SHEN Weiliang , LIU Cheng , NI Jie GAO Xinming NI Junjie WANG JianpingJIN Shan HOU Congcong WU Xiongfei and ZHU Junquan

1) Key Laboratory of Applied Marine Biotechnology of Ministry of Education, School of Marine Sciences, Ningbo University,Ningbo 315211, China

2) Ningbo Academy of Oceanology and Fishery, Ningbo 315012, China

Abstract Phascolosoma esculenta is an intertidal organism that has recently attracted attention because of its ability to survive at relatively low temperatures. However, the gene regulation in P. esculenta in relation to its response to low temperatures is unclear. To explore the low temperature adaptability of P. esculenta, this study analyzed the changes in the morphology and hsp70 and hsp90 gene expression of P. esculenta exposed to a low temperature gradient. At 5℃, P. esculenta stretched and softened, and some individuals moved apart from the group. Histological analysis revealed cuticle breaches, myofiber scattering, disruption of the body wall,and epithelial layer dispersion and muscle fiber rupturing in the nephridium. Furthermore, the mRNA expression levels of hsp70 and hsp90 increased under acute low temperature stress, suggesting that these genes function in low temperature tolerance. Overall, low temperature stress causes morphological changes and histological damage in P. esculenta, and hsp70 and hsp90 potentially function in the low temperature adaptability of P. esculenta. Our results provide new insights into the adaptive strategies of P. esculenta under low temperature environments.

Key words Phascolosoma esculenta; low temperature; morphological alteration; hsp70; hsp90

1 Introduction

Temperature is a key environmental factor regulating the physiology, energy expenditure, and overall fitness of aquatic organisms (Cherkasovet al., 2006; Killenet al.,2010; Liuet al., 2019). The intertidal zone, an important habitat of aquatic organisms, is affected by many factors,such as rising and falling tides, short sunshine duration,and large temperature differences between day and night.These factors, especially the variability in day/night temperature gradient, form complex and changing environments (Ansart and Vernon, 2003; Rongeset al., 2012; Chibaet al., 2016). Therefore, the distribution of intertidal organisms is often strongly affected by the temperature of the air and of the substrate surface in their habitat (Loomis, 1995; Helmuthet al., 2002; Marshallet al., 2010;Somero, 2010; Wetheyet al., 2011). In winter, the surface of the marine floor in intertidal zones is often found at temperatures as low as 4℃; many intertidal organisms endure such low temperatures to survive by hibernating,burrowing, or moving slowly (Ansart and Vernon, 2003;Rongeset al., 2012; Chibaet al., 2016). Furthermore, organisms respond to environmental stress by expressing a particular set of stress proteins that preserve the stability of vital proteins (Li and Zhang, 2018).

Heat shock proteins (HSPs) are a highly conserved family of proteins with important biological functions under abrupt environmental changes (Srivastavaet al., 1998; Aliet al., 2003; Zhaoet al., 2012; Luoet al., 2017; Kimet al.,2019). Under normal conditions, HSPs act as molecular chaperones, participating in metabolism, apoptosis, and cell cycle regulation (Luoet al., 2017; Wanget al., 2017; Kimet al., 2019). HSPs are largely expressed to preserve cellular homeostasis by preventing or correcting stress-induced abnormal protein folding, such as during abrupt environmental changes, heavy metal stress, or bacterial infection (Morimoto, 1993; Srivastavaet al., 1998; Aliet al.,2003; Zhaoet al., 2012; Luoet al., 2017). Among HSPs,HSP70 and HSP90 are the most widely studied high-molecular-weight HSPs. The expression levels of these HSPs are markedly upregulated upon stress exposure; these proteins help eliminate abnormal or denatured proteins in cells and prevent cell death caused by ATP depletion (Morimotoet al., 1997; Mathew and Morimoto, 1998). The upregulated expression levels of thehsp70andhsp90genes in aquatic animals under low temperature stress protect tissues and cells from damage caused by low temperature(Brunet al., 2008; Jiet al., 2016; Chenet al., 2019). However, up to date, few studies have examined the expression characteristics of thehsp70andhsp90genes in intertidal organisms in response to low ambient tem- perature.

