1 Introduction

Heat shock proteins (HSPs) ubiquitously distribute and highly conserve, behaving as molecular chaperones and exerting a protective effect against different stressful stimuli and cell damage (Lindquist et al., 1988; Joly et al.,2010; Mjzhed et al., 2012). According to their molecular sizes and functions, HSPs have been classified into several families including HSP100, HSP90 (85–90 kDa),HSP70 (68–73 kDa), HSP60 and low molecular mass HSPs (16–24 kDa) (Georgopoulos and Welch, 1993;Moseley, 1997). Among the different HSPs, HSP70 and HSP90 play essential roles in protein folding, membrane translocation, degradation of misfolded proteins and other regulatory processes (Erbse et al., 2004; Multhoff, 2007;Ming et al., 2010; Pratt and Toft, 2003; Sreedhar et al.,2004; Pearl and Prodromou, 2006). Recent researches indicated that HSPs function as regulators in the immune response and activators in the innate immune system(Bausinger et al., 2002; Pockley, 2003; Tsan and Gao,2004). The expression of HSP70 gene was transiently induced by bacterial and environmental stimuli in shellfish (Song et al., 2006; Cellura et al., 2007). Several members of the HSP70 family have been identified in various Crustacea (Cheng et al., 2003; Jiao et al., 2004;Liu et al., 2004; Lo et al., 2004; Chuang et al., 2007;Leignel et al., 2007; Ravaux et al., 2007; Wu et al., 2008;Luan et al., 2010; Cui et al., 2010; Fu et al., 2013). Previous studies of HSP90s in various invertebrates demonstrated that the expression of HSP90 gene could be stimulated by a variety of environmental stresses such as heat shock, heavy metals, osmotic stress, pH and ammonia-N stresses (Gao et al., 2007; Li et al., 2009; Pan et al.,2000; Li et al., 2012). HSP90 not only functions as a molecular chaperone, but also plays an important role in innate immunity of invertebrates (Wang et al., 2006; Gaoet al., 2008; Fu et al., 2011; Xie et al., 2015; Chaurasia et al., 2016). The ridgetail white prawn Exopalaemon carinicauda is one of the most important commercial shrimps, and mainly cultured in the coastal area of Zhejiang, Jiangsu, Shandong, Hebei Provinces of China (Li et al., 2015). Due to multiple merits of good reproductive performance, fast growth and good environmental adaptability, the annual culture area and yield of E. carinicauda in China are more than 20000 ha and 45000 t, respectively, according to an uncompleted statistic. Due to the high commercial value of E. carinicauda, the prevention of ‘milky shrimp’ disease caused by Hematodinium infections was conducted (Xu et al., 2010). The immunity gene discovery was conducted by expressed sequence tags (ESTs) (Duan et al., 2013b) and transcriptome analysis (Li et al., 2015). The identification of immunity-related genes such as selenium dependent glutathione peroxidase (GPx) (Duan et al., 2013a), farnesoic acid O-methyltransferase (FAMeT) (Duan et al., 2014a), calreticulin(CRT) (Duan et al., 2014b) and oncoprotein NM23 (Duan et al., 2015) of E. carinicauda were conducted recently.However, various diseases caused by shrimp pathogens,such as the white spot syndrome virus (WSSV) and bacteria from the genus Vibrio, have led to massive mortality and great loss in the shrimp cultivation industry (Lightner,2011; Tassanakajon et al., 2013). Therefore, better understanding of the immune mechanism of the ridgetail white prawn will be beneficial to the diseases prevention.

