The experimental shrimps, with an average weight of 5.4 ± 0.7 g, were obtained from a commercial farm in Panyu (Guangdong, China). The shrimps were immediately transported to the lab and acclimated in 500 L filtered, aerated (oxygen pump, HAP-120, HAILEA, Guangdong, China) seawater tanks (Guanzhong, K500 L, Jiangsu, China) at least 4 days before experiments. During the acclimation stage, the water salinity and temperature in tanks were consistent with that of the culture ponds (salinity 5‰, pH 8.3 ± 0.1 and temperature 28 ± 1°C). Commercial shrimp feeds (Haida Group Feed, Jieyang, China) were given two times per day (5% of shrimp body weight per time).

Sixty-three healthy shrimps were randomly divided into three replicate tanks. They were placed in an artificial climate incubator (Laifu, Ningbo, China), and the water temperature was decreased from acclimation temperature (AT, 28°C) to 13°C with a cooling rate of 7.5°C/day (2.5°C/8 h). After 13°C for 24 h, the water temperature rose back to 28°C at the same rate.

Total RNA was extracted from hemocytes and hepatopancreases using RNAiso Plus reagent (TaKaRa, Japan) following the manufacturer’s protocol. RNA quality was assessed by electrophoresis on 1.0% agarose gel, and concentration was tested by mySPEC (VWR, USA). Total RNA was purified, and first-strand cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO, Shanghai) according to the manufacturer’s instructions.

The SYBR Green real-time Polymerase Chain Reaction (PCR) assays were carried out on a CFX Connect™ Real-Time System (Bio-Rad) using THUNDERBIRD® SYBR® qPCR Mix (TOYOBO). Previous studies showed that the expressions of β-actin were constant after environmental stimuli such as ammonia (Duan et al., 2018a), dissolved oxygen (Han et al., 2018b), and pH stress (Han et al., 2018a). Therefore, we used β-actin as the housekeeping gene, and specific primer sequences were designed based on the coding sequence of the target genes using Primer Premier 6.0 software (Table 1). The real-time PCR program was 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s, followed by 1 step of 95°C for 10 s. Melting curves were obtained by increasing the temperature from 65 to 95°C (0.5°C/s) to denature the double-stranded DNA. The relative mRNA expressions were calculated by the comparative Ct method (2^−ΔΔCt).

All the data were presented as mean ± SD of triplicates. Data were statistically analyzed by SPSS 19.0 with one-way ANOVA and Tukey test. p < 0.05 was significant difference.

According to the results of hepatopancreas with HE staining, the hepatocytes in 28°C exhibited the well-organized tubules. With the decrease of temperature, stellate tubule lumen appeared dilatation, and some vacuoles appeared and ruptured to make the epithelial cell layer thinner. The secretory cells (“blasenzellen”, B-cells), which are the main site for synthesis of digestive enzymes, typically contain a single large secretory vesicle. The number and volume of B-cells significantly increased during the cooling process. All these symptoms got remission during the temperature return process (Figure 1).

Analysis of plasma metabolite concentrations is shown in Figure 2. Compared to the AT group, the contents of glucose (Glu) decreased to the minimum value (50.78 mmol/L) at 13°C for 24 h. After temperature rose back to 28°C, it recovered to the level nearly of AT (r28°C = 62.82 mmol/L, AT = 64.81 mmol/L) (Figure 2A). The contents of triglyceride (TG) and total cholesterol (TC) decreased after cooling and reached the lowest (TG = 0.25 mmol/L, TC = 0.39 mmol/L) at 23°C, and then increased gradually and reached the peak of 0.49 mmol/L and 1.12 mmol/L, respectively, in 13°C for 24 h (Figure 2B), while the change of TG contents was not statistically different with the AT group. Changes of total protein (TP) content during the cooling process were similar to the TC and TG. During the return process, the content of TP reduced to 20.67 g/L in r28°C (Figure 2C). Alkaline phosphatase (ALP) activities in plasma decreased significantly after cooling and reached the lowest (2 U/L) at 13°C. After temperature rose back to 28°C, ALP activities increased to 15.67 U/L, which is near the activities at 18°C (17.67 U/L) in the cooling process. Alanine aminotransferase (ALT) activities remained stable from AT to 18°C and then significantly increased and reached the highest level (70.33 U/L) at 13°C. During the return process, ALT activities decreased to the level near AT (43.67 U/L) (Figure 2D).