Phascolosoma esculenta(Sipuncula: Phascolosomatidae),an economically important species in the Chinese intertidal zone, has a delicious taste and is high in protein. It lives in high-tide areas with rich sediment, sand, and organic matter.P.esculentais widely distributed in the coastal shoals of Southeast China (Zhuet al., 2007; Suet al.,2010; Gaoet al., 2019) and can adapt to a wide temperature range; in fact, it can survive at 0℃. However, to date,few studies have explored the adaptation mechanism ofP.esculentato low temperatures. Therefore, this study investigated the low temperature adaptation ofP. esculentaby studying changes in its morphology andhsp70andhsp90expression under controlled experimental conditions. This study may serve as a theoretical basis for the artificial cultivation and breeding ofP. esculenta.

2 Materials and Methods

2.1 Experimental Animals

P. esculenta(4 – 6 cm body length, 3.0 – 5.5 g body weight)were collected from Xiangshan (29?46?N, 121?61?E), in Ningbo, Zhejiang Province, China in October 2017. At this time, the sea temperature was approximately 20℃.The experiment was conducted at the Science and Technology Innovation Base of the Ningbo Academy of Oceanology and Fishery, in Ningbo, Zhejiang, China. The collected specimens ofP. esculentawere acclimated for a week in seawater at 20 ± 1℃, pH 7.6 ± 0.5, and a salinity of 24 ± 0.8. Two-thirds of the seawater was renewed twice daily.

2.2 Acute Low Temperature Stress Experiment

The temperature of the seawater was set at 5℃ to simulate the temperature for the natural survival ofP. esculentaduring winter. Before the experiment, the water temperatures of glass tanks (60 cm × 40 cm × 50 cm) in the control and the 3 low temperature groups were set at 20 ± 0.5,15 ± 0.5, 10 ± 0.5, and 5 ± 0.5℃, respectively, using a controlled ecological-experiment system (Huixin Titanium Equipment Development Co., Ltd., Dalian, China). Then,480 healthyP. esculentaindividuals were distributed randomly in equal numbers among 4 groups of 3 replicates,each of which included 40 individuals. Six individuals from each group were dissected at 6, 12, 24, 48, 72, and 96 h after low temperature stress initiation, respectively. First,the coelomic fluid was drawn from the tail position with a 1 mL sterile syringe and then stored in a 1.5 mL RNasefree Eppendorf (EP) tube. Then, sampled individuals were dissected on ice, and the tissue samples, including body walls, coelomic fluids, intestines, contracted muscles, and nephridia were placed in 1.5 mL RNase-free EP tubes. Finally, the samples were subjected to quick freezing with liquid nitrogen and stored at ?80℃. In addition, morphological changes ofP. esculentain water tanks were observed at 96 h after low temperature stress treatment initiation.

2.3 Paraffin Sectioning

The body wall, coelomic fluid, intestine, and nephridium tissues of the control and low temperature groups were fixed in Bouin’s fluid (Solarbio). Subsequently, the samples were cut into slices for dehydration, clearing, encapsulation,and hematoxylin-eosin staining for observation under a light microscope (Olympus BX51, Japan). Images were acquired using Image-Pro Plus 6.0, image analysis software.

2.4 Total RNA Extraction and Real-Time Quantitative PCR (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Tiangen, Beijing, China) following the manufacturer’s instructions and then reverse transcribed into cDNA by using the Prime Script? RT reagent kit (TaKaRa, Dalian, China).

The specific primers ofP. esculenta hsp70(GenBank:EU416330.1) andhsp90(GenBank: GQ503177.1)used for RT-qPCR were designed by Primer Premier 5.0 software.These primers and the specific primers of the reference genegapdh(Suet al., 2010)are shown in Table 1. The expression levels ofhsp70andhsp90in various tissues (body wall, coelomic fluid, intestine, contractive muscle, and nephridium) ofP. esculentawere analyzed by RT-qPCR for the control group. In addition, RT-qPCR was performed for the temporal-spatial expression analysis ofhsp70andhsp90mRNA. RT-qPCR was run utilizing SYBR green Master I (Roche, Basel, Switzerland) on a Roche Light-Cycler 480 (Bioplastics, Holland). The PCR steps were as follows: 94℃ for 5 min; 40 cycles (94℃ for 20 s, 60℃ for 20 s, and 72℃ for 20 s); and a final extension at 72℃ for 10 min. The relative quantity ofhsp70andhsp90mRNA expression was calculated using the 2?ΔΔCTmethod.