采用个别分舱治疗，实施常规治疗干预。①升压：关紧舱门，打开供氧阀，调节氧流量，电脑控制均速升压，常规升压时间15 min，升压速率<0.01 MPa/min，必要时根据病人情况，酌情延长加压时间，减慢加压速率。②稳压：病人先吸氧30 min，休息10 min后再次吸氧30 min，吸氧浓度100%。③减压：电脑控制均速减压，减压时间25 min。治疗时注意，对中度耳痛病人应常规暂停3 d～5 d后再行高压氧治疗，或根据病人需要于第2 天治疗前10 min给予羟甲唑啉鼻腔喷雾喷双侧鼻腔，帮助调压。如实施上述措施后病人依旧无法耐受高压氧，则继续暂停治疗，或根据需要行鼓膜穿刺后，再行治疗。

HSP70 (GenBank accession number: HQ185257) and HSP90 (GenBank accession number: HQ162267) in E.carinicauda have been identified and played important roles after pH and ammonia-N stresses (Han et al., 2011;Li et al., 2012). However, little is known about the potential roles of HSP70 and HSP90 in E. carinicauda against pathogens challenge. The aim of this study was to investigate the expression profiles of HSP70 and HSP90 genes in E. carinicauda after pathogens (WSSV and Vibrio anguillarum) challenge, and compare different expression patterns of HSP70 and HSP90 genes challenged by different pathogens. The results will be essential to understand the roles of HSP70 and HSP90 in resisting different pathogens in E. carinicauda.

2 Materials and Methods

2.1 Animals and Rearing Conditions

Healthy adult E. carinicauda with an average weight of 1.85 ± 0.23 g were collected from a commercial farm in Rizhao, China. They were reared in 200 L polyvinyl chloride polymer (PVC) tanks with filtered aerated seawater(salinity 30, pH 8.2) at 20 ± 0.5℃ for 7 d before the pathogens infection. There were 100 shrimps in each group.During the acclimation period, the shrimps were fed daily with a ration of 10% of body weight, and two-thirds of the water in each group was renewed daily.

2.2 Experimental Design of WSSV and V. anguillarum Challenges

The shrimps were divided into the virus (WSSV) challenged group, the bacteria (V. anguillarum) challenged group and the control group, and there were three replicates each group. WSSV crude extract and V. anguillarum strain were provided by the Mariculture Disease Control and Pathogenic Molecular Biology Laboratory, Yellow Sea Fisheries Research Institute. WSSV crude extract was obtained from 10 g WSSV-infected carapace from Litopenaeus vannamei, the carapace were homogenized separately in 10 mL sterile 0.9% saline solution and centrifuged at 1200 r min−1, 4℃ for 20 min, and then the supernatant was filtered through a 0.45 mm filter. Quantitative detection of WSSV was performed by RT-qPCR on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, USA), the methods referred to Durand and Lightner (Durand and Lightner, 2002). The RT-qPCR was carried out in a total volume of 20 µL, containing 12.5 µL of Perfect Real Time premix (1) (RR039A, Ta-KaRa), 50−100 ng of virus DNA, 0.25 mmol L−1 each of F3 and R3 primers, 0.125 mmol L−1 of probes, and added sterile ddH2O to total volume of 20 µL. Recombinant plasmid PUCm-T/WSSV69 containing purpose fragment was used as standard. The PCR program was 94℃ for 10 s, then 40 cycles of 95℃ for 5 s and 60℃ for 34 s. V. anguillarum strains (no. 20130513003S03) was obtained from the infected L. vannamei and activated on marine agar 2611E.

RT-qPCR data from three replicate samples were analyzed with the ABI 7500 system SDS Software (Applied Biosystems, USA) to estimate the transcript copy numbers for each sample. The comparative CT method was to analyze the relative expression levels of HSP70 and HSP90 genes. The CT for the target amplified products of HSP70, HSP90 genes and internal control Actin was determined for each sample. The difference in the CT between the target and the internal control, called ΔCT, was calculated to normalize the differences in the amount of template and the efficiency of the RT-qPCR. In the same challenge time, the ΔCT of the control group was used as the calibrator, and the difference between the ΔCT of the challenged group and the control group was called ΔΔCT.The expression levels of HSP70 and HSP90 genes were calculated by the 2−ΔΔCT comparative CT method (Livak and Schmittgen, 2001). Statistical analysis was performed using SPSS software (Ver 17.0). Statistical significance was determined using one-way ANOVA (González-Rodríguez et al., 2012) and post hoc Duncan multiple range tests. Significance was set at P ＜ 0.05.

