Growth performance
Selenium is an essential trace element in P. fulvidraco. Previously, an 8-week feeding trial showed that the addition of 0.23 mg/kg of selenium to the diet significantly improved the growth performance of P. fulvidraco (Hu et al. 2016). In this study, WGR and SGR were higher in the T0 group than that in the G0 group. However, the difference was not significant, which may attributable to the short breeding period (only 6 weeks). Nevertheless, these results suggest that selenium can improve the growth performance of P. fulvidraco.
Lipid metabolism
Liver Lipid metabolism is crucial for the conversion of macronutrients into metabolic intermediates for membrane biosynthesis, energy storage, signal transduction, and resistance to low temperature stress (Hsieh et al. 2003; Dietricha et al. 2018). Maintaining the relative fluidity and integrity of cell membranes for aquatic animals under low temperature stress is vital for maintaining normal cellular physiological functions and improving stress resistance (Sonna et al. 2002; Ibarz et al. 2005; Hsieh et al. 2005). To adapt to low temperatures, poikilotherms use different mechanisms, including enhanced sphingolipid metabolism, steroid biosynthesis, cholesterol transport, and increased accumulation of unsaturated fatty acids, to maintain the fluidity and integrity of cell membranes (Wang et al. 2014; Hu et al. 2014; Meng et al. 2019). Glycerophospholipids and sphingolipids are the first and second major classes of membrane lipids, respectively, and together form the main components of the biological membrane matrix to maintain the stability of cell membranes (Jin et al. 2020; Ren et al. 2020). We found that lipids and lipid-like molecules accounted for the highest percentage of selenium-induced DEMs, while some glycerophospholipids and sphingolipids were also significantly up-regulated, and may be attributed as a mechanism to maintain the stability of P. fulvidraco cell membranes under low temperature stress. Moreover, the target gene of selenium-regulated miR-143 is involved in lipids, fatty acids, sphingolipids, and glycerophospholipids metabolism in the mesenteric tissue of P. fulvidraco have been confirmed (Cui et al. 2020). Furthermore, on mammals, selenium supplementation increased intestinal lactic acid bacteria content in mice, causing significant enrichment of the metabolite phosphatidylglycerol (Callejon-Leblic et al. 2022).
The fatty acid composition of the cell membrane affects its fluidity, and the proportion and content of unsaturated fatty acids are closely related to the resistance of fish to low temperatures. Increasing the content of unsaturated fatty acids and controlling fatty acid desaturation promotes the resistance of fish to low temperature stress (Hsieh et al. 2003). In this study, the portion of glycerophospholipids was significantly reduced in the cell membranes of fish in the T0 group. Glycerophospholipids are hydrolyzed by phospholipases A1, A2, C, and D to produce glycerol, fatty acids, and phosphoric acid; and phospholipase A2 helps release highly unsaturated fatty acids with important physiological functions bound at the sn-2 site of the phospholipid backbone (Henderson et al. 1987). We found that the contents of the polyunsaturated fatty acids EPA and DPA in the T0 group were increased. We hypothesize that the consumed glycerophospholipids in our study may have been catabolized to generate unsaturated fatty acids for maintaining cell membrane fluidity in P. fulvidraco under low-temperature stress. The role of selenium in regulating unsaturated lipid production has been long reported. In broilers, selenium resulted in significantly high levels of the long-chain polyunsaturated fatty acids such as EPA (20:5), DPA (22:5), and DHA (22:6) in muscle (Haug et al. 2007). However, in this study, we found the levels of serum unsaturated fatty acid DHA were significantly reduced in P. fulvidraco in the T0 group. Analyzing this result, we speculated that unsaturated fatty acids may have differentially catabolism or anabolism to response to low temperature stress generated by selenium. Similarly, this deduction was consistent with the report that selenium reduced the adverse effects of heat stress on lipid metabolism of rainbow trout (Li et al. 2022).
Carbohydrate metabolism
The decarboxylation reaction is exergonic and may occur in two forms—simple decarboxylation and oxidative decarboxylation (Wang et al. 2002). We found that the number of down-regulated carboxylic acid and its derivatives (13 DEMs) was higher than that of up-regulated (6 DEMs) in the T0 group compared to the G0 group, indicating that selenium promoted decarboxylation and energy release under low temperature stress in P. fulvidraco. In addition, we also observed the significant enrichment of central carbon metabolic pathways which primarily include the glycolytic pathway pentose phosphate pathway and tricarboxylic acid cycle that supply the energy and precursors for other metabolic pathways (Sauer et al. 1999; Bennett et al. 2009). These findings suggest that selenium regulates the central carbon metabolic pathways to provide energy for improving P. fulvidraco resistance to low temperature stress.
Amino acid metabolism
Free amino acids play several functions, including protein synthesis, degeneration, growth, osmotic pressure homeostasis, and energy metabolism, in poikilotherms (Karanova and Andreev 2010). The increase in certain amino acids is positively correlated with the decrease in water temperature, which enhances resistance to low temperature stress (Karanova 2006). We found that valine, leucine and isoleucine degradation/biosynthesis, D-arginine and D-ornithine metabolism, as well as protein digestion and absorption pathways, were significantly enriched in the T0 group under low temperature stress. In addition, the serum levels of ornithine and L-valine were significantly up-regulated and L-isoleucine were significantly down-regulated in the T0 group. These above results showed the levels of different amino acids exhibited different trends under the influence of selenium. Selenium metabolism is closely linked to the 1C metabolic pathway (Daniels 1996). The folate cycle and methionine cycle are the two principal components of 1C metabolism; selenium supplementation increased the levels of cysteine and cysteinyl-glycine and decreased the levels of some other essential free amino acids in the muscle tissues of rainbow trout (Wischhusen et al. 2022). The effect of selenium on amino acid metabolism may be evident during the synthesis of selenoproteins. The insufficient intake of exogenous selenium or a stressful state leads to the increased participation of amino acids in the synthesis of antioxidant enzymes to promote the metabolism of antioxidant-like amino acid derivatives, resulting in a decrease in certain amino acids (Ge et al. 2011).