Table 1 Primers used for the expression analysis of the hsp70 and hsp90 genes in Phascolosoma esculenta by real-time quantitative PCR

2.5 Statistical Analysis

Data were analyzed using SPSS 17.0 and Microsoft Excel software. All data are expressed as means ± SE. One-Way ANOVA was performed to determine significant differences between the low temperature and control groups,followed by Tukey’s multiple comparison test. Values were considered statistically significant at theP＜ 0.05 level. All graphs were created using OriginPro 9.1 (OriginLab, Northampton, MA).

3 Results

3.1 Low Temperature Induced Morphological Changes in P. esculenta

As shown in Fig.1, the morphology ofP. esculentachanged significantly after 96 h under the 5℃ treatment.In the control group,P.esculentaindividuals were fullbodied, vigorous, and closely intertwined (Figs.1A1 and 1B1). Similarly, individuals incubated at 10 and 15℃showed the same characteristics (Figs.1A2, A3, B2 and B3). By contrast, at 5℃, the body of the specimens appeared long and soft, and the introvert elongated (shown in Fig.1B4), with some individuals distanced from the group(Fig.1A4).

Fig.1 Morphological characteristics of Phascolosoma esculenta after 96 h under low temperature stress. (A) P. esculenta individuals were maintained at 20, 15, 10, and 5℃, respectively; red circle shows individuals that have moved aside from the group. (B) P. esculenta appearance at 20, 15, 10, and 5℃, respectively. (C) Anatomical drawing of P. esculenta based on previous reports (Ying et al., 2005; Mu et al., 2013). IN, introvert; NE, nephridium; OE, oesophagus; WM, wingshaped muscle; PH, pharynx; INT, intestine; SM, spindle muscle; VR, ventral retractor muscle.

3.2 Tissue Damage Caused by Low Temperature

The apparent morphological characteristics ofP. esculentaat different low temperatures prompted us to explore whether or not the abnormal behavior observed at 5℃was caused by tissue and cell damage. After 96 h of 5℃low temperature stress, no significant changes were observed in the coelomic fluid or in the intestine, whereas the body wall and the nephridia showed obvious damage(Figs.2 and 3).

The body wall ofP.esculentais composed of the cuticle, the epithelium, the circular muscle layer, the longitudinal muscle layer, and the parietal peritoneum (Fig.2A1).The cuticle is the outermost layer of the body wall, and the innermost layer closer to the cuticle is the epithelium,which consists of columnar epithelial cells that are closely arranged with unclear cell boundaries. The inner side of the epithelium is known as the circular muscle layer. The longitudinal muscle layer is significantly thicker than the circular muscle layer. In turn, the innermost layer of the body wall is the parietal peritoneum, which separates the body wall from the body cavity. In the control group, the outer membrane of the cuticle remained intact, and the myofibers of the circular muscle layer were orderly arranged(Fig.2A1). Similarly, the myofibers of the longitudinal muscle layer were intact and arranged in an orderly manner(Fig.2B1). Furthermore, the single columnar epithelial cells on the epithelium were arranged closely and pressed against the inside of the cuticle (Fig.2C1). After 96 h of stress at 5℃, the outer membrane of the cuticle appeared broken, and the myofibers of the circular muscle layer were scattered (Fig.2A2). Furthermore, the myofibers of the longitudinal muscle layer were broken (Fig.2B2), and part of the epithelium was separated from the cuticle (Fig.2C2).

