Effects of decreasing temperature on phospholipid fatty acid composition of different tissues and hematology in Atlantic salmon (Salmo salar)

Journal Pre-proof Effects of decreasing temperature on phospholipid fatty acid composition of different tissues and hematology in Atlantic salmon (Salmo salar) Chengyue Liu, Jian Ge, Yangen Zhou, Ramasamy Thirumurugan, Qinfeng Gao, Shuanglin Dong PII:

S0044-8486(19)31245-1

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734587

Reference:

AQUA 734587

To appear in:

Aquaculture

Received Date: 22 May 2019 Revised Date:

7 August 2019

Accepted Date: 8 October 2019

Please cite this article as: Liu, C., Ge, J., Zhou, Y., Thirumurugan, R., Gao, Q., Dong, S., Effects of decreasing temperature on phospholipid fatty acid composition of different tissues and hematology in Atlantic salmon (Salmo salar), Aquaculture (2019), doi: https://doi.org/10.1016/ j.aquaculture.2019.734587. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Effects of decreasing temperature on phospholipid fatty acid composition of different tissues and hematology in Atlantic salmon (Salmo salar) Chengyue Liu1, 2, Jian Ge1, Yangen Zhou1, 3*, Ramasamy Thirumurugan4, Qinfeng Gao1, 3, Shuanglin Dong1, 3

1

Key Laboratory of Mariculture, Ministry of Education, Ocean University of China,

Qingdao, Shandong Province 266100, China 2

Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea

Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, Guangdong province 510301, China 3

Function Laboratory for Marine Fisheries Science and Food Production Processes,

Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong Province 266235, China 4

Laboratory of Aquabiotics/Nanoscience, Department of Animal Science, School of

Life Sciences, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India.

*Corresponding Author: Yangen Zhou, Key Laboratory of Mariculture (Ocean University of China), Qingdao 266100, China. Tel: +86 532 8203 1590; E-mail address: [email protected].

1

Abstract: This study investigated the phospholipid fatty acid (PLFA) composition of the dorsal muscle, heart, brain, and spleen and hematology of Atlantic salmon (Salmo salar) (initial weight 72.89 ± 3.12 g) under decreasing water temperature from 16 °C to 12 °C, 8 °C, 6 °C, 4 °C, 2 °C, and 1 °C. Results showed the PLFA composition of the muscle was comparatively similar to that of the heart, whereas the unsaturated index (UI) and ratio of unsaturated to saturated fatty acids (U/S) of the spleen were the lowest. The proportion of monounsaturated fatty acids (MUFAs) of phospholipids was significantly higher in the brain than in the other tissues. The U/S, UI, UFAs (MUFAs and PUFAs) of phospholipids in each tissue increased with the temperature above 8 °C, whereas saturated fatty acids decreased. Moreover, PLFA in the muscle, heart, and spleen were more sensitive to temperature variations. Hematology parameters were unaltered except for triacylglycerol. The results demonstrate that homeostasis could be maintained by compensatory restructuring of the biological membranes in the appropriate temperature range. Homeostasis was broken when tissues responded strongly once the temperature was declined to 6 °C. When the temperature was

below 4 °C, aspartate aminotransferase (AST), alanine

aminotransferase (ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) increased significantly, and all reached a maximum at 1 °C, especially AST and ALT. When the temperature dropped to below 2 °C, the fish stopped eating, and the compensatory restructuring of PLFA in tissues failed under the combined stressors of low temperature and starvation, causing organ failure, especially in the muscle and spleen. 2

Keywords: Atlantic salmon, declining temperature, different tissues, hematology, phospholipid fatty acid

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1. Introduction According to a report by the Food and Agriculture Organization of the United Nations (FAO, 2016), annual worldwide production of Salmonidae has exceeded 2 million tons, and Salmonidae are ranked as the world’s third largest farmed fish, following Cyprinidae and Tilapia. Atlantic salmon (Salmo salar), a representative of the family Salmonidae, is rich in high quality protein and essential polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid, which has increased demand for Atlantic salmon, especially in China (Bou et al., 2017; Penney and Moffitt, 2015). Even though Salmonidae belongs to cold-water fish, it still faces a series of challenges during overwintering such as nutritional status (Waagbø, 1994), viral disease (Rimstad and Mjaaland, 2002) and low water temperature (Handeland et al., 2013). Water temperature is recognized as a primary abiotic factor influencing the entire life history of fish and other poikilotherms, including growth, development, and reproduction (Atwood et al., 2015; Beitinger et al., 2000; Tan et al., 2016). In the non-lethal temperature range, fish respond to gradual temperature changes in nature (such as those associated with tides and seasons) with a series of physiological and biochemical responses (Donaldson et al., 2008; Han et al., 2016; Ji et al., 2016). Hematology is representative of the fishes’ defense against pathogens, immunology, humoral regulation, and maintenance of the internal environment (Fazio et al., 2018). As the main pathway for the transportation of enzymes and hormones, the blood circulatory system guarantees the effectiveness of the regulatory functions 4

of the neuroendocrine system (Fazio et al., 2018; Ji et al., 2016; Lermen et al., 2004). Donaldson et al. (2008) found that changes in external variables (e.g., temperature, salinity, pollutants, etc.) quickly initiated neuroendocrine and hematological responses in fish. Řehulka et al. (2004) reported that temperature variations significantly affected red blood cell counts and mean corpuscular volume in rainbow trout (Oncorhynchus mykiss). Phospholipids play an essential role in the cellular structure of animals because it, together with protein, comprises the biological membrane (Kostal and Simek, 1998; Liu et al., 2018; Liu et al., 2019; Pettegrew et al., 2001; Snyder and Hennessey, 2003). Phospholipids are known to be the only structural component in the biological membrane that responds to ambient temperature (Jobling and Bendiksen, 2015). Variations in the composition of phospholipid fatty acids (PLFA) are particularly important during the process by which fish acclimatize to changes in temperature. This process depends on the compensation recombination of membrane phospholipid (Cengiz et al., 2017; Liu et al., 2018). Liu et al. (2018) reported that decreasing ambient temperature resulted in significant increases in monounsaturated fatty acid (MUFA) and PUFA (mainly n-3 fatty acids) levels of liver PLFA in Atlantic salmon and rainbow trout. Snyder and Hennessey (2003) analyzed the differences in the fatty acid (FA) composition of alewives (Alosa pseudoharengus) during overwintering, and found decreased PUFA levels (specifically 18:1n9, 22:6n3, and 20:5n3) in the overwintering mortalities. Recently, with the rapid improvement of engineering technology, offshore 5

breeder vessels and far offshore salmon mariculture in the Yellow Sea cold water mass are emerging (Han et al., 2016). However, abiotic factors, including low water temperature during winter, are constraining mariculture development. To our hypothesis, both PLFA and hematological could indicate the ability of Atlantic salmon to adapt to temperature changes. Hence, the purpose of the present study was to analyze the variations of hematological parameters and PLFA compositions in the tissues of Atlantic salmon under decreasing water temperatures, in order to identify the minimum tolerant temperature for fish, and to determine the mechanism by which Atlantic salmon acclimatize to low temperatures.

