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Expression and distribution of three heat shock protein genes under heat shock stress and under exposure to Vibrio harveyi in Penaeus monodon
Developmental and Comparative Immunology 34 (2010) 1082–1089
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Expression and distribution of three heat shock protein genes under heat shock stress and under exposure to Vibrio harveyi in Penaeus monodon Wanilada Rungrassamee a,∗ , Rungnapa Leelatanawit a , Pikul Jiravanichpaisal b , Sirawut Klinbunga b , Nitsara Karoonuthaisiri a a Microarray Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Pathumthani, 12120, Thailand b Aquatic Molecular Genetics and Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Pathumthani, 12120, Thailand
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Article history: Received 12 April 2010 Received in revised form 24 May 2010 Accepted 24 May 2010 Available online 8 June 2010 Keywords: Heat shock response Heat shock protein Gene expression Black tiger shrimp Penaeus monodon Vibrio harveyi
a b s t r a c t A sudden increase in temperature results in heat shock stress of the cultured shrimp. To cope with the stress, shrimp has to overcome by triggering a response known as heat shock response. To understand the heat shock response in the black tiger shrimp (Penaeus monodon), we examined expression patterns and distribution of three heat shock protein (hsp) genes in P. monodon juveniles. The expression levels of hsp21, hsp70 and hsp90 were determined by quantitative real-time PCR in nine tissues (gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocyte) under untreated and heat shock conditions. Under untreated condition, all three hsp genes were differentially expressed in all examined tissues where the hsp70 transcript showed the highest basal level. Under heat shock condition, only hsp90 was inducible in all nine tissues when comparing to its untreated level. The timecourse induction experiment in gill and hepatopancreas revealed that the transcriptional levels of hsp21, hsp70 and hsp90 were inducible under the heat shock condition and in time-dependent manner. To determine the response of the hsp genes upon bacterial exposure, we further determined transcript levels of the hsp genes in gill of P. monodon after Vibrio harveyi injection. The expression levels of hsp70 and hsp90 were significantly increased after a 3-h exposure to V. harveyi where the hsp21 transcript was induced later after a 24-h exposure. This evidence suggests for putative roles and involvement of the hsp genes as a part of immunity response against V. harveyi in P. monodon. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Natural and farming environments can be variable and unpredictable. An organism needs mechanisms to adapt to a wide range of the stressful conditions. In some habitats, a stress from daily or seasonal fluctuation of environmental temperature causes an organism to respond by inducing sets of proteins including heat shock proteins (HSPs) and this process is known as a heat shock stress response (Parsell and Lindquist, 1993). The HSPs are ubiquitous, highly conserved and found in all organisms (Kregel, 2002). They play critical roles as molecular chaperones in heat tolerance by repairing and refolding denatured proteins (Morimoto, 1998; Sharma et al., 2009). In addition to heat shock stress, HSPs respond to other factors such as pathogen infection, oxidative stress, heavy metals and xenobiotics stresses (Feder and Hofmann, 1999; Moseley, 2000). The HSPs are found in various forms and categorized into different families based on their molecular weights
∗ Corresponding author. Tel.: +66 2 564 6700x3255; fax: +66 2 564 6707. E-mail address: [email protected] (W. Rungrassamee). 0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2010.05.012
(kDa) such as HSP110, HSP100, HSP90, HSP70, HSP60 and small HSPs (Parsell and Lindquist, 1993; Feder and Hofmann, 1999). The HSP70 family has been extensively characterized as a primary family of heat shock proteins, and the members in HSP70 family can be constitutive or inducible expressed under heat shock stress (Nover and Scharf, 1997). The heat shock responses are well characterized in many model organisms such as Drosophila melanogaster, mouse and Arabidopsis; however, the responses can vary in different organisms (Feder and Hofmann, 1999; Cotto and Morimoto, 1999). In black tiger shrimp (Penaeus monodon), one of the important aquaculture species, a complete understanding of how the animal responds to heat shock stress will help improving farming condition to boost the shrimp immunity and protecting them from a risk of disease outbreak. In an attempt to understand heat shock stress response in P. monodon, heat shock proteins have previously been characterized by identifying full-length cDNA sequences of hsp21, hsp70 and hsp90 (Huang et al., 2008; Lo et al., 2004; Jiang et al., 2009). The hsp21 transcript has been shown to be heat inducible in pleopods and was down-regulated during the infection by White Spot Syndrome Virus (Huang et al., 2008). The hsp70 was inducible in hemocytes
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Fig. 1. Schematic representation of experiments performed in this study: (A) heat shock and (B) V. harveyi experiments. (A) In heat shock (HS) experiment, a group of 4-month old P. monodon juveniles was maintained at the room temperature (RT) where tissues were collected as the untreated control. The juvenile shrimp were then placed to a 35 ◦ C seawater tank and tissues were collected at 1 and 2.5 h of exposure time (1 h HS and 2.5 h HS, respectively). For the recovery phase, the juvenile shrimp were returned to the room temperature seawater tank, and tissues were collected immediately after 0 h RT, while the remainders were allowed to recover in the seawater tank for 1 and 3 h (1 h RT and 3 h RT, respectively) before tissue collection. Gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocyte were collected at the sampling time with asterisk (*) (N = 5). Otherwise, only gill and hepatopancreas were collected (N = 5). (B) In V. harveyi experiment, a group of 4-month old juveniles was injected with 100 l of heat-killed V. harveyi 1526 in the saline solution (∼2.0 OD600 ) at the lateral side of abdominal muscles, where a control group was injected with 100 l of the saline solution but without bacterial cells (control). The gill tissues were dissected out from shrimp at 3, 12, 24 and 72 h after the injection (N = 3 for each time point).