In the hepatopancreas, the relative expressions of GRP78 increased significantly at 18°C and reached the peak at 13°C, which is about fourfold that at AT. During the next 24 h maintaining in 13°C and the return process, GRP78 expressions significantly decreased and they were near to the level of AT in r28°C (Figure 3A). Expressions of apoptosis related genes including CASP3 and ASK1 showed the same trend with the PERK sub-pathway and their highest expression levels appeared in 13°C (Figure 3B). In UPR, expressions of ATF6 showed the same trend found in GRP78 (Figure 3C). In the IRE1 sub-pathway, expressions of IRE1 and XBP1 reached the peak at 18°C (Figure 3D). In the PERK sub-pathway, expressions of PERK, eIF2α, and ATF4 increased gradually during the cooling process, and the highest expressions (16.67, 9.74, and 6.21 folds compared with that at AT, respectively) appeared at 13°C and then decreased significantly. There was no obvious difference among the expressions of eIF2α and ATF4 between r28°C and AT (Figure 3E).

In the hemocyte, the expression level of GRP78 remained stable from AT to 13°C and then significantly increased in 13°C for 24 h, which is more than twofold the level in AT. After temperature rose back, expressions of GRP78 were approximate to the level of AT in r28°C (Figure 4A). For genes related to apoptosis, expressions of CASP3 and ASK1 showed the same trend with the PERK sub-pathway (Figure 4B). In UPR, the expressions of ATF6 decreased significantly after cooling in 23°C and then increased gradually and reached the highest level in 13°C for 24 h compared with the expression in AT. After temperature rose back, it returned to nearly the level of AT (Figure 4C). In the IRE1 sub-pathway, IRE1 and XBP1 showed a similar trend as ATF6 (Figure 4D). In the PERK sub-pathway, expressions of PERK, eIF2α, and ATF4 increased gradually after cooling, reaching at 13°C for 24 h approximately seven-, four-, and twofold, respectively, of the levels found in AT, but after temperature rose back the expressions decreased (Figure 4E).

The hepatopancreas as a vital organ of crustaceans is involved in excretion, molting, diverse metabolic activities, and storage of energy reserves (Yepiz-Plascencia et al., 2000; Verri et al., 2001). In this study, the number and volume of B-cells in hepatopancreas tubules was significantly increased after suffering cold stress. This may be related to the fact that B-cells are the main site of absorption and digestion of nutrients (Almohanna and Nott, 1989; Wang et al., 2016). We suspected that the high rate of synthesis and release of digestive enzymes in B-cells accelerated the mobilization of nutrients in hepatopancreas tubules, by which shrimp can adapt to environmental stress.

The hepatopancreas of shrimp has high self-repairing ability. L. vannamei can repair hepatopancreas injury after long-term exposure to low zinc (Jui-Pin et al., 2008) and low pH (Han et al., 2018a). The hepatopancreas weight of L. vannamei significantly declined after fasting, but then increased immediately after re-feeding (Pascual et al., 2006; Sanchez-Paz et al., 2007). In the present study, histological damage of the hepatopancreas got remission after temperature return, confirming the shrimp’s self-repair ability.