2. Materials and methods 2.1 Experimental design The experiment utilized juvenile Atlantic salmon (S. salar) obtained from Shandong Oriental Sea Technologies (Yantai, China), and was conducted from December 20th, 2016 to January 27th, 2017 at the Key Laboratory of Mariculture, Ocean University of China (Qingdao, China). Before the experiments commenced, the fish were acclimated for two weeks at an optimal water temperature (16 °C ± 0.5 °C) that was maintained using a semi-recirculating system. Juvenile Atlantic salmon (initial weight: 72.89 ± 3.12 g) were stocked at a density of 25 fish per tank (270 L; 0.58 m height × 0.78 m diameter). Each treatment contained four replicates. During the experimental period, the fish were fed with the same commercial trout feed twice a day (at 08:00 and 18:00) at a daily ration of approximately 2% wet body 6

weight. The FA composition of the feed is the same as described by Liu et al. (2018). Uneaten feed residue and feces were removed by siphoning after 2 h of feeding. Approximately 50% of the water in each tank was changed daily. Water temperature was controlled using a temperature control system (ZKH-WK 2000, Zhongkehai, Qingdao, China), with fluctuations within ± 0.5 °C. The temperature, pH, salinity, and dissolved oxygen con were measured twice daily (08:00 and 18:00) using a multi-parameter YSI professional-plus probe (Yellow Spring Instrument Co., Yellow Spring, Ohio, USA). Photoperiod was 12:12-h (light/dark). After the acclimation period, water temperature was decreased from 16 °C to 12 °C, 8 °C, 6 °C, 4 °C, 2 °C, and 1 °C at a rate of 0.5 °C h-1. Each temperature was maintained for one week to facilitate temperature acclimation, because it was previously reported that fish adapt to cold shock within one week (Donaldson et al., 2008). Three fish were collected from each tank at each designated temperature and euthanized using MS-222 (70 mg L-1). The brain, dorsal muscle, spleen, and heart were collected from each fish, and frozen intact in liquid nitrogen. Blood was collected from the caudal vasculature with a needle and heparinized syringe, and centrifuged for 20 min at 1160 × g after 24 h storage at 4 °C. Serum and tissue samples were stored at -80 °C in an ultra-low temperature freezer (New Brunswick Scientific, Edison, New Jersey, USA) until subsequent analyses of serum biochemistry, serum enzyme, and FA composition.

2.2 Hematology analyses 7

Serum biochemical parameters (glucose, triacylglycerol, total protein, and albumin) and serum enzyme including aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were analyzed using a Cobas C-311 analyzer for clinical chemistry (Roche Diagnostics, Shanghai, China) with commercial assay kits (GLUC HK Gen 3; TP Gen 2; LDHI Gen 2; ALTL; ASTL; ALP Gen 2; TRIGL; LDL-C Gen2, Roche Diagnostics, Shanghai, China).

2.3 Lipid extraction and FA analyses Tissue samples from three fishes collected from four replicate tanks at each temperature were analyzed. Each tissue sample (0.1 g) was homogenized for 1 min and the lipid fraction was extracted using chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene as an antioxidant following the methodology previously described by Hsieh et al. (2003). The chloroform layer was separated from the methanol and dried to a constant weight under a stream of nitrogen to obtain the lipids. Phospholipids were separated on one-dimensional thin-layer hybrid silica gel plates (100 × 50 mm) (Yinlong Company, Yantai, China) with N-hexane/ether/acetic acid (84:15:1, v/v/v) as the developing solvent, according to the method described by Hazel (1979). Fatty acid methyl esters (FAMEs) were obtained by esterification with 2 mL methyl esterification reagent (hydrochloric acid/methanol, 1:5, v/v) at 90 °C for 3 h following the methodology described by Fadhlaoui and Couture (2016). The upper phase was dried under nitrogen and resuspended in hexane. 8

The FAMEs were quantified by injecting 1 µL of sample into a gas chromatograph (GC-2010 Plus; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector instrument (GC-2010; Shimadzu, Kyoto, Japan) and an RTX-WAX fused silica capillary column (30 m in length × 0.25 mm internal diameter × 0.25 µm thickness; Phenomenex, Torrance, California, USA). The gradient temperature program was set as follows: initial temperature of 60 °C for 1.0 min; increased at a rate of 10 °C min-1 to 190 °C, followed by an increase of 2.0 °C min-1 to 260 °C; and held at 260 °C for 0.6 min. FAME identification and quantification were performed by the comparison of retention times (identification) and peak areas (quantification) with 37-FAME Mix calibration solution (Supelco, Bellefonte, Pennsylvania, USA).

2.4 Statistical analyses The SAS statistical software program, version 9.4 (SAS Institute, Cary, North Carolina, USA) was used for statistical analyses. All data were evaluated by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple comparisons test to identify significant differences (P < 0.05) between the means of different treatment groups. The amounts, orders of magnitude, and units of serum biochemical parameters and serum enzymes are many and complicated, which create barriers to comparing the results properly in one graph. Hence, all data were standardized based on multivariate statistical analysis. The formula was as follows: 9

`

where,

is the standard error,

− ̅

=

is the mean value, and j = 1, 2, 3, …, m,

This process facilitated data analyses, with the ANOVA results between processed and original data found to be identical.

3. Results Throughout the whole experiment, the water quality was maintained at optimal conditions. Atlantic salmon stopped feeding at 4 °C, and two fish died when the temperature was decreased to 1 °C.

3.1 Hematology parameters The variation of hematology parameters of Atlantic salmon with decreasing temperature were shown as Table 2 and Fig.1. Serum glucose levels increased significantly when the temperatures reduced from 16 °C to 4 °C and then decreased significantly. During the early phase of decreasing temperature, triacylglycerol (TAG) concentrations increased significantly; however, they changed insignificantly when the temperature fell below 4 °C. Total protein and albumin showed a similar trend, where concentrations significantly decreased at 8 °C, but changed insignificantly below 4 °C (Table 2). Serum enzyme activities (AST, ALT, LDH, and ALP) showed no apparent change during the early phase of the experiment, but increased significantly when the temperatures reduced to 4 °C, and reached a maximum level at

10

1 °C, especially in AST and ALT.