2.3 RNA Extraction and cDNA Synthesis

Hemocytes were collected with a syringe which contained an equal volume of anti-coagulant buffer (1.59 g sodium citrate, 3.92 g sodium chloride, 4.56 g glucose,0.66 g EDTA-2Na, 200 mL ddH2O) (Soderhall et al.,1983), and centrifuged at 800 g, 4℃ for 15 min. Total RNA was extracted from hemocytes and hepatopancreas using Trizol Reagent (Invitrogen, USA) following the manufacturer’s protocol. Contaminating genomic DNA was eliminated using RQ1 RNase-free DNase (Promega)following the manufacturer’s instruction. The RNA sam-ples were analyzed in 1.0% agarose electrophoresis and quantified at 260 nm, all OD260/OD280 were between 1.8 and 2.0. The 3’ and 5’ ends RACE cDNA template were synthesized using SMART™ cDNA Kit (Clontech, USA)following the protocol of the manufacturer.

2.4 Expression of HSP70 and HSP90 Genes After WSSV and V. anguillarum Challenges

The expression patterns of HSP90 gene in hemocytes and hepatopancreas of E. carinicauda after V.anguillarum challenge are shown in Fig.4. The HSP90 gene mRNA abundance of hemocytes in V. anguillarum challenge group increased significantly and reached the peak value at the first 3 h after challenge, which was 7.26 fold of that in the control group (P ＜ 0.05). Afterwards, the HSP90 gene mRNA abundance in challenge groups gradually decreased and then increased to high level at 48 h(4.50-fold of the control group, P ＜ 0.05). Finally they recovered to the control level at 72 h after challenge(Fig.4A). In the hepatopancreas of E. carinicauda, the expression levels of HSP90 gene were gradually increased to peak level at 3 h after V.anguillarum challenge (2.57-fold of the control group, P ＜ 0.05). Then the expression levels decreased significantly at 72 h (P ＜ 0.05) (Fig.4B).

The mRNA abundance of HSP70 gene in hemocytes and hepatopancreas of E. carinicauda after V.anguillarum challenge was shown in Fig.2. Compared to the control group, the expression of HSP70 gene in hemocytes increased significantly and reached the highest level at 12 h (9.09-fold of the control group, P ＜ 0.05), then it decreased gradually and reached to the control group level at 72 h (Fig.2A). However, HSP70 gene transcripts in hepatopancreas decreased to the lower level at 3 h (0.88-fold of the control group, P ＞ 0.05). Then it increased to a peak at 6 h (2.2-fold of the control group, P ＜ 0.05), which was higher than that of control, and maintained a high level at 12 h. Then it obviously decreased to the lowest level at 24 h (0.48-fold of the control group, P ＜ 0.05).Afterwards it reached the highest level at 48 h (2.41-fold of the control group, P ＜ 0.05) (Fig.2B).

Table 1 Primer sequences used in this study

Primer name Sequence (5’-3’)HSP70 R1 (reverse) TTGGTGGGGATGGTGGTGTT F1 (forward) GGACCTGTTGCTGTTGGATG F2 (forward) CAGGGTCGTTGTCACCTCTAA HSP90 R2 (reverse) CTTGACCACCTCCTTGATACG Actin-F CCGAGACATCAAGGAGAAGC Actin Actin-R ATACCGCAAGATTCCATACCC F3 (forward) ACAATGGTCCCGTCCTCATC WSSV R3 (reverse) TGCCTTGCCGGAAATTAGTG Probe (T) TET-CAGAAGCCATGAAGAAT GCCGTCTATCAC-TAMRA