under heat shock conditions and the increase in the hsp70 expression was correlated with the reduction of Gill-Associated Virus (GAV) replication (Vega et al., 2006). Similarly, hsp90 was heat inducible in brain, stomach and heart and may play a role in ovary maturation (Jiang et al., 2009). Nonetheless, the gene expression patterns for hsp21, hsp70 and hsp90 in P. monodon were investigated under different heat shock treatment conditions in their studies. In this study, we aimed to characterize gene expression patterns of all three hsp21, hsp70, and hsp90 transcripts under the same heat shock treatment conditions to give a better understanding of the response to the stress. We also investigated if hsp genes were inducible upon the presence of a heat-killed shrimp pathogen. The expression patterns of the three major hsp genes (hsp21, hsp70 and hsp90) in nine tissues of P. monodon under both untreated and heat shock conditions were examined to validate for inducibility of hsp genes under the stress condition. We provided the first report on the time dependency of expression patterns of hsp21, hsp70 and hsp90 in hepatopancreas and gill tissues during and after heat shock conditions. Moreover, expression patterns of these genes were examined in P. monodon upon an exposure to the heat-killed shrimp pathogen, Vibrio harveyi, to determine the possible roles of the hsp genes as part of the immune response in shrimp.
in Chachoengsao province (Eastern Thailand) were transferred to seawater tanks with aeration and maintained at room temperature (27 ± 2 ◦ C, RT) with salinity at 20 parts per thousand (ppt). In the heat shock experiment, a group of 50 juvenile shrimp was transferred from the RT seawater tank to a 35 ± 2 ◦ C seawater tank. Tissue samples were collected from t = 0 (untreated at room temperature (RT), prior to heat shock treatment), heat shock (HS) = 1 and 2.5 h for heat shock treatment duration (N = 5 for each time point). Then, the group of remaining juveniles were returned to the RT seawater tank, the tissue samples (N = 5 for each time point) were collected at RT = 0 (immediate after placing back to room temperature), RT = 1 and RT = 3 h (1 and 3 h, respectively, after placing back to room temperature). The collected tissues were quickly placed in liquid nitrogen (−80 ◦ C). For V. harveyi infection (Fig. 1B), a group of 25 juvenile shrimp was injected with 100 l of heat-killed V. harveyi 1526 (Rengpipat et al., 2003) (∼2 × 108 cells/ml) at the lateral side of abdominal muscles, where the control group of 25 juvenile shrimp was injected with the same diluent but without bacterial cells (100 l of saline solution). The gill tissues were dissected out from shrimp at 3, 12, 24 and 72 h after injection (N = 3 for each time point). 2.2. RNA extraction and cDNA synthesis
2. Materials and methods 2.1. Experimental animals, heat shock treatment and Vibrio harveyi infection The heat shock treatment was depicted in Fig. 1A. Sixty of 4month-old P. monodon juveniles (13.4 ± 2.0 g) obtained from a farm
Each tissue sample was ground using a mortar in liquid nitrogen and transferred to TriReagent® (Molecular Research Center). RNA extraction was performed according to supplier’s instruction. The RNA pellets were resuspended in 50 l of RNase-free water and treated with DNaseI (0.5 unit/g, Promega) for 30 min at 37 ◦ C to remove DNA contamination. The DNA-free RNA was purified by phenol: chloroform extraction and precipitated with 1/10 volume
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Table 1 Oligonucleotides used in this study.