3.2 FA compositions of different tissues’ phospholipids At a normal temperature (16 °C), composition of tissue PLFAs showed tissue specificity. As illustrated in Tables 3-6, a total of 14-18 FA species were identified from the four tissues, including four to six SFAs species, three to five MUFAs species, and seven to eight PUFAs species. With the exception of the brain tissue, SFAs of other tissue phospholipids accounted for approximately 40%. The predominant SFA, MUFA, and PUFA were 16:0, 18:1n9, and 22:6n3, respectively. The levels of 16:0 and 22:6n3 were the highest PLFAs found in the heart, muscle, and spleen, with their sum accounting for over 50%. Regarding the brain phospholipids, the proportion of SFAs was comparatively low (27.23%); however, the proportion of 18:1n9 was higher than those of the other tissues. In the tissues’ PLFAs of the Atlantic salmon at normal temperature (16 °C), the unsaturated index (UI) was the highest in the muscle (2.77), followed by the heart (2.74), brain (2.41), and spleen (2.27). The ratio of unsaturated to saturated FAs (U/S) was highest in the brain (2.67), followed by the muscle and heart (both were 1.65), and finally the spleen (1.53). In addition, 15:0, 21:0, 16:2n4, 18:4n3, 22:5n6, and 22:4n6 were detected in tissue phospholipids; however, they were ignored because their concentrations were below 0.1%.

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3.3 FA compositions of different tissues’ phospholipids at decreasing temperatures The composition of the muscle, brain, heart, and spleen PLFAs in the Atlantic salmon acclimated to different temperatures is presented in Tables 3-6. Overall, PLFAs in each tissue showed a pattern that was dependent on the decreasing temperature, including a decrease in the proportions of SFAs, primarily due to reductions in the proportions of 16:0 and 18:0, as well as an increase in the proportion of PUFAs. The proportions of MUFAs (mainly 18:1n9) and n-3 PUFAs (mainly 20:5n3 and 22:6n3) significantly increased in the four tissues, whereas the proportion of n-6 PUFAs was unaltered, except for in the muscle tissue. During the early stage of the experiment (when the water temperature was decreased from 16 °C to 12 °C), there were decreases in SFAs and increases in unsaturated fatty acids (UFAs) in all tissues except for brain, which resulted in an elevation of the UI and U/S. When the water temperature was decreased to 8 °C, the proportion of PUFAs (mainly 20:5n3 and 22:6n3) in the brain increased, whereas the proportion of SFAs (mainly 16:0) significantly decreased. There were no significant fluctuations in fatty acid composition of the spleen at 6 °C, nor of the brain and muscle at 4 °C. The UI and U/S of muscle phospholipids decreased at 1 °C due to the decrease in MUFAs, the increase in SFAs, and the unaltered PUFA. During the entire experiment, only in the heart phospholipids, PUFAs, MUFAs, U/S, and UI increased, whereas SFAs decreased.

4. Discussion 12

4.1 Effects of decreasing temperature on hematology Serum biochemistry and enzymes, considered to be the primary parameters of fish physiology and pathology, directly reflect the status of metabolism, tissues, and organs (Dewilde and Houston, 2011; Fazllolahzadeh et al., 2011). In the present research, we found that decreasing temperature could affect not only serum biochemistry but also serum enzymes.

4.1.1

Effect of decreasing temperature on serum biochemistry Acclimation to environmental stress in fish requires a lot of energy, which is

provided by oxidation of glucose and TAG, which are the major sources for energy production from metabolism (Cho et al., 2015). The present study showed that TAG and glucose concentrations increased constantly as the temperature decreased. At 2 °C, the serum glucose concentration decreased significantly. The increase of glucose indicated the initial stress response, resulting from enhanced metabolism and heat. The rapid decrease of glucose concentrations was due to insufficient energy supply after the fish stopped eating. Similarly, Ning et al. (2017) reported that the hematology profiles in dusty rabbit fish (Siganus fuscescens) under different rates of temperature reductions, and found that glucose concentrations rapidly increased because of drastic stressors in both acute and chronic treatments. Chang et al. (2006) found that the glucose concentrations of common carp (Cyprinus carpio) increased with time under low temperature stress. Based on our finding, during the entire process of decreasing temperature, serum albumin and total protein concentrations of 13

the Atlantic salmon decreased significantly below 6 °C. This significant decrease during the later phase of the experiment might be caused by the decrease in food and protein intake.

4.1.2

Effect of decreasing temperature on serum enzyme activities AST, ALT, LDH, and ALP are indispensable for maintaining basic physiological

functions (Fazllolahzadeh et al., 2011). ALT and AST catalyze glutamic acid to pyruvic acid and oxaloacetic, respectively, via transamination (Cho et al., 2015). LDH and ASP, which are abundant in muscle, the skeleton, and kidneys, are metabolic regulatory enzymes, the former of which is a sensitive index of the calcium-phosphorus metabolic balance, and directly participates in the transportation of phosphate groups and the metabolism of calcium and phosphorus (Larsen et al., 2001), whereas the latter is a glycolytic enzyme (Collazos et al., 1998). In the present study, when temperature dropped to below 4 °C (especially at 1 °C), the activities of the four enzymes in serum significantly increased, reflecting that essential tissues such as the heart, kidneys, and liver were damaged.

4.2 Effect of decreasing temperature on the composition of tissues’ PLFAs The biological membrane comprises phospholipids and proteins, which are combined by hydrophobic interaction and slight static. Different compositions of cell membrane PLFAs vary in membrane-associated physical and biological function, including fluidity, membrane phase behavior, membrane permeability, and 14