From the inside to the outside, the nephridia ofP. esculentaconsist of an epithelial layer, a muscular layer, an extracellular matrix, and an outer membrane. In the control group, the simple columnar epithelium of the epithelial layer was arranged closely and was integrated (Figs.3A1 and B1), and the apical portions of columnar cells expanded to form vacuoles (Fig.3A1). The flask-shaped in-folding of the outer membrane formed the lumen of flask-shaped protrusion. The myofibrils of the circular muscle became interwoven to form a network structure at the base of the columnar epithelial cells (Fig.3A1). However, after 96 h at 5℃, part of the epithelial layer ruptured, and the simple columnar epithelium was disorderly arranged, whereas the lumen of the flask-shaped protrusion was enlarged (Fig.3A2).Furthermore, the myofiber of the circular muscle was partially loose, appearing as a fault (Figs.3A2 and B2).

Fig.2 Morphological changes in the body wall of Phascolosoma esculenta after 96 h under low temperature stress, at 5℃.A1, B1, and C1: morphology of the body wall in the control group; A2, B2, and C2: morphology of the body wall in the 5℃-treated group. Cu, cuticle; Ep, epithelium; CML, circular muscle layer; LML, longitudinal muscle layer; PP, parietal peritoneum.

3.3 Hsp70 and hsp90 mRNA Expression Under Control Conditions

As indicated by mRNA abundance, Fig.4 showed that thehsp70andhsp90genes were widely expressed inP.esculentatissues, including the coelomic fluid, the intestine, the contractive muscle, the nephridium, and the body wall. The expression level ofhsp70mRNA was the highest in the coelomic fluid, followed by the intestine, the body wall, and the contractive muscle, and the lowest in the nephridium (Fig.4A). Similarly, the expression level ofhsp90mRNA was the highest in the coelomic fluid,followed by the intestine, the body wall, and the contractive muscle, and the lowest in the nephridium (Fig.4B).

Fig.3 Morphological changes in the nephridium of Phascolosoma esculenta after 96 h under 5℃. A1 and B1: morphology of the nephridium of an individual in the control group. A2 and B2: morphology of the nephridium in the 5℃ low temperature group. FI, flask-shaped in-folding; LF, lumen of flask-shaped protrusion; CM, circular muscle; SCE, simple columnar epithelium; AP, apical portion; ‘*’ indicates the rupture of the epithelial layer.

Fig.4 mRNA expression for (A) hsp70 and (B) hsp90 in different tissues of Phascolosoma esculenta. CF, coelomic fluid; I,intestine; CM, contractive muscle; N, nephridium; BW, body wall.

3.4 Changes in the Expression of hsp70 and hsp90 Genes Under Low Temperature Stress

The mRNA levels ofhsp70andhsp90in the body wall,the coelomic fluid, the intestine, and the nephridium were quantified by RT-qPCR to determine whether or not low temperature influences the expression ofhsp70andhsp90in low temperature-treatedP. esculenta. Overall, the expression levels ofhsp70andhsp90inP. esculentainitially increased and then decreased across tissues under low temperature stress.

In the body wall, thehsp70andhsp90mRNA expression levels in the individuals maintained at 15℃ peaked at 48 h (approximately 1.6-fold of the control group,P＜0.05) and 24 h (approximately 1.8-fold of the control group,P＜ 0.01), respectively, and the expression levels of both genes were significantly lower than the corresponding expression levels in the control group at other sampling time points (P＜ 0.01) (Fig.5). At 10℃,hsp70mRNA expression increased significantly after 12 h and peaked at 72 h (approximately 78.9-fold of the control group,P＜ 0.01) (Fig.5A).By contrast,hsp90mRNA expression peaked at 48 h (approximately 15.2-fold of the control group,P＜ 0.01), and it was lower than that in the control group at other sampling time points (Fig.5B). At 5℃,hsp70mRNA expression peaked at 12 h (approximately 9.4-fold of the control group,P＜ 0.01) and then returned to the control level (P＞0.05) (Fig.5A); however, in this case,hsp90mRNA expression was significantly lower than that in the control group (P＜ 0.01) (Fig.5B). These findings showed that the expression level ofhsp70mRNA was higher than that ofhsp90mRNA in the body wall at their respective peak levels under low temperature stress, especially at 10℃.