It has been widely accepted that protein acts as the main energy source for shrimp (New, 1976; Zhang et al., 2006; Cuzon et al., 2010). Research has shown that lipids are the main energy source of tilapia (Oreochromis niloticus) during long-time hypoxia stress (Li et al., 2018). In this study, the results showed that lipids (TC, the major components of lipids, supply and store energy) and protein (TP provides energy and transports various metabolites) in plasma responded more rapidly to temperature fluctuation, while Glu remained stable before 13°C and recovered to AT levels after temperature rose back to 28°C. It has been reported that the hepatopancreas is typically high in lipids and appears to be the main site for gluconeogenesis in decapod crustaceans (Hervant et al., 1999; Vinagre and Silva, 2011; Reyes-Ramos et al., 2018; Berry et al., 2019). Thus, combined with hepatopancreas histology and plasma results, we deduced that the increase of B-cells facilitates the gluconeogenesis to synthesize glucose from protein and lipid, by which shrimps maintain glucose demand under cold stress. However, after temperature dropped to 13°C, the rupture of hepatopancreas tubules causes lipids and proteins to enter hemolymph, resulting in an increase of lipid and protein content in plasma. The glucose content decreased at the same time due to the damage of the hepatopancreas.

It is known to all that nonspecific immunity plays an important role in the immune defense of aquatic animals. L. vannamei depends entirely on cellular and humoral immunity to prevent external injury (Iwanaga and Lee, 2005). ALP is directly involved in the transfer and metabolism of phosphoric acid groups in organisms and plays a significant role in the immune system against pathogens. The present study showed that ALP played a major role during the cold stress response in Sparus aurata and L. vannamei, and this is probably because ALP can help protect the hepatopancreas and hemolymph from cold-stress damage (Mateus et al., 2017; Peng et al., 2018). The activity of ALT in plasma can reflect the damage of the hepatopancreas (Jiang et al., 2014; Yan et al., 2016). It has been shown that various forms of stress can cause an increase in plasma ALT activity in fish (Cho et al., 1994), and it is responsive to temperature change in fish (Costas et al., 2012). In the present study, the ALP activity decreased to the lowest level, and the ALT activity increased to the highest level at 13°C, indicating that the shrimp was damaged at this temperature. However, there was no obvious change in the next 24 h when the temperature was kept at 13°C, but it increased significantly after the temperature rose back to 28°C. Thus, we deduced that shrimp has the ability to adapt low-temperature stress to a certain extent, and these results were consistent with those found in the hepatopancreas histological analysis.

UPR is a feedback regulatory system, capable of controlling the elimination of misfolded proteins in the ER, thereby maintaining the homeostasis of the endoplasmic reticulum. Appropriate ERS can activate UPR to improve the ER function and protect cells. But if the imbalance exceeds its regulating ability, it will lead to apoptosis. In invertebrates, apoptosis is also an effector factor of immune response. Environmental stresses such as temperature stimulation, pH changes, and toxic substances can induce apoptosis. In this study, the relative mRNA expressions of all UPR pathway- and apoptosis-related genes in the hepatopancreas and hemocytes were significantly changed during the cooling and re-warming process, which indicated that the UPR pathway and apoptosis participated in this process.

Previous studies indicated that shrimp can adapt to the environmental changes by self-regulation to a certain degree. In these studies, it was observed that the glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase activities increased after shrimp were exposed to Zn for 7 days but declined after exposure for 14 and 28 days (Jui-Pin et al., 2008). Additionally, the lipid peroxidation levels in shrimp had no significant changes between 10 and 15 days after Cd exposure (Chiodi Boudet et al., 2015). In our study, the expressions of genes (GRP78, ATF6, IRE1, XBP1, PERK, eIF2α, and ATF4) in the hepatopancreas reached their highest level at 13°C instead of 13°C for 24 h. The plasma metabolites concentration analysis also showed that ALT activity got its highest point at 13°C, and the activity of ALT in plasma is inversely proportional to the health of hepatopancreas. This finding is consistent with previous studies and confirms the self-repair ability of shrimp. In addition, all these related gene expressions reached their highest level in 13°C in hepatopancreas, while in hemocytes their peak appeared in 13°C for 24 h. Thus, we deduced that the shrimp response to temperature fluctuation in the hepatopancreas may be relatively rapid compared to that that in hemolymph.

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.