membrane-related enzymes (Fadhlaoui and Couture, 2016; Ji et al., 2016; Wijekoon, 2012). Phospholipids play an essential role in the cell structure of insects (Kostal and Simek, 1998), fish (Tocher et al., 2008), and mammals (Renooij et al., 1976). Different physiological and biochemical functions in tissues are related to the differences in PLFA composition. 4.2.1 PLFA composition of tissues in Atlantic salmon Proportions of oleic acid (18:1n9) of the brain were much higher than those of the other tissues, resulting in a higher U/S. The proportion of SFA in the spleen was the highest, whereas PUFA was the lowest relative to the other tissues, resulting in the lowest U/S and UI in the spleen (Table 3). The brain is rich in neurons and is the central nervous system of fish. By analyzing the PLFA composition of the brain and retina in rainbow trout, Bell and Tocher (1989) found that the proportions of 16:0-22:6n3 were the highest; however, the proportion of 18:1-22:6n3 was comparatively high in phosphatidylethanolamine and phosphatidylcholines. In addition, the PLFA composition was found to have the same pattern in marine fish (Buda et al., 1994; Kreps et al., 1975). Zhang et al. (2010) compared FA compositions among various tissues (red muscle, white muscle, spleen, and liver) in pompano (Trachinotus ovatus) and found that PUFAs affected macrophage aggregates, resulting in relatively high proportions of SFAs, which consequently affected the immune system. 4.2.2 Effect of decreasing temperature on PLFA composition of different tissues In the Atlantic salmon, the U/S and UI of PLFA in each tissue became 15

progressively higher with decreasing temperatures, which can be attributed to the decrease in the proportions of SFAs (16:0 and 18:0) and the concomitant increase in UFAs (such as 18:1n9, 20:5n3, and 22:6n3) (Tables 3-6). Hazel (1979), Farkas et al. (2001), and Donaldson et al. (2008) reported that Atlantic salmon can adapt to low temperatures by compensatory restructuring of tissue phospholipids, which comprised two aspects. First, the FA level in the acyl chain decreases while the UFA level and length of the acyl chain increases. Second, the ratio of 1-bit MUFA and 2-bit highly UFA increase (Hazel, 1979; Hazel and Williams, 1990; Snyder et al., 2012; Wijekoon, 2012; Wodtke, 1978). Compared to SFAs, homologous UFAs have a lower melting point and occupy a larger space in the membrane lipid bilayer, and form a wedge structure with phospholipids, which enhances their fluidity and stability, because it removes the wrapping by the lamella structure (Cossins, 1977; Hazel, 1979). In the present study, the increased phospholipid UI in the Atlantic salmon guaranteed the proper fluidity of the liquid-crystalline phase biological membrane, thereby allowing for better adaptation to low temperatures. Hence, variation in PLFA composition was the main mechanism to achieve physiological compensation during low temperature acclimation. This mechanism can explain by temperature acclimation in poikilotherms, described as lipid phase transition (Sinensky (1974). The U/S and UI of each tissue increased with decreasing temperatures; however, the pattern of change was tissue-specific. During the early stage of the experiment (when the water temperature decreased from 16 °C to 12 °C), PLFA composition 16

changed significantly in the muscle, heart, and especially the spleen, but did not change in the brain. The muscle and heart can react to temperature changes with sensitive perception of the ambient environment (Aho and Vornanen, 2001; Ingemansson et al., 1993; Skuladottir et al., 1990). Cold shock for hours stimulates expression of the long chain PUFA synthase gene (Wijekoon, 2012). Aho and Vornanen (2001) reported that the basic heart rate of rainbow trout was increased significantly after exposure to low temperature in winter for 96 h. As the water temperature continued decreasing (from 6 °C to 4 °C), the amount of UFAs in the muscle and heart increased rapidly, whereas increased decelerated in the spleen. The amount of unsaturated PLFA in the brain began to increase significantly. Previous studies have reported that the optimum growth temperature for salmonids (e.g., rainbow trout, steelhead trout, and Atlantic salmon) ranges from 18 °C to 8 °C (Biro et al., 2004; Sigholt and Finstad, 1990; Wijekoon, 2012). If ambient temperature is below the optimal growth temperature, all tissues will respond thoroughly under the coordination of the central nervous system, including metabolism, hematology, and ion osmoregulation (Donaldson et al., 2008), which was confirmed in our findings by the significant change in hematology (e.g., serum glucose concentrations). When the temperature was below 6 °C, PLFA in the spleen were unaltered. When the temperature dropped to below 2 °C, the PLFA in the muscle and spleen changed abnormally. MUFAs and PUFAs in the brain and heart increased continuously, whereas the MUFA (18:1n9) in the muscle and spleen began to decrease 17

(Tables 3 and 6). When the temperature decreased to 6 °C, the Atlantic salmon gradually entered a dormant state, and stopped eating at 4 °C. An explanation for this is that the muscle and spleen were stressed by double stressors (i.e., low temperature and hunger), whereas important tissues such as the brain and heart were merely stressed by the low temperature. Stubhaug et al. (2007) reported that Atlantic salmon tended to protect long chain PUFAs (such as eicosapentaenoic acid and docosahexaenoic acid) from oxidation by consuming SFAs (16:0) and MUFAs when stressed by starvation. Xu et al. (2015) reported similar results where the expression of scd1 mRNA (which synthesize MUFAs) in large yellow croaker (Larimichthys crocea) increased in the brain and decreased in the liver under low temperature and starvation conditions. Skuladottir et al. (1990) found that the U/S of FA in Atlantic salmon decreased under extremely lower temperature (-3 ℃) in the muscle and liver, whereas it only slightly increased in the heart.

5. Conclusions The present study showed that when the temperature was above 8 °C, the U/S, UI, PUFAs and MUFAs of PLFAs in Atlantic salmon increased, whereas SFA decreased. PLFA in the muscle, heart, and spleen were more sensitive to temperature than that in the brain. Hematology parameters were unaltered, except for TAG. To maintain homeostasis, the Atlantic salmon adapted to low temperatures by compensatory restructuring of their biological membranes. When the temperature was below 6 °C, the fish were unable to maintain homeostasis, resulting in comprehensive 18

responses from tissues. When the temperature was below 4 °C, AST, ALT, LDH, and ALP increased significantly and reached a maximum at 1 °C, especially AST and ALT. When the temperature decreased to 2 °C, fish stopped eating, and the compensatory restructuring of PLFA in tissues failed under the combined stressors of low temperature and starvation, causing organ failure, especially in the muscle and spleen.

Acknowledgments The authors would like to express our gratitude for those who have critically review this manuscript as well as to help collect samples. This work was jointly supported by the National Natural Science Foundation of China (NSFC) (Nos. 31702364, 31572634, and 31872575), the Primary Research and Development program of Shandong Province (Nos. 2017CXGC0102, 2017CXGC0106, and 2018CXGC0101).