In the coelomic fluid,hsp70mRNA expression at 15℃was significantly lower than that in the control group (P＜0.01) (Fig.6A), whereashsp90mRNA expression peaked at 48 h and was significantly upregulated (approximately 2.3-fold of the control group,P＜ 0.01) (Fig.6B). At 10℃,hsp70mRNA expression peaked at 48 h (approximately 34.5-fold of the control group,P＜ 0.01) and then markedly decreased at 96 h (P＜ 0.01) (Fig.6A); similarly,hsp90mRNA expression peaked at 48 h (approximately 49.7-fold of the control group,P＜ 0.01) and then returned to the control level at 96 h (P＞ 0.05) (Fig.6B). However, at 5℃,hsp70mRNA expression returned to the control level at 72 h (P＞0.05) (Fig.6A), whereashsp90mRNA expression was significantly lower than that in the control group in 96 h (P＜0.01) (Fig.6B). These results revealed that the peak mRNA expression ofhsp90in the coelomic fluid was higher than that ofhsp70at the same low temperature.

Fig.5 Changes in the expression levels of (A) hsp70 and (B) hsp90 mRNA in the body wall under low temperature stress.Values are expressed as means ± standard error (n = 3). Asterisks above the bars indicate statistically significant difference relative to the 20℃ control group. ‘*’ indicates significance at the 0.05 level of probability, and ‘**’ indicates significance at the 0.01 level of probability.

Fig.6 Same as Fig.5 but for (A) hsp70 and (B) hsp90 mRNA in the coelomic fluid under cold stress.

In the intestine, the expression levels ofhsp70andhsp90mRNA in the 15℃ low temperature group peaked at 48 h(approximately 7.3-fold of the control group,P＜ 0.01 and approximately 2.6-fold of the control group,P＜ 0.01, respectively) and then significantly decreased at 96 h (P＜0.01) (Fig.7). Under the 10 and 5℃ low temperature stress,hsp70mRNA expression peaked at 24 h (approximately 11.1-fold of the control group,P＜ 0.01 and approximately 2.6-fold of the control group,P＜ 0.01, respectively) (Fig.7A);meanwhile,hsp90mRNA expression peaked at 48 h (approximately 46.7-fold of the control group,P＜ 0.01 and approximately 0.3-fold of the control group,P＜ 0.01, respectively) (Fig.7B). Clearly, the maximum expression level ofhsp90mRNA in the intestine was markedly higher than that ofhsp70mRNA at 10℃, whereas the opposite was true at 5 and at 15℃.

As for the nephridium, the expression levels ofhsp70andhsp90mRNA at 15℃ peaked at 48 h, although thehsp70mRNA expression did not significantly differ from that in the control group at 48 h (P＞ 0.05), whereashsp90mRNA expression increased significantly (approximately 1.7-fold of the control group,P＜ 0.05) (Fig.8). At 10℃,hsp70mRNA expression significantly increased at 12 h and peaked at 48 h (approximately 43.3-fold of the control group,P＜ 0.01) (Fig.8A); however,hsp90mRNA expression peaked at 48 h (approximately 9.8-fold of the control group,P＜ 0.01), and it was lower than that in the control group at the other sampling time points (Fig.8B). As for the 5℃ low temperature group,hsp70mRNA expression peaked at 12 h (approximately 5.7-fold of the control group,P＜ 0.01) and then returned to the control level at 96 h (P＞0.05) (Fig.8A), whereashsp90mRNA expression peaked and did not significantly differ from that in the control group at 72 h (P＞ 0.05) (Fig.8B).These results revealed that the peak level ofhsp70mRNA in the nephridium was higher than that ofhsp90mRNA at the same temperature.

Fig.7 Same as Fig.5 but for (A) hsp70 and (B) hsp90 mRNA in the intestine under cold stress.

Fig.8 Same as Fig.5 but for (A) hsp70 and (B) hsp90 mRNA in the nephridium under cold stress.