For WSSV challenged group, shrimps were injected individually with 20 µL live WSSV crude extract (106 copies mL−1) as described above. The control group received individually an injection of 20 µL sterile 0.9% saline solution. For V.anguillarum challenged group, shrimps were injected individually with 20 µL live V.anguillarum suspension (1×109 CFU mL−1) in sterile 0.9% saline solution, resulting in 2×107 CFU shrimp−1. Then both the challenged groups and the control group were returned to the PVC tanks with aerated seawater at 20℃ as described above. The seawater waste was treated to make it harmless for environment. Through the separation and identification of pathogens from shrimps in the WSSV and V.anguillarum challenged group, respectively, the results showed that the shrimp was infected with particular pathogen. Hemocytes and hepatopancreas of three shrimps from each treatment (the challenged group and the control group) were randomly sampled at 0, 3, 6, 12, 24, 48 and 72 h post-injection respectively, then the samples were snap-frozen in liquid nitrogen. There were three replicates at each time point.

3 Results

3.1 mRNA Abundance of HSP70 Gene in Hemocytes and Hepatopancreas After WSSV Challenge

The temporal expressions of HSP90 gene in hemocytes and hepatopancreas of E. carinicauda after WSSV challenge were shown in Fig.3. Compared to the control, the expression of HSP90 gene in hemocytes from WSSV challenged group increased significantly and reached the higher level at the first 3 h, which was 3.29 fold of that in the control group (P ＜ 0.05). Afterwards, the expression of HSP90 gene in WSSV challenged group tended to increase to the highest level at 12 h (6.87-fold of the control group, P ＜ 0.05), and then gradually decreased till the end of the experiment (72 h) (Fig.3A). Similarly, the transcripts of HSP90 gene in hepatopancreas in WSSV challenged groups increased gradually to the maximum at 6 h(2.73-fold of the control group, P ＜ 0.05), then decreased continuously to the minimum at 72 h (0.16-fold of the control group, P ＜ 0.05). During the experimental time interval, HSP90 gene transcript in the control group fluctuated slightly, but showed no significant difference (P ＞0.05) (Fig.3B).

Fig.1 The mRNA abundance of HSP70 gene in hemocytes (A) and hepatopancreas (B) of E. carinicauda at different times after WSSV challenge. The reference gene is actin gene. Vertical bars represented the mean ± SD (n = 3). Significant differences (P ＜ 0.05) of HSP70 gene expression between the challenged and the control group were indicated with asterisks.

3.2 mRNA Abundance of HSP70 Gene in Hemocytes and Hepatopancreas After V. anguillarum Challenge

例3、例4和例5中，将“斗技”“绣花枕头”具有中国特色含义的词汇英译时采取解释加注的处理方法，方便读者阅读。“炎儿”这种称呼，也是采取解释加注的方式，避免引起读者的阅读障碍。

Fig.2 The mRNA abundance of HSP70 gene in hemocytes (A) and hepatopancreas (B) of E. carinicauda at different times after V.anguillarum challenge. The reference gene is actin gene. Vertical bars represent the mean ± SD (n = 3). Significant differences (P ＜ 0.05) of HSP70 gene expression between the challenged and the control group were indicated with asterisks.

3.3 mRNA Abundance of HSP90 Gene in Hemocytes and Hepatopancreas After WSSV Challenge

Expression profiles of HSP70 gene in hemocytes and hepatopancreas of E. carinicauda after WSSV challenge were shown in Fig.1. Compared with the control group,HSP70 gene transcripts in hemocytes were up-regulated significantly at 3 h (4.75-fold of the control group, P ＜0.05) and reached to the peak level at 6 h (5.0-fold of the control group, P＜0.05). Then it was down-regulated gradually from 6 h to 72 h, but was still significantly higher than that of control (P ＜ 0.05) at 24 h (Fig.1A). In hepatopancreas, the HSP70 gene mRNA abundance increased significantly at 6 h (10.53-fold of the control group, P ＜0.05) post injection. After a decrease from 12 h to 24 h, it increased to the maximum at 48 h (10.58-fold of the control group, P ＜ 0.05) (Fig.1B), and then decreased to two times of the control 72 h after challenge.