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parr. Aquac. Res. 34, 1423-1441. Kostal, V., Simek, P., 1998. Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa. J. Comp. Physiol. B. 168, 453-460. Kreps, E., Avrova, N., Chebotarëva, M., Chirkovskaya, E., Krasilnikova, V., Kruglova, E., Levitina, M., Obukhova, E., Pomazanskaya, L., Pravdina, N., 1975. Phospholipids and glycolipids in the brain of marine fish. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 52, 283-292. Larsen, D.A., Beckman, B.R., Dickhoff, W.W., 2001. The Effect of Low Temperature and Fasting during the Winter on Metabolic Stores and Endocrine Physiology (Insulin, Insulin-like Growth Factor-I, and Thyroxine) of Coho Salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 123, 308-323. Lermen, C.L., Lappe, R., Crestani, M., Vieira, V.P., Gioda, C.R., Mrc, S., Baldisserotto, B., Moraes, G., Morsch, V.M., 2004. Effect of different temperature regimes on metabolic and blood parameters of silver catfish Rhamdia quelen. Aquaculture. 239, 497-507. Liu, C., Zhou, Y., Dong, K., Sun, D., Gao, Q., Dong, S., 2018. Differences in fatty acid composition of gill and liver phospholipids between Steelhead trout Oncorhynchus mykiss and Atlantic salmon Salmo salar under declining temperatures. Aquaculture. 495, 815-822. Liu, C., Dong, S., Zhou, Y., Shi, K., Dajiang, S., Qinfeng, G., 2019. Temperature-Dependent Fatty Acid Composition Change of Phospholipid in Steelhead Trout Oncorhynchus mykiss Tissues. J. Ocean Univ. China. 18, 519-527. Ning, J., Qin, Y., Hu, L., Zhang, W., Li, L., Chang, Y., Song, J., 2017. Effects of abrupt and gradual decreases in water temperature on blood physiological and biochemical parameters in dusty rabbit fish Siganus fuscescens. J. Dalian Ocean Univ. 32, 294-301 (in Chinese with English Abstract). Penney, Z.L., Moffitt, C.M., 2015. Fatty-acid profiles of white muscle and liver in 23

stream-maturing steelhead trout Oncorhynchus mykiss from early migration to kelt emigration. J. Fish Biol. 86, 105-120. Pettegrew, J.W., Panchalingam, K., McClure, R.J., Gershon, S., Muenz, L.R., Levine, J.,

2001.

Effects

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lithium

administration

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phosphatidylinositol cycle constituents, membrane phospholipids and amino acids. Bipolar Disord. 3, 189-201. Řehulka, J., Minařík, B., Řehulková, E., 2004. Red blood cell indices of rainbow trout Oncorhynchus mykiss Walbaum in aquaculture. Aquac. Res. 35, 529-546. Renooij, W., Van Golde, L.M., Zwaal, R.F., Van Deenen, L.L., 1976. Topological Asymmetry of Phospholipid Metabolism in Rat Erythrocyte Membranes: Evidence for Flip-Flop of Lecithin. Eur. J. Biochem. 61, 53-58. Rimstad, E., Mjaaland, S., 2002. Infectious salmon anaemia virus. An orthomyxovirus causing an emerging infection in Atlantic salmon Review article. Acta. Pathol. Microbiol. Immunol. Scand. 110, 273-282. Sigholt, T., Finstad, B., 1990. Effect of low temperature on seawater tolerance in Atlantic Salmon Salmo salar Smolts. Aquaculture. 84, 167-172. Sinensky, M., 1974. Homeoviscous adaptation—a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli. Proc. Natl. Acad. Sci. 71, 522-525. Skuladottir, G., Schiöth, H., Gudmundsdottir, E., Richards, B., Gardarsson, F., Jonsson, L., 1990. Fatty acid composition of muscle, heart and liver lipids in Atlantic salmon, Salmo salar, at extremely low environmental temperature. Aquaculture. 84, 71-80. Snyder, R.J., Hennessey, T.M., 2003. Cold tolerance and homeoviscous adaptation in freshwater alewives Alosa pseudoharengus. Fish Physiol. Biochem. 29, 117-126. Snyder, R.J., Schregel, W.D., Wei, Y., 2012. Effects of thermal acclimation on tissue fatty acid composition of freshwater alewives Alosa pseudoharengus. Fish Physiol. Biochem. 38, 363-373. 24

Stubhaug, I., Lie, Ø., Torstensen, B., 2007. Fatty acid productive value and β-oxidation capacity in Atlantic salmon Salmo salar L. fed on different lipid sources along the whole growth period. Aquac. Nutr. 13, 145-155. Tan, E., Kinoshita, S., Suzuki, Y., Ineno, T., Tamaki, K., Kera, A., Muto, K., Yada, T., Kitamura, S., Asakawa, S., Watabe, S., 2016. Different gene expression profiles between normal and thermally selected strains of rainbow trout, Oncorhynchus mykiss, as revealed by comprehensive transcriptome analysis. Gene. 576, 637-643. Tocher, D.R., Bendiksen, E.Å., Campbell, P.J., Bell, J.G., 2008. The role of phospholipids in nutrition and metabolism of teleost fish. Aquaculture. 280, 21-34. Waagbø, R., 1994. The impact of nutritional factors on the immune system in Atlantic salmon, Salmo salar L.: a review. Aquac. Res. 25, 175-197. Wijekoon, M.P.A., 2012. Effect of water temperature and diet on cell membrane fluidity and fatty acid composition of muscle, liver, gill and intestine mucosa of adult and juvenile steelhead trout, Oncorhynchus mykiss. Memorial University of Newfoundland, Newfoundland, Canada. Wodtke, E., 1978. Lipid adaptation in liver mitochondrial membranes of carp acclimated to different environmental temperatures: phospholipid composition, fatty acid pattern, and cholesterol content. Biochim. Biophys. Acta. 529, 280-291. Xu, H., Zhang, D.L., Lv, C.H., Luo, H.Y., Wang, Z.Y., 2015. Molecular cloning and expression analysis of scd1 gene from large yellow croaker Larimichthys crocea under cold stress. Gene. 568, 100-108. Zhang, S., Xu, J., Hou, Y., Xu, S., Miao, M., Yan, X., 2010. Comparison of fatty acid composition among muscles and visceral organs of Trachinotus ovatus. Food Sci. 31, 192-195 (in Chinese with English Abstract).