4 Discussion

The temperature of the environment is the most important factor affecting the physiological processes of aquatic organisms. Frequent temperature changes can result in changes in cellular physical and chemical properties, energy budget, and immune function, thus hampering normal physiological functions of the body (Liuet al., 2019).Owing to the unique biotopes of intertidal zones, the daily and seasonal environmental changes in these areas are relatively large, whereby conditions can be extremely harsh for survival in intertidal zones, especially low temperatures in winter (Helmuthet al., 2002; Ansart and Vernon,2003; Marshallet al., 2010; Somero, 2010; Rongeset al.,2012; Chibaet al., 2016).

Intertidal organisms have evolved different adaptive characteristics to cope with low temperatures, including hibernation, burrowing the marine floor, and reducing speed of movement (Ansart and Vernon, 2003; Rongeset al., 2012;Chibaet al., 2016). However, a temperature low enough or an extended cold period can result in transient or permanent damage. For example, Zenget al. (2010) found thatSipunculus nudusshows poor vitality, and some individuals stop peristalsis after 24 h at temperatures below 8℃. Similarly, Fenget al. (2014) found that the body ofPerinereisaibuhitensissuffers atrophy and hardens at 5℃,with slow swimming. A previous study onP. esculentarevealed that the body becomes soft and shows slow contraction after stimulation without death when the temperature is between 0 and 10℃; this result may be related to the fact that it is often affected by the variable temperature in the intertidal zones, especially low temperature in winter, and therefore develops a high degree of resistance to low temperature (Zenget al., 2006). In this study, we confirmed the observation that the body ofP. esculentastretched and softened, with some individuals moving away from their group when the temperature was 5℃. This result suggests that a relatively low temperature exerts adverse effects on the physical functions ofP. esculenta.

The body wall of sipunculans is in direct contact with the environment and the first tissue to perceive the low temperature stimulus. The surface of the body wall is composed of a cuticle that can prevent water evaporation in vivo and reduce the damage to the body caused by moving, thus playing a protective role (Cutler, 1986; Deng et al., 2006).In addition, the contraction of muscle fibers in the body wall promotes the flow of the coelomic fluid, thus playing an important role in the transport of nutrients and oxygen in P. esculenta (Deng et al., 2006). In the present study,after 96 h at 5℃, the outer membrane of the cuticle in P.esculenta appeared broken and the myofiber arrangement of the circular muscle layer was scattered. Part of the epithelium was separated from the cuticle owing to myofiber scattering; this result implied that damage to the body wall was caused by low temperature, which may lead to a decrease in the defense ability of the P. esculenta body wall in response to low temperature stress. In addition, the damage to the body wall may be an important factor in the softening of P. esculenta. Consistent with our results, Ou et al. (2018) observed that the myofibril gap in Eleutheronema tetradactylum increases and a small number of myofibrils becomes separated from the fiber bundle under low temperature stress, thus resulting in the fragmentation of muscle fibers. This result suggests that the degree of lipid peroxidation in the muscles increases under temperature stress.

The nephridia are the excretory organs of sipunculans;they accumulate and excrete gametes and are involved in phagocytosis, secretion of sex hormones, osmoregulation,and volume regulation of body fluids (Adrianov and Maiorova, 2002; Bartolomaeus and Quast, 2005). The muscle tissue of the nephridia plays an important role in regulating their functions. The contraction of muscle fibers helps the nephridia filter coelomic fluid and prevents the backflow of the filtrate (Adrianov et al., 2002; Long et al., 2014).Therefore, the integrity of the nephridia is important in preserving the normal physiological functions of sipunculans. Nevertheless, our study showed that the simple columnar epithelium of the epithelial layer in P. esculenta was arranged disorderly after 96 h at 5℃, showing the rupture of the partial epithelial layer. Furthermore, the myofibrillar reticular structure of the circular muscle was partially loose, appearing as a fault after 96 h at 5℃. This result suggests that the damage in the nephridium may obstruct filtration and osmoregulation, and eventually leads to osmolarity disorders and softening of the body.