林志气不打一处出，这回可真是下了狠心，一把将紫云拖过来，面对墙壁。抽出裤腰带，朝紫云背上，屁股上，大腿上猛抽，白裙上刷过一条条血印。紫云毫不在乎，静静地等着他打。打了这一顿，一切就结束了。

Fig.3 The mRNA abundance of HSP90 gene in hemocytes (A) and hepatopancreas (B) of E. carinicauda at different time intervals after WSSV challenge. The reference gene is actin gene. Vertical bars represent the mean ± SD (n = 3). Significant differences (P ＜ 0.05) of HSP90 gene expression between the challenged and the control group are indicated with asterisks.

3.4 mRNA Abundance of HSP90 Gene in Hemocytes and Hepatopancreas After V. anguillarum Challenge

Real time quantitative RT-PCR was performed on an ABI PRISM 7500 Sequence Detection System (Applied Biosystems) to investigate the HSP70 and HSP90 gene expression patterns in hemocytes and hepatopancreas of E.carinicauda. The specific primers F1 and R1 were used to amplify a HSP70 gene fragment of 102 bp, and the specific primers F2 and R2 were used to amplify a HSP90 gene fragment of 187 bp (Table 1). The primers Actin-F and Actin-R were used to amplify an actin gene fragment of 195 bp as an internal control for RT-PCR (Table 1). The RT-PCR amplifications were carried out in triplicate in a total volume of 20 µL containing 10 µL SYBRR Premix Ex TaqTM II (2 ×) (TaKaRa), 2 µL of the cDNA, 0.8 µL each of primers (10 m mol−1) (or 18S-HF and 18S-HR to amplify the 18S), 0.4 µL ROX Reference Dye II (50×)×3 and 6.0 µL DEPC treated water. The PCR program was 95℃ for 30 s, then 40 cycles of 95℃ for 5 s and 60℃ for 34 s, followed by 1 cycles of 95℃ 15 s, 60℃ for 1 min and 95℃ for 15 s. DEPC-treated water for the replacement of template was used as negative control.

Fig.4 The mRNA abundance of HSP90 gene in hemocytes (A) and hepatopancreas (B) of E. carinicauda at different times after V.anguillarum challenge. The reference gene is actin gene. Vertical bars represent the mean ± SD (n = 3). Significant differences (P ＜ 0.05) of HSP90 expression between the challenged and the control group are indicated with asterisks.