25

Figure legends Fig. 1 The change tendency of UI, U/S, SFA, PUFA and MUFA of fatty acids from gill and liver phospholipids in Steelhead trout and Atlantic salmon acclimated at different temperatures. Note: SFA: saturated fatty acids, UFA: unsaturated fatty acids, UI: unsaturation index, U/S: the ratio of unsaturated to saturated fatty acids, PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids

26

Table 1 The important water quality parameters of different temperatures (mean ± S.E., n=3). Temperature (°C)

16

12

8

6

4

2

1

DO (mg/L)

7.4±0.4c

7.7±0.2bc

8.2±0.3b

8.7±0.4ab

8.9±0.2a

9.1±0.4a

9.1±0.2a

pH

7.6±0.2a

7.7±0.0a

7.7±0.1a

7.7±0.5a

7.7±0.5a

7.5±0.0a

7.8±0.3a

TAN (mg/L)

0.05±0.01a

0.04±0.02a

0.05±0.03a

0.05±0.03a

0.04±0.00a

0.04±0.03a

0.06±0.02a

0.05±0.01a

0.05±0.01a

0.05±0.02a

0.05±0.01a

0.05±0.02a

0.04±0.01a

0.04±0.02a

1.30±0.2a

1.32±0.5a

1.29±0.2a

1.30±0.2a

1.32±0.5a

1.31±0.3a

1.33±0.3a

NO2-N (mg/L) NO3-N (mg/L)

Note: TAN: total ammonia nitrogen, DO: dissolved oxygen content, NO2--N: nitrite, NO3--N: nitrate. In each row, different superscript letters indicate significant differences at the P < 0.05 level.

28

Table 2 The content of serum biochemical parameters and serum enzymes in Atlantic salmon at different temperatures Temperature (°C)

16

12

8

6

4

2

1

P value

PSE

GLU (mmol/L)

3.21c

3.27c

3.31c

4.31b

4.81a

4.41b

4.38b

<.0001

0.1153

TRI (mmol/L)

2.81d

3.28c

3.54bc

3.52cd

4.38ab

5.06a

4.89a

<.0001

0.1420

TP (g/L)

36.57a

34.73a

30.70b

27.04c

27.56c

28.64c

27.34c

<.0001

0.7126

ALB (µmol/L)

225.00a

225.71a

218.14b

191.86c

197.57c

205.29bc

193.86c

<.0001

2.5748

413.71d 418.17d

753.70c

1623.01b

2074.24a <.0001

50.2061

AST (U/L)

459.71d 429.09d

ALT (U/L)

8.92d

9.49d

14.23d

16.79d

44.97c

110.55b

171.66a

<.0001

2.0859

LDH (U/L)

670.57c

697.14c

663.71c

680.43c

1344.86b

2121.86a

2129.71a <.0001

1.9897

ALP (U/L)

50.29c

56.11c

57.00c

64.43b

110.47a

111.71a

114.71a

1.9897

<.0001

Note: Values are means of twelve replicates. Different letter indicates significant different (P < 0.05) in the same parameter at temperatures. GLU: Glucose, TRI: Triacylglycerol, TP: Total protein, ALB: Albumin, AST: Aspartate transaminase, ALT: Alanine transaminase, LDH: Lactate dehydrogenase, ALP: Alkaline phosphatase, PSE: Pooled standard error.

29

Table 3 Fatty acid composition of muscle phospholipids from Atlantic salmon acclimated to different temperatures (%) Temperature (°C)

16

12

8

6

4

2

1

P value

PSE

C14:0

1.31

1.33

1.32

1.19

1.1

1.25

1.33

0.1367

0.0633

C16:0

26.28a

25.35b

24.26c

23.15d

23.08d

23.03d

23.45d

<.0001

0.1167

C18:0

9.17a

8.36b

7.11c

6.61d

6.36d

6.50d

7.00d

<.0001

0.0728

C20:0

0.99

0.93

0.93

0.91

0.82

0.92

0.83

0.3483

0.0538

∑SFA

37.75a

35.97b

33.62c

31.86e

31.36f

31.71ef

32.60d

<.0001

0.1342

Saturated fatty acids

Monounsaturated fatty acids C16:1n7

1.18

1.27

1.32

1.35

1.37

1.32

1.25

0.0722

0.0437

C18:1n9

7.71bc

7.77abc

7.95ab

8.09ab

8.17a

7.82abc

7.47c

0.0013

0.0930

C24:1n9

1.11

1.15

1.10

0.94

0.93

1.15

1.09

0.2054

0.0726

∑MUFA

10.00ab

10.19ab

10.37ab

10.38ab

10.48a

10.29ab

9.81b

0.0493

0.1365

Polyunsaturated fatty acids C18:2n6

6.67b

7.19a

7.39a

7.46a

7.48a

7.29a

7.22a

0.0002

0.0984

C18:3n3

0.55b

0.82a

1.07a

0.91a

0.88a

0.92a

0.87a

0.0004

0.0597

C18:3n6

1.62a

1.44ab

1.34b

1.25b

1.24b

1.25b

1.28b

0.0106

0.0712

C20:3n3

1.18ab

1.24ab

1.38a

1.15ab

1.10b

1.18ab

1.22ab

0.0383

0.0522

C20:4n6

2.25d

2.51c

2.95b

3.34a

3.36a

3.23ab

3.18ab

<.0001

0.0767

C20:5n3

5.59c

5.61c

6.10b

6.20ab

6.43a

6.50a

6.45a

<.0001

0.0860

C22:6n3

34.39d

35.04c

35.78b

37.44a

37.69a

37.64a

37.36a

<.0001

0.1188

∑PUFA

52.25d

53.84c

56.01b

57.76a

58.16a

58.00a

57.59a

<.0001

0.1440

U/S

1.65f

1.78e

1.97d

2.14b

2.19a

2.15ab

2.07c

<.0001

0.0116

UI

2.77e

2.83d

2.94c

3.04ab

3.07a

3.06a

3.04b

<.0001

0.0069

n-3 PUFA

41.71e

42.71d

44.33c

45.71b

46.09ab

46.24a

45.91ab

<.0001

0.1306

n-6 PUFA

10.54c

11.14b

11.68a

12.05a

12.07a

11.76a

11.68a

<.0001

0.1436

n3/n6

3.97

3.84

3.8

3.79

3.82

3.93

3.93

0.2006

0.0569

30

Note: Values are means of four replicates. Means within lines with the same letter are significantly different (P < 0.05) based on the analysis of One-way ANOVA by the Student-Newman-Keuls (SNK) test. PSE: Pooled standard error, SFA: saturated fatty acids, UFA: unsaturated fatty acids, UI: unsaturation index, U/S: the unsaturated to saturated

fatty

acids

ratio,

PUFA:

polyunsaturated

fatty

acids,

MUFA:

monounsaturated fatty acids, n-3 PUFA: Omega-3 series polyunsaturated fatty acids, n-6 PUFA: Omega-6 series polyunsaturated fatty acids, n-3/n-6: The Omega-3 to Omega-6 series polyunsaturated fatty acids ratio.