Nucleotide metabolism
Nucleotides are small molecules having diverse biological functions and are involved in genetic material synthesis, biochemical reaction catalysis, and energy provision and transformation. Low temperature stress causes significant alterations in nucleotide metabolism in aquatic animals such as Gilthead sea (Melis et al. 2017), Nibea albiflora (Xu et al. 2018), and Litopenaeus vannamei (Fan et al. 2019). Research fundings showed that selenium affected the metabolism of urinary purine derivatives in sheep and cattle (Shi et al. 2011; Ferreira et al. 2020). We found purine nucleoside (Deoxyhypoxanthine) was significantly down-regulated while pyrimidine nucleoside (cytosine) was significantly up-regulated, furthermore, the pyrimidine metabolic pathway was significantly enriched in the T0 group under low temperature stress. Inosine and hypoxanthine are oxidized to xanthine and uric acid via the degradation pathway of purine metabolism, and higher levels of uric acid may cause adverse effects on liver and kidney function (Fournier et al. 2003). We found reduced contents of purine nucleosides in the T0 group, suggesting that selenium play a protective effect on the liver and kidney function of P. fulvidraco under low temperature stress.
Oxidation products and other metabolites
Low temperature stress disrupts animal homeostasis and produces large amounts of free radicals. Remove failure to scavenge these excessive oxygen radicals leads to oxidative damage to proteins, lipids, and other biomolecules, and the production of oxidation products. The antioxidant system and non-specific immune system are vital defense mechanisms and exert a mitigating effect on environmental stress-induced oxidative damage. Selenium is a central element for GSH-Px activity; GSH-Px uses reduced GSH as a substrate to reduce the generation of harmful hydroperoxides and free radicals, reducing peroxide damage to biomacromolecules and cell membranes (Levander 1983). Previous studies showed that the addition of selenium to the diet significantly increased the activity of liver GSH-Px and superoxide dismutase enzymes in P. fulvidraco (Hu et al. 2019) and reduced lipid peroxide malondialdehyde and reactive oxygen species production in rainbow trout (Li et al. 2022). We found that organic oxygen compounds, nitrogen-containing organic compounds, lotaustralin, and coumarin and their derivatives were significantly down-regulated in T0 group, suggesting that selenium is beneficial in scavenging free radicals produced by low temperature stress and reducing organism damage.
Intestinal microbes diversity
The intestinal microbes, a crucial microbial barrier in the fish intestine, has diverse physiological functions, including regulating fish nutrient metabolism, immune response and inhibiting the proliferation of harmful bacteria. Increased intestinal microbes diversity is beneficial in maintaining intestinal health, while the lack of diversity can cause intestinal dysbiosis (Al-Masqari et al. 2022). The nutritional composition and feeding environment can affect the abundance of intestinal microbes and metabolite levels. Low temperature stress significantly reduced the intestinal microbial diversity in rainbow trout (Cruz-Flores et al. 2022), Seriola lalandi (Soriano et al. 2018) and Apostichopus japonicus (Rong 2012), in our previous study, the intestinal microbes diversity of P. fulvidraco was also significantly reduced, and the pathogenic bacteria of plesiomonas was rapidly appreciation (Hu et al. 2022). In this study, we found selenium supplementation was helpful to increase the diversity of P. fulvidraco intestinal microbes and reduce the relative abundance of Plesiomonas while, other microbes included Brevundimonas, Chryseobacterium, Sphingobium, Sphingomonas, and Flavobacterium were significient proliferation. Similarly, Baia et al. (2018) observed that selenomethionine supplementation at a dose of 5.03 mg/kg significantly affected the intestinal microbes diversity in Carassius auratus, increasing the abundance of Cetobacterium and decreasing that of Ralstonia Cetobacterium, a species of anaerobic bacteria present in the intestine of many fish, is involved in synthesizing vitamin B12 (Tsuchiya et al. 2008; Wu et al. 2010). Plesiomonas and Ralstonia are present in the intestinal tract of fish and are opportunistic pathogens of fish (Kim et al. 2007; Yilmaz 2019 ). However, a study on rainbow trout observed that selenium had less effect on intestinal microbes diversity under normal feeding conditions (Baia et al. 2018). These studies suggested that selenium supplementation for different fish has differential effects on intestinal microbes and the health of fish. This may be attributed to the intestinal environment in which microbial colonization species in fish gut act with selenium. Currently, few studies have been conducted on Brevundimonas, Chryseobacterium, Sphingobium, Sphingomonas, and Flavobacterium, the function of these microbes is not exactly clear, further more, we found there was no association between the intestinal microbes and the metabolites in this experiment. So, the selenium-induced changes in the metabolome of P. fulvidraco under low temperature stress may primarily related to the antioxidant capacity and the function of regulating lipid and protein metabolism of selenium, but not so much related to the changes of the intestinal microbes.