Organisms respond by activating stress proteins to prevent stress-induced body damage (Feder and Hofmann,1999; Luo et al., 2017; Wang et al., 2017). HSP70 and HSP90, the most highly conserved and important proteins in the HSPs family, regulate cytoskeleton dynamics, apoptosis, antioxidation, immunity, cell structure stabilization,and embryo development (Morimoto, 1993; Srivastava et al., 1998; Ran et al., 2007; Zhao et al., 2012; Kim et al.,2019). When an organism is exposed to stress, HSP70 and HSP90 can recognize the hydrophobic region of denatured proteins, assist denatured or folding proteins in restoring their natural conformation, and prevent irreversible aggregation and precipitation of protein particles, thereby preventing stress-induced cellular damage (Srivastava et al., 1998; Ran et al., 2007; Kim et al., 2019). Under normal conditions, tissue expression analysis of the hsp70 and hsp90 genes in P. esculenta revealed their ubiquitous expression in five tissues examined herein; moreover, the expression patterns of hsp70 and hsp90 were similar in all tissues, suggesting that these genes serve similar functions in P. esculenta. Other studies confirmed that the functions of the hsp70 and hsp90 genes are closely related(Wegele et al., 2004; Peng et al., 2016). Furthermore, the expression levels of the hsp70 and hsp90 genes in the body wall, the coelomic fluid, the intestine, and the nephridium of P. esculenta markedly increased under low temperature stress, suggesting that hsp70 and hsp90 may improve the low temperature tolerance of P. esculenta via protecting tissues and cells from cold damage. Similar results have been reported for other aquatic species, such as Argopecten irradians (Brun et al., 2008), Scophthalmus maximus(Ji et al., 2016), Ostrinia furnacalis (Chen et al., 2019),Oryzias melastigma (Li et al., 2015), and Huso dauricus(Peng et al., 2016). Notably, our study demonstrated that the expression levels of hsp70 and hsp90 increased, whereas the damage of body wall and nephridium occurred,which was speculated that the protective ability of hsp70 and hsp90 was not enough to deal with long time acute low temperature stress, eventually leading to different degrees of damage to body wall and nephridium in P. esculenta.

In the present study, we found that hsp70 and hsp90 mRNA levels in P. esculenta increased most obviously under 10℃ low temperature stress, implying that hsp70 and hsp90 in P. esculenta are sensitive to 10℃. By contrast,Peng et al. (2016) recorded that the hsp70 and hsp90 expression levels in H. dauricus are the highest at 4℃. In addition, low temperature treatment exerts no effect on stimulating the expression of hsp70 and hsp90 in Pomacea canaliculata (Xu et al., 2014). Similarly, Ji et al. (2016)found that the hsp70 gene expression in S. maximus decreases to baseline level as temperature decreases to 1℃.These results suggest that low temperature induces the species-specific expression of hsp70 and hsp90. Furthermore,the hsp70 and hsp90 mRNA abundance levels in P. esculenta at low temperature were tissue-specific. In specific,the expression of hsp70 mRNA in the body wall and nephridium was higher than that of hsp90 mRNA, but the opposite was true in the coelomic fluid and the intestine.This finding indicated a certain tissue specificity in the main functions of hsp70 and hsp90 in P. esculenta under low temperature stress. At present, our research is limited to the expression of the hsp70 and hsp90 genes. In the future, we will further study the molecular mechanisms of hsp70 and hsp90 in P. esculenta exposed to low temperature, which is expected to provide insights into the low temperature adaptability of P. esculenta.

5 Conclusions

Upon exposure to low temperature stress, P. esculenta exhibited evident morphological changes. Its body stretched and softened at 5℃, which may be due to damage of the body wall and the nephridium at low temperature. Thehsp70andhsp90genes were widely expressed in all five tissues under study and significantly increased in the body wall, the coelomic fluid, the intestine, and the nephridium under cold stress. This finding indicates that these genes play an important role in improving the adaptation ofP.esculentato low temperature, thus providing a theoretical basis for studying the adaptation strategy ofP.esculentato low temperature.

Acknowledgements

This project was supported by the Ningbo Science and Technology Plan Projects (Nos. 2019B10016, 2016C10004)and the Collaborative Innovation Center for Zhejiang Marine High-Efficiency and Healthy Aquaculture, the K.C.Wong Magna Fund in Ningbo University.