31

Table 4 Fatty acid composition of heart phospholipids from Atlantic salmon acclimated to different temperatures (%) Temperature (°C)

16

12

8

6

4

2

1

P value

PSE

C14:0

0.75

0.79

0.78

0.71

0.78

0.71

0.69

0.4065

0.0374

C16:0

26.86a

26.28b

25.79c

24.97d

24.25e

23.86f

23.45g

<.0001

0.1117

C17:0

0.43

0.42

0.46

0.46

0.46

0.46

0.46

0.8437

0.0245

C18:0

9.62a

9.45a

9.09b

8.62c

8.28d

7.80e

7.54e

<.0001

0.1129

∑SFA

37.67a

36.94b

36.12c

34.76d

33.77e

32.84f

32.15g

<.0001

0.1495

Saturated fatty acids

Monounsaturated fatty acids C16:1n7

1.05

0.91

0.99

0.88

0.99

0.92

0.89

0.3923

0.0591

C17:1n7

0.19

0.29

0.22

0.21

0.24

0.21

0.24

0.6949

0.0378

C18:1n9

6.61c

6.75c

7.31b

7.71ab

7.95ab

8.14a

8.31a

<.0001

0.1783

C20:1n9

0.5

0.37

0.37

0.37

0.39

0.35

0.36

0.167

0.0378

C24:1n9

1.72a

1.64ab

1.67ab

1.49bc

1.38c

1.43bc

1.45bc

0.0013

0.0547

∑MUFA

10.07b

9.97b

10.55ab

10.66ab

10.95ab

11.06ab

11.25a

0.0151

0.2497

Polyunsaturated fatty acids C18:2n6

6.84b

7.21ab

7.50a

7.63a

7.62a

7.78a

7.73a

0.0125

0.1732

C18:3n3

0.39

0.45

0.36

0.36

0.4

0.39

0.41

0.9012

0.0482

C18:3n6

1.30a

1.15a

0.97b

0.91b

0.90b

0.88b

0.92b

<.0001

0.0512

C20:2n6

0.81

0.74

0.65

0.7

0.72

0.67

0.65

0.2385

0.0469

C20:3n3

1.4

1.32

1.14

1.22

1.18

1.13

1.1

0.3032

0.0932

C20:4n6

2.5

2.55

2.6

2.58

2.66

2.81

2.74

0.223

0.0865

C20:5n3

4.48e

4.75de

5.04cd

5.34bc

5.55b

5.77ab

6.05a

<.0001

0.1226

C22:6n3

34.53e

34.92de

35.06d

35.84c

36.24b

36.67a

37.00a

<.0001

0.1289

∑PUFA

52.25d

53.09d

53.33d

54.58c

55.27bc

56.10ab

56.60a

<.0001

0.3242

U/S

1.65g

1.71f

1.77e

1.88d

1.96c

2.04b

2.11a

<.0001

0.0145

UI

2.74e

2.78d

2.80d

2.87c

2.91b

2.95a

2.99a

<.0001

0.0109

32

n-3 PUFA

40.80f

41.43e

41.61e

42.77d

43.38c

43.96b

44.56a

<.0001

0.1791

n-6 PUFA

11.45

11.65

11.72

11.81

11.90

12.14

12.04

0.2663

0.1979

n3/n6

3.56

3.56

3.55

3.62

3.65

3.62

3.70

0.2842

0.0432

Note: Values are means of four replicates. Means within lines with the same letter are significantly different (P < 0.05) based on the analysis of One-way ANOVA by the Student-Newman-Keuls (SNK) test. PSE: Pooled standard error, SFA: saturated fatty acids, UFA: unsaturated fatty acids, UI: unsaturation index, U/S: the unsaturated to saturated

fatty

acids

ratio,

PUFA:

polyunsaturated

fatty

acids,

MUFA:

monounsaturated fatty acids, n-3 PUFA: Omega-3 series polyunsaturated fatty acids, n-6 PUFA: Omega-6 series polyunsaturated fatty acids; n-3/n-6: The Omega-3 to Omega-6 series polyunsaturated fatty acids ratio.

33

Table 5 Fatty acid composition of brain phospholipids from Atlantic salmon acclimated to different temperatures (%) Temperature (°C)