3. Results
Primer
Sequence 5 –3
Amplicon size (bp)
hsp21 F hsp21 R hsp70 F hsp70 R hsp90 F hsp90 R ef1˛ F ef1˛ R
AAT TCA TTG CGG AAG CGA GCC A ACT TCA GCG TGA TCG ACC AGG AAT AGA AGT CAC TCC GTG ATG CCA AGA ACT CCT TGC CGT TGA AGA AGT CCT GCA TGA AGG AGA ACC AGA AGC ACA TGA ACG CAG TAT TCG TCG ATG GGT GGC GTA CTG GTA AGG AAC TGG AA GAG GAG CAT ACT GTT GGA AGG TCT C
100 123 145 123
of 3 M sodium acetate and 1 volume of isopropanol. The concentration of each RNA sample was measured by using a NanoDrop UV–vis Spectrophotometer (ND-8000). Treated RNA samples were further confirmed for an absence of DNA contamination by PCR. A RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas) was used to synthesize the first strand cDNA using total RNA (1.5 g) as a template in a total reaction volume of 20 l. Aliquots of 0.5 l of cDNA preparation were used for quantitative real-time PCR reactions. 2.3. Quantification of hsp21, hsp70 and hsp90 expression by real-time qRT-PCR The abundance of the hsp21, hsp70 and hsp90 transcripts was measured by real-time quantitative reverse transcription PCR (real-time qRT-PCR). The reactions were carried out in an iCycler (BIORAD) using IQTM SYBR® Green Supermix (BIORAD) with primer pairs for hsp21 (the amplicon size = 100 bp), hsp70 (123 bp), and hsp90 (145 bp), respectively (Table 1). All reactions were carried out according to the supplier’s instructions. The housekeeping gene, ef1˛, was used as an internal control for all real-time PCR experiments (primers ef1˛ F + R, 123 bp; Table 1). The cycling parameters used were as follows: an initial denaturation at 95 ◦ C for 3 min, 40 cycles at 95 ◦ C for 20 s, 57 ◦ C for 30 s and 72 ◦ C for 30 s. The fluorescent signal intensities were recorded at the end of each cycle. Melting curve analysis was performed from 55 to 95 ◦ C with continuous fluorescence reading every 0.5 ◦ C increment. Standard curves for the quantification of number of mRNA transcripts per g of total RNA in the quantitative real-time PCR were plotted from the result of amplification reactions using each primer pair and plasmid templates containing the partial sequences of the hsp21, hsp70 or hsp90 genes. In order to construct these templates, a DNA fragment containing hsp21 (100 bp), hsp70 (123 bp) or hsp90 (145 bp) partial sequence was amplified by PCR using the same primer pairs in the real-time PCR reaction. The PCR products were gel-purified (Qiagen Gel Purification Kit) and subsequently cloned into a plasmid vector pGEM-T (Promega). Each resulting vector was transformed into E. coli DH5␣ and the transformants were selected on LB plates containing ampicillin (50 g/ml). Similarly, a plasmid containing the ef1˛ segment was constructed using a TOPO-TA cloning kit (Invitrogen) and the TOPO cloning reaction and transformation were done according to supplier’s instruction. All plasmids were extracted from the 4 ml overnight cultures using a plasmid miniprep kit (Favorgen). Plasmid concentrations were quantified using a Nanodrop (ND-8100) at a wavelength of 260 nm and the copy numbers were calculated. Five concentrations of 10-fold serial dilutions (106 to 102 copies) of each plasmid were used as templates to construct standard curves for hsp21, hsp70, hsp90 and ef1˛. Real-time PCR samples were set up in SYBR green reactions with primers for hsp21, hsp70, hsp90 and ef1˛ (Table 1). The cycling parameters used were the same as described earlier. The standard curve allowed a quantification of mRNA in samples. Data was analyzed by iCycler software. All real-time PCR experiments were performed in five independent trials.
3.1. Tissue distribution of hsp21, hsp70 and hsp90 in P. monodon To validate the inducibility of hsp genes in heat shock condition, the expression levels of the hsp21, hsp70, and hsp90 transcripts were measured by quantitative real-time PCR in nine tissues (gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocytes) from P. monodon juveniles under untreated and heat shock conditions (Fig. 2). The elevated temperature at 35 ◦ C for 2.5 h was chosen for the heat shock condition because at this temperature the heat shock stress response can be induced with a minor effect on shrimp survival rates (data not shown). The transcripts of hsp21, hsp70 and hsp90 transcripts were detected with varied expression levels in all examined tissues. Under untreated condition, the basal expression level of the hsp21 transcript was highest in heart but lowest in hemocytes (Fig. 2A), whereas the hsp70 basal expression level was highest in hepatopancreas and lowest in thoracic ganglia (Fig. 2B). On the other hand, the basal levels of the hsp90 transcript were detected with marginal differences in all tissues (Fig. 2C). Among the three hsp transcripts, the hsp70 transcript was expressed at the highest level under untreated condition in all tissues except in thoracic ganglia. Under the heat shock condition, hsp21 was significantly induced in gill, heart, hepatopancreas, pleopod and thoracic ganglia (1.9, 2.0-, 1.8-, 2.5- and 2.4-fold of induction, respectively) while its expression levels remained relatively the same in stomach, intestine, eyestalