Environmental temperatures and salinities are two critical abiotic factors that affect the physiological responses and behavior of aquatic organisms. Under optimal temperature and salinity conditions, individuals have reduced energy needs to satisfy normal physiological demands and demonstrate high growth performance (Jana et al., 2006; Kang et al., 2015; Dahlke et al., 2020). According to different salinity environments, energetic costs for ionic homeostasis, appetite and absorption ability, and swimming behavior are important factors affecting the growth rate of fishes. Studies on several euryhaline teleosts have suggested that improved growth performance was found in brackish water conditions (8–20‰) because the ingestion of feed with some external water affects the appetite and energy absorption in fishes (Boeuf and Payan, 2001; Deane and Woo, 2009). Additionally, fishes are aquatic ectotherms; therefore, water temperature fluctuations directly affect the activities of metabolic enzymes, which leads to physiological responses in the whole individual (Barnes et al., 2014). Responses to temperature changes also affect the appetite and hormone secretion, which are the major factors that affect the energy obtained (Donaldson et al., 2008; Deane and Woo, 2009).

Gills, a specialized organ in aquatic species, have several important roles, including osmoregulation, acid-base regulation, nitrogen extraction, and gas exchange. The ionocytes distributed widely in the gill epithelium are mitochondria-rich (MR) cells that contain a large abundance of Na⁺, K⁺-ATPase (NKA), which is the primary driving force for other transporters to maintain osmotic homeostasis in fishes (Hiroi and McCormick, 2012; Yan and Hwang, 2019). In euryhaline fishes, acclimation to biotic or abiotic fluctuations should utilize large amounts of energy to maintain several ion transporters in gills for normal physiological responses (De Boeck et al., 2013; Chang et al., 2016; Roa and Tresguerres, 2019). The glycogen-rich (GR) cell is an accessory cell for the acute energy supply to ionocytes in teleosts. Branchial glycogen is predominantly stored in GR cells and is utilized under acute salinity treatment (Chang et al., 2007; Tseng et al., 2007; Hwang et al., 2011). Glycogen phosphorylase is a rate-limiting enzyme in glycogenolysis and an ideal indicator for identifying the localization of GR cells in the gill epithelium (Tseng et al., 2007). Branchial glycogen utilization has been studied in other fish species under different stress conditions, including Amazonian oscars (Astronotus ocellatus) under hypoxia, (De Boeck et al., 2013), blue-spotted mudskippers (Boleophthalmus pectinirostris) under low-salinity conditions (Guo et al., 2020), leopard shark (Triakis semifasciata) during starvation (Roa and Tresguerres, 2019), and tilapia (Oreochromis mossambicus) during seawater acclimation (Chang et al., 2007) or under cadmium exposure (Lin et al., 2011). In addition, glucose transporters have an energy supply function from circulating plasma glucose to support GR cells for glycogen storage and energy demands of ion homeostasis of mitochondria-rich ionocytes (Tseng et al., 2008; Tseng and Hwang, 2008; Balmaceda-Aguilera et al., 2012). In zebrafish, glucose transporter isoforms, including GLUT1a, GLUT6, and GLUT13.1, have been identified in NKA-, GR-, and H⁺-ATPase-rich (HR)-immunoreactive cells, respectively. These glucose transporters play different physiological roles in energy metabolism in the gills (Tseng et al., 2011). Furthermore, GR cells produce lactate as an acute-phase energy source for ionocytes through monocarboxylate cotransporters (MCTs) (Hwang et al., 2011; Tresguerres et al., 2020).

Aerobic metabolism in mitochondria requires an oxygen supply for optimal energy release. Citrate synthase (CS) and cytochrome c oxidase (COX) are two important factors for assessing the aerobic capacity in the citrate acid cycle (CAC) and the electron transport chain, respectively. Because ionocytes are MR cells, the abundance of these aerobic markers is high in these cells. CS is the first and rate-limiting enzyme in the citric acid cycle in mitochondria matrix and relative to provide intermediate for electron transporter chain and precursors of certain amino acids. COX is the complex IV of the electron transport chain located in the mitochondrial membrane. Moreover, CS requires oxygen to generate a proton electrochemical potential, in order to supply ATP synthase for ATP synthesis (Gagnon and Holdway, 1999; Webb et al., 2005). Studies have reported the existence of an energy supply system for ionocytes in stenohaline zebrafish and euryhaline tilapia (Chang et al., 2007; Tseng et al., 2008). The energy supply system plays an important role via aerobic metabolism to be the major source of energy for ionic homeostasis in branchial ionocytes of fishes upon adverse environmental temperature and salinity conditions.

Milkfish (Chanos chanos), an important aquaculture species in Southeast Asia, can be cultured in environments ranging from fresh water (FW) to seawater (SW). The optimal culture temperature in milkfish ranges from 20°C to 33°C, with the best results observed at 28°C (Martinez et al., 2006). In Taiwan, a large number of milkfish die during cold snap in winter, leading to enormous economic losses (Liao, 1991). The cold-tolerance ability of SW-acclimated milkfish was found to be better than that of FW-acclimated milkfish (Kang et al., 2015). Previous studies have reported that under hypothermal stress (18°C), branchial NKA activity of FW- and SW-acclimated milkfish was significantly decreased, leading to an imbalance in plasma chloride and sodium concentrations (Kang et al., 2015). Hepatic glycogen depletion and hepatocyte apoptosis occurring in hypothermal FW-acclimated milkfish might be the reasons why FW-acclimated milkfish exhibit lower tolerance to cold than SW-acclimated milkfish (Chang et al., 2018; Chang et al., 2021). Therefore, the energy supply system in milkfish is critical to support the energy demands of ionocytes in gills for osmoregulation under hypothermal stress, even though ionic homeostasis also includes several cellular physiological requirements. In this study, the localization of GR and MR cells in milkfish gills was specifically identified, and differential strategies for energy supply in gills between FW- and SW-acclimated milkfish under hypothermal stress were described.

In this study, several indicators were used to identify MR and GR cells in milkfish gills. Among them, NKA is a traditional marker for MR cells (ionocytes). The mitochondrial markers COX and CS were co-localized with NKA in the gill filaments of milkfish. Immunolocalization of GP, a marker of GR cells, revealed that GR cells were localized next to the ionocytes. Notably, similar studies on immunostaining of glycogen particles in sharks showed that the content of glycogen was high in branchial NKA- and H⁺-ATPase-immunoreactive cells, indicating that glycogen was stored in MR cells in elasmobranch gills (Roa and Tresguerres, 2019). Compared to the findings in sharks, the accessory GR cells were found to supply energy to ionocytes in milkfish, zebrafish, and tilapia, which may be specific to teleostean gills (Chang et al., 2007; Tseng et al., 2011).

Glucose transporters (GLUTs) have glucose uptake capacity, as well as other functions that play a critical role in the energy supply system. Several isoforms of GLUTs are expressed in different cell types, such as NKA, HR, and GR cells, in gills. In zebrafish, the isoform GLUT6 identified in branchial GR cells had higher values of Michaelis constant (K _m) and maximal velocity (V _max) compared to those of the GLUT isoforms in ionocytes, GLUT1α and GLUT13.1. This result indicates that GLUT6 exhibits lower glucose absorption affinity to avoid competing with GLUT1α and GLUT13.1 for the energy substrate in ionocytes (Tseng et al., 2011). In this study, serial cryosectioning revealed the presence of the isoform GLUT1 in the gills of milkfish. GLUT1 was co-localized with GP and distributed in branchial GR cells ( Figure S1A ). In situ hybridization of glut1 in gill filaments was used to confirm the expression of GLUT1, and the results showed that glut1 mRNA was localized near NKA-IR cells ( Figure S1B ). Isoforms of GLUT have different distribution patterns in different species of teleosts. Interestingly, in mice, the K _m and V _max values of GLUT1 were higher than those of other GLUTs (Zhao and Keating, 2007). Further research is needed to assess if the isoforms of GLUTs in milkfish branchial cells have variable enzyme kinetics (K _m and V _max), indicating specific physiological importance.

Energy supply is required to support metabolic costs in the gills of teleosts when exposed to environmental fluctuations. Upon salinity challenge, glycogen utilization for energy demands of osmoregulation was identified in the gills of tilapia (Oreochromis mossambicus) and blue-spotted mudskippers (Boleophthalmus pectinirostris) (Chang et al., 2007; Tseng et al., 2007; Guo et al., 2020). Increasing requirements for glucose uptake and glycogen utilization in gills have been reported in moonlight gourami (Trichogaster microlepis), largemouth bass (Micropterus salmoides), and white shrimp (Litopenaeus vannamei) during the acute response to hypoxia (Martínez-Quintana et al., 2014; Hu et al., 2015; Yang et al., 2017). In this study, the increase in GLUT1 expression and glucose content in gills ( Figures 2A and 3A ) implied that branchial glucose uptake capacity in both FW- and SW-acclimated milkfish was upregulated under hypothermal stress. Moreover, the findings indicated that the branchial glucose uptake capacity in SW milkfish was greater than that in FW milkfish. Meanwhile, the increased expression of GP and degradation of branchial glycogen identified in SW-acclimated milkfish indicated that branchial glycogen was utilized in SW individuals, but not in FW individuals ( Figures 2B , 3B ). On the other hand, our previous study indicated that only FW individuals were found to utilize hepatic glycogen to maintain normal energy demands for the whole body under hypothermal stress (Chang et al., 2018). Taken together, these results suggested that when exposed to low-temperature condition, branchial and hepatic glycogen might be the source of energy supply for gills in SW-acclimated milkfish and FW-acclimated milkfish, respectively. Different environmental salinities could lead to diverse metabolic requirements of milkfish against hypothermal stress, thereby triggering different energy utilization strategies. Chang et al. (2018) also revealed that different environmental salinities did not influence the feed intake, which of both FW- and SW-acclimated milkfish was reduced under hypothermal stress, indicating that different energy utilization strategies between FW- and SW-acclimated milkfish were not induced by dietary intake. Meanwhile, this study showed that the lactate content in both FW and SW individuals under hypothermal stress was downregulated, suggesting that lactate was also used as an energy source in the branchial cells of milkfish. Moreover, Tseng et al. (Tseng et al., 2008) reported that lactate could be used as a fuel to maintain aerobic metabolism in the branchial ionocytes of tilapia upon salinity challenge. These findings with similar evidence illustrate that lactate is the form of energy transported to ionocytes from GR cells to supply energy demands for ionic homeostasis (Tseng and Hwang, 2008).