4 Discussion

Various diseases caused by shrimp pathogens, especially WSSV and bacteria from the genus Vibrio, have led to massive mortality and great loss in the shrimp cultivation industry (Li and Xiang, 2013). Previous studies have demonstrated that hemocytes and hepatopancreas are the main cells and tissue involved in the immune responses,and the major site for the synthesis of immune defense molecules eliminating pathogens or other particulates in crustaceans (Iwanaga and Lee, 2005; Lemaitre and Hoffmann, 2007; Du et al., 2013). To know the roles of HSP70 and HSP90 in the immune response of shrimp, the transcriptional profile of HSP70 and HSP90 genes showed different patterns in hemocytes and hepatopancreas after E. carinicauda were challenged with WSSV and V. anguillarum. HSPs are constitutively expressed due to pathogenic infection and can be considered as potential biomarkers against viral and bacterial infections in Crustacea. Several studies have confirmed the rapid responses of HSP70 family in aquatic organisms as a result of pathogen challenge (Song et al., 2006; Cellura et al.,2007; Cui et al., 2010; Wang et al., 2008). In the present study, the transcription of HSP70 gene showed a clear time dependent response in hemocytes and hepatopancreas after the shrimps were challenged with WSSV,which was significantly induced to the highest level at 6 h and 48 h after post-injection, respectively, indicating that HSP70 could be induced by WSSV. The results were in accordance with previous studies in F. chinensis (Wang et al., 2006), M. japonicas (Threechada et al., 2011), L.vannamei (Chen et al., 2013; Chen et al., 2016) and M.rosenbergii (Chaurasia et al., 2016), and suggested that HSP70 was involved in the response to WSSV infection.Meanwhile, the abundance of HSP70 gene transcripts in hemocytes and hepatopancreas of E. carinicauda after V.anguillarum challenge were stimulated to the peak value at 12 h and 6 h respectively. Similar expression pattern of HSP70 gene after bacterial challenges were also reported in M. rosenbergii (Liu et al., 2004; Chaurasia et al., 2016),Scylla serrate (Fu et al., 2013), Portunus trituberculatus(Cui et al., 2010), Botia reevesae (Qin et al., 2013) and Pelodiscus sinensis (Dang et al., 2015). The time and tissue differences of HSP70 gene expression after WSSV and V. anguillarum challenges in E. carinicauda might be caused by the pathway of infection and different pathogenic types (Xu et al., 2009).

Heat shock protein 90 (HSP90) works as a multifunctional chaperone and is involved in the regulation of many essential cellular pathways (Terasawa et al., 2005).To further understand the possible biological function of HSP90 in immune response, mRNA abundance was examined at different time points and in different tissues of E. carinicauda after WSSV and bacterial challenges. Realtime PCR analysis showed that the mRNA abundance of HSP90 gene in hemocytes and hepatopancreas of E.carinicauda after WSSV challenge could be significantly induced at specific time point. The peak value of HSP90 gene transcripts existed at 12 h in hemocytes and 6 h in hepatopancreas after WSSV challenge, which were in agreement with earlier reports in M. rosenbergii (Chaurasia et al., 2016), F. chinensis (Wang et al., 2006), M. japonicas (Threechada et al., 2011) and L. vannamei (Chen et al., 2016). Compared to the WSSV challenged group,both the mRNA abundance of HSP90 gene in hemocytes and hepatopancreas of E. carinicauda reached the peak value at the first 3 h after V.anguillarum challenge. Our results are almost in agreement with the expression pattern of HSP90 gene in hemocytes of Crassostrea hongkongensis after Vibrio alginolyticus challenge (Fu et al.,2011) and those in hepatopancreas of Penaeus monodon after Vibrio harveyi challenge (Rungrassamee et al., 2006),whereas the highest value of HSP90 gene mRNA in hepatopancreas of M. rosenbergii was obtained at 24 h after Aeromonas hydrophila and V. harveyi challenges (Chaurasia et al., 2016). The exact reason of the expression differences needs further indepth researches. In summary,the expressions of HSP70 and HSP90 genes in hemocytes and hepatopancreas of E. carinicauda with WSSV and V.anguillarum challenges suggested that they might be a part of disease-response genes or a part of disease-defense genes against viral and bacterial infections. These results could contribute to better understand the defense mechanisms against pathogens in E. carinicauda. Further studies will focus on the mechanisms of the regulation of HSPs in host defenses against infectious pathogens.

Acknowledgements

This study was supported by the earmarked fund for Modern Agro-industry Technology Research System (No.CARS-48), the Program of Shandong Leading Talent (No.LNJY2015002), the National Natural Science Foundation of China (No. 31472275), and Qingdao Industrial Development Program (Science and Technology Benefit Special Project, No. 17-3-3-62-nsh).

酵母菌：“什么糖我们都喜欢吃。但我说的糖是指糖类，有葡萄糖、果糖、麦芽糖等，还有一些名称里不带‘糖’字的糖，如淀粉等。怎么样，我们爱好广泛吧！”

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