salinity preferences.

In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H + -ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

Cortisol regulation of Na+, K+-ATPase ß1 subunit transcription via the pre-receptor 11ß-hydroxysteroid dehydrogenase 1-like (11ß-Hsd1L) in gills of hypothermal freshwater milkfish, Chanos chanos.

Hypothermal stress changes the balance of osmoregulation by affecting Na+, K+-ATPase (Na-K-ATPase) activity or inducing modulation to epithelium permeability in fish. Meanwhile, cellular concentrations of cortisol can be modulated by the pre-receptor enzymes 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-Hsd1 and 2). In fish, increasing levels of exogenous cortisol stimulate Na+ uptake via specific interaction with cortisol. This study investigated cortisol effects on expression of Na-K-ATPase subunit proteins and activity in gills of milkfish under hypothermal stress and revealed that the plasma cortisol contents as well as gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance were decreased in fresh water (FW) milkfish. Meanwhile, in the seawater (SW) milkfish, the plasma cortisol contents and gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance was increased under hypothermal stress. On the other hand, the abundance of 11ß-hsd2 mRNA increased in both FW and SW. In addition, 11ß-hsd1l expression increased in FW milkfish but decreased in SW milkfish after cortisol injection. Accordingly, the results that gill Na-K-ATPase activity of FW milkfish was affected by environmental temperatures as well as cortisol-dependent Na-K-ATPase ß1-subunit levels might be due to increased expression of 11ß-hsd1l that elevated intracellular cortisol contents. In hypothermal SW milkfish, decreasing abundance of Na-K-ATPase ß1 protein due to reduced expression of 11ß-hsd1l was found after cortisol injection. Thus, under hypothermal stress, 11ß-HSD1L in FW milkfish gills was used to modulate cortisol and the following effects on increasing the transcription of Na-K-ATPase ß1 protein.

Positive correlation of gene expression between branchial FXYD proteins and Na+/K+-ATPase of euryhaline milkfish in response to hypoosmotic challenges.

FXYD proteins are crucial regulators of Na+/K+-ATPase (NKA), which plays an important role in ion exchange by providing the driving force for other ion-transporting systems in the osmoregulatory organs, including the gills. In milkfish (Chanos chanos), gill NKA has been widely investigated and found to alter its expression (both mRNA and protein) and activity in response to environmental salinity changes. However, the expression and roles of the regulatory proteins of NKA, the FXYD proteins, in milkfish gills upon salinity challenge is not yet clear. Hence, this study illustrated the potential roles of milkfish branchial FXYD proteins in modulating NKA expression via identification and tissue distributions of FXYD proteins, as well as the effects of salinity on expression of gill fxyd and nka mRNA. Six milkfish FXYD proteins (CcFXYD) were identified. In milkfish gill, gill-specific Ccfxyd11 was the predominant member, followed by Ccfxyd9 and Ccfxyd8. Upon hypoosmotic challenges, increases in gill Ccfxyd11, Ccfxyd8, Ccnka α1, and Ccnka ß1 mRNA as well as significantly positive correlations were observed. Moreover, after acute salinity changes, expression of gill Ccfxyd11 and Ccnka was found to change with ambient salinity, and significant positive correlations were also exhibited between Ccfxyd11 and Ccnka α1. Overall, these results revealed close relationships between CcFXYD11 and CcNKA α1 in milkfish gills, highlighting the potential roles of CcFXYD11 in osmoregulation.

An assay of optimal cytochrome c oxidase activity in fish gills.

Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 µg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish.

Na+, K+-ATPase ß1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na+, K+-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and ß-subunits. Among the NKA genes, α1-ß1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-ß1 isozyme. Based on the sequenced milkfish transcriptome, an NKA ß1 subunit gene was identified that had the highest amino acid homology to ß233, a NKA ß1 subunit paralog originally identified in the eel. Despite this high level of homology to ß233, phylogenetic analysis and the fact that only a single NKA ß1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA ß1 subunit gene. The results of accurate domain prediction of the ß1 subunit, co-localization of α1 and ß1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and ß1 subunits, indicated the formation of a α1-ß1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and ß1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA ß1 subunit and provided the first evidence for an NKA α1-ß1 association in gill ionocytes of euryhaline teleosts.

Transcriptomic Analysis of Metabolic Pathways in Milkfish That Respond to Salinity and Temperature Changes.

Milkfish (Chanos chanos), an important marine aquaculture species in southern Taiwan, show considerable euryhalinity but have low tolerance to sudden drops in water temperatures in winter. Here, we used high throughput next-generation sequencing (NGS) to identify molecular and biological processes involved in the responses to environmental changes. Preliminary tests revealed that seawater (SW)-acclimated milkfish tolerated lower temperatures than the fresh water (FW)-acclimated group. Although FW- and SW-acclimated milkfish have different levels of tolerance for hypothermal stress, to date, the molecular physiological basis of this difference has not been elucidated. Here, we performed a next-generation sequence analysis of mRNAs from four groups of milkfish. We obtained 29669 unigenes with an average length of approximately 1936 base pairs. Gene ontology (GO) analysis was performed after gene annotation. A large number of genes for molecular regulation were identified through a transcriptomic comparison in a KEGG analysis. Basal metabolic pathways involved in hypothermal tolerance, such as glycolysis, fatty acid metabolism, amino acid catabolism and oxidative phosphorylation, were analyzed using PathVisio and Cytoscape software. Our results indicate that in response to hypothermal stress, genes for oxidative phosphorylation, e.g., succinate dehydrogenase, were more highly up-regulated in SW than FW fish. Moreover, SW and FW milkfish used different strategies when exposed to hypothermal stress: SW milkfish up-regulated oxidative phosphorylation and catabolism genes to produce more energy budget, whereas FW milkfish down-regulated genes related to basal metabolism to reduce energy loss.

Comparisons of two types of teleostean pseudobranchs, silver moony (Monodactylus argenteus) and tilapia (Oreochromis mossambicus), with salinity-dependent morphology and ion transporter expression.

There are essentially four different morphological types of pseudobranchs in teleosts, including lamellae-free, lamellae semi-free, covered, and embedded types. In the euryhaline silver moony (Monodactylus argenteus), the pseudobranch belongs to the lamellae semi-free type, which is characterized by one row of filaments on the opercular membrane and fusion on the buccal edge. The pseudobranchial epithelium of the moony contains two types of Na(+), K(+)-ATPase (NKA)-rich cells: chloride cells (CCs) and pseudobranch-type cells (PSCs). Our results revealed increased expression of NKA, the Na(+), K(+), 2Cl(-) cotransporter (NKCC), and the cystic fibrosis transmembrane conductance regulator (CFTR) for Cl(-) secretion and CCs profiles in the pseudobranchs of seawater (SW)-acclimated silver moonies, which indicates the potential role of pseudobranchs containing CCs in hypo-osmoregulation. In contrast, the pseudobranch of the Mozambique tilapia (Oreochromis mossambicus) belongs to the embedded type, which is covered by the connective tissues and only contains PSCs but not CCs. No sign of NKCC and CFTR-immunoreactivity (IR) was found in the pseudobranchs of SW and freshwater (FW) tilapia. However, higher NKA protein expression and larger sizes of NKA-IR PSCs were found in the pseudobranchs of FW-acclimated tilapia. Moreover, in the FW-acclimated moony, NKA-IR PSCs also exhibited higher numbers and larger sizes than in the SW individuals. Taken together, similar responses in low-salinity environments in different types of pseudobranchs indicated that the salinity-dependent morphologies of PSCs might be involved in critical functions for FW teleosts.