16

12

8

6

4

2

1

P value

PSE

C14:0

0.42

0.47

0.39

0.44

0.37

0.43

0.37

0.1931

0.0267

C16:0

18.36a

18.29a

17.69b

16.50c

15.55d

15.09d

15.09d

<.0001

0.1405

C17:0

0.17

0.17

0.18

0.19

0.16

0.18

0.19

0.5389

0.0098

C18:0

7.84a

7.78a

7.46a

7.34a

6.70b

6.41b

6.42b

<.0001

0.1875

C21:0

0.24

0.23

0.21

0.23

0.19

0.22

0.16

0.4243

0.0270

C22:0

0.19

0.24

0.16

0.18

0.19

0.26

0.15

0.2164

0.0306

∑SFA

27.23a

27.18a

26.08b

24.88c

23.16d

22.59d

22.38d

<.0001

0.2515

Saturated fatty acids

Monounsaturated fatty acids C16:1n7

1.54

1.53

1.43

1.37

1.38

1.38

1.48

0.2539

0.0595

C18:1n9

24.03c

24.48bc

25.11ab

25.65a

26.11a

25.96a

26.07a

<.0001

0.2530

C20:1n9

1.58

1.57

1.61

1.33

1.38

1.49

1.61

0.2546

0.0886

C22:1n9

0.55

0.56

0.49

0.5

0.6

0.55

0.52

0.3981

0.0340

C24:1n9

8.14a

8.19a

7.75b

7.46b

7.48b

7.48b

7.33b

<.0001

0.1087

∑MUFA

35.84

36.33

36.39

36.31

36.96

36.86

37.01

0.0998

0.2851

Polyunsaturated fatty acids C18:2n6

1.29

1.28

1.29

1.28

1.17

1.23

1.38

0.3643

0.0576

C18:3n3

0.22a

0.21a

0.18ab

0.19ab

0.16b

0.18ab

0.18ab

0.0078

0.0102

C20:2n6

0.38

0.37

0.38

0.37

0.34

0.37

0.38

0.7536

0.0196

C20:3n3

0.48

0.41

0.42

0.43

0.37

0.41

0.41

0.1135

0.0240

C20:4n6

1.46bc

1.38c

1.56abc

1.70ab

1.76a

1.60abc

1.63ab

0.0015

0.0538

C20:5n3

5.07bc

5.02c

5.20abc

5.35abc

5.56a

5.58a

5.51ab

0.0045

0.1072

C22:6n3

28.01c

27.83c

28.49c

29.49b

30.52a

31.18a

31.13a

<.0001

0.2094

∑PUFA

36.92cd

36.49d

37.53c

38.82b

39.88a

40.55a

40.61a

<.0001

0.2437

U/S

2.67e

2.68e

2.83d

3.02c

3.32b

3.43ab

3.47a

<.0001

0.0370

UI

2.41d

2.39d

2.45c

2.52b

2.60a

2.63a

2.63a

<.0001

0.0112

34

n-3 PUFA

33.78cd

33.46c

34.30c

35.46b

36.61a

37.36a

37.22a

<.0001

0.2101

n-6 PUFA

3.14

3.03

3.23

3.35

3.27

3.20

3.39

0.2102

0.0944

n3/n6

10.77

11.06

10.62

10.57

11.20

11.69

10.97

0.2938

0.3211

Note: Values are means of four replicates. Means within lines with the same letter are significantly different (P < 0.05) based on the analysis of One-way ANOVA by the Student-Newman-Keuls (SNK) test. PSE: Pooled standard error, SFA: saturated fatty acids, UFA: unsaturated fatty acids, UI: unsaturation index, U/S: the unsaturated to saturated

fatty

acids

ratio,

PUFA:

polyunsaturated

fatty

acids,

MUFA:

monounsaturated fatty acids, n-3 PUFA: Omega-3 series polyunsaturated fatty acids, n-6 PUFA: Omega-6 series polyunsaturated fatty acids; n-3/n-6: The Omega-3 to Omega-6 series polyunsaturated fatty acids ratio.

35

Table 6 Fatty acid composition of spleen phospholipids from Atlantic salmon acclimated to different temperatures (%) Temperature (°C)

16

12

8

6

4

2

1

P value

PSE

C14:0

1.03c

1.28a

1.25ab

1.18abc

1.12abc

1.14abc

1.08c

0.0054

0.0405

C16:0

26.93a

26.33b

25.26c

24.76c

24.77c

24.97c

25.36c

<.0001

0.1386

C17:0

0.62

0.64

0.65

0.67

0.64

0.66

0.63

0.4333

0.0348

C18:0

10.89a

9.14b

8.19c

8.12c

8.17c

8.39bc

8.62bc

<.0001

0.2167

∑SFA

39.68a

37.40b

35.35c

34.73c

34.69c

35.15c

35.68c

<.0001

0.2353

Saturated fatty acids

Monounsaturated fatty acids C16:1n7

1.15

1.41

1.31

1.31

1.26

1.24

1.17

0.2421

0.0726

C18:1n9

11.75ab

12.32a

12.29a

11.68ab

11.84ab

11.64ab

11.36b

0.0040

0.1587

C20:1n9

0.60

0.58

0.50

0.54

0.54

0.55

0.55

0.3146

0.0304

C24:1n9

1.97

2.11

1.94

2.04

1.93

1.99

2.00

0.6382

0.0724

∑MUFA

15.47ab

16.42a

16.03ab

15.58ab

15.57ab

15.42ab

15.07b

0.0131

0.2267

Polyunsaturated fatty acids C18:2n6

7.44a

7.49a

7.40a

6.76b

6.73b

6.62b

6.61b

<.0001

0.1144

C18:3n3

0.83a

0.78a

0.61b

0.60b

0.55b

0.49b

0.52b

<.0001

0.0384

C20:2n6

1.40a

1.07b

1.03b

1.01b

0.96b

0.88b

0.97b

<.0001

0.0444

C20:3n6

1.04

1.19

1.20

1.19

1.16

1.12

1.13

0.838

0.0806

C20:4n6

4.56b

4.88b

5.31a

5.79a

5.85a

5.78a

5.67a

<.0001

0.1278

C20:5n3

4.36b

4.80ab

5.02a

5.41a

5.34a

5.33a

5.28a

0.0037

0.1588

C22:2n6

0.96

0.99

0.86

0.98

0.78

0.77

0.71

0.0755

0.0730

C22:6n3

24.25d

24.99c

27.19b

27.93a

28.37a

28.44a

28.36a

<.0001

0.1974

∑PUFA

44.84d

46.18c

48.62b

49.68a

49.74a

49.43ab

49.25ab

<.0001

0.2482

U/S

1.52d

1.67c

1.83ab

1.88a

1.88a

1.85ab

1.80b

<.0001

0.0174

UI

2.26d

2.35c

2.50b

2.56a

2.58a

2.57a

2.56a

<.0001

0.0110

n-3 PUFA

29.44d

30.57c

32.82b

33.95a

34.26a

34.26a

34.16a

<.0001

0.2062

36

n-6 PUFA

15.4

15.61

15.8

15.74

15.48

15.17

15.09

0.2309

0.2151

n3/n6

2.22c

2.34b

2.46ab

2.54ab

2.60a

2.57a

2.65a

0.0003

0.0527

Note: Values are means of four replicates. Means within lines with the same letter are significantly different (P < 0.05) based on the analysis of One-way ANOVA by the Student-Newman-Keuls (SNK) test. PSE: Pooled standard error, SFA: saturated fatty acids, UFA: unsaturated fatty acids, UI: unsaturation index, U/S: the unsaturated to saturated

fatty

acids

ratio,

PUFA:

polyunsaturated

fatty

acids,

MUFA:

monounsaturated fatty acids, n-3 PUFA: Omega-3 series polyunsaturated fatty acids, n-6 PUFA: Omega-6 series polyunsaturated fatty acids; n-3/n-6: The Omega-3 to Omega-6 series polyunsaturated fatty acids ratio.

37

Fig.1

27

Highlights Atlantic salmon can adapt to low temperatures by a compensatory reconstruction of phospholipid fatty acid of biological membrane. The muscle, heart and spleen phospholipid fatty acid of Atlantic salmon were more sensitive to temperature decline. With declining water temperature, the phospholipid fatty acid composition of Atlantic salmon significantly changed in muscle, heart, brain, and spleen.

Statement of relevance In this study, we investigated the effects of descending temperatures on phospholipid fatty acid composition of different tissues and hematology in Atlantic salmon (Salmo salar). Our findings showed the differences of low temperature tolerance and acclimation mechanisms of the Atlantic salmon. This manuscript provides useful information about low-temperature-induced impacts of environmental stresses on the acclimation of salmonids.