Changes in ambient temperatures affect the enzymatic activity of ectothermic teleosts. According to the Q₁₀ temperature coefficient of the chemical reaction, enzyme activity should be reduced by half when the ambient temperature decreases by 10°C (Lefevre, 2016). Therefore, compensatory responses in protein abundance or enzyme activity are crucial for teleosts to maintain normal physiological functions and individual behavior upon hypothermal challenge (Hardewig et al., 1999). To enhance aerobic capacity in response to low-temperature stress, several species exhibit an increase in the mitochondrial density or aerobic enzyme content (Hardewig et al., 1999; Orczewska et al., 2010; Strobel et al., 2013). In addition, fluctuations in environmental salinities also affect aerobic metabolism and oxygen consumption of teleosts because less energy is required for the osmoregulation of various creatures under optimal habitat environments (Ern et al., 2014; Li et al., 2019). Our previous studies reported that although the oxygen consumption rate in SW milkfish exposed to 28°C was higher than that in FW individuals, SW milkfish exhibited lower branchial NKA protein expression and activity, which could reflect the energetic requirement for maintaining ionic homeostasis (Kang et al., 2015; Chang et al., 2019). In this study, CS and COX activities as indicators of aerobic metabolism and energy production were analyzed in the gills of milkfish under hypothermal stress. The branchial COX activity in gills of milkfish exposed to 28°C revealed similar patterns to NKA activity, indicating that the energy demands of ion regulation were lower in SW milkfish than in FW milkfish at normal temperature. The branchial COX protein abundance of both FW and SW milkfish was increased under hypothermal stress, whereas the branchial COX activity of SW milkfish was elevated to a similar capacity as that of FW milkfish, revealing that milkfish have compensatory responses in gills to increase the energy supply. However, a significant decrease in NKA activity was found in the gills of both FW and SW milkfish under hypothermal stress (Kang et al., 2015). The results reflected insufficient energy supply for ion regulation, which might be due to the enhanced energy requirement of gills to maintain other normal physiological functions, including antioxidant responses against low temperature-induced damages (Orczewska et al., 2010; Kang et al., 2015; Wu et al., 2015). Notably, in our previous study (Chang et al., 2019), we performed a similar analysis in the liver of milkfish and revealed different trends compared to those in gills. First, unlike in the gills of SW milkfish, COX activity and protein content in the liver remained stable under hypothermal stress. Next, in contrast to the stabilized CS protein abundance in livers of FW milkfish under hypothermal stress, CS protein abundance in FW milkfish gills was increased as a compensatory response to support activity in gills of hypothermal milkfish, despite its CS activity being slightly reduced. Furthermore, the CS : COX activity ratio, which reflects anabolic demand in mitochondria, increased in hypothermal FW milkfish livers but did not change in gills. Previous studies revealed that CS activity was upregulated in different tissues of teleosts at low temperatures, playing an important role in the biosynthesis of amino acids and lipids as well as maintaining aerobic metabolism to stabilize physiological mechanisms (McClelland et al., 2006; Orczewska et al., 2010; Strobel et al., 2013; Chang et al., 2019). Lipids and amino acids may be considered as supplementary fuels to support the normal demands of aerobic metabolism (Soengas et al., 2007; Speers-Roesch et al., 2016).

In conclusion, the present results suggested that compared to SW milkfish, FW milkfish have higher energy demands to maintain morphological features, mitochondria biosynthesis, and physiological functions in gills under hypothermal stress. The glycogen stored in livers and gills were found to be the source to support energy demands. However, under hypothermal stress, FW milkfish mainly used hepatic glycogen while SW milkfish used branchial glycogen for energy supply. The responses differed between FW and SW milkfish implied that FW- and SW-acclimated milkfish performed different energy utilization strategies under hypothermal stress. Notably, the muscle reserves in milkfish might be the other source of energy supply which was not analyzed under different conditions yet. Hence, more investigations are needed to check if the other source in milkfish, such as muscle reserves, was also involved in energy supply of gills.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Chung Hsing University, Taiwan (IACUC Approval No. 107-165 to T-HL).

C-HC and C-JL contributed to manuscript writing. C-HC, W-JL, L-YW, and T-HL conceived discussed and designed this study. W-JL, L-YW, and Y-TL carried out the fish culture and experiments. C-JL and T-HL reviewed and edited the manuscript. T-HL supervised the projected. All authors read and approved the final manuscript for publication.

This research was partly supported by a grant to T-HL from the Ministry of Science and Technology (MOST) of Taiwan (108-2313-B-005-006-MY3) and in part by the integrative Evolutionary Galliform Genomics (iEGG) and Animal Biotechnology Center from The Feature Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (109-S-0023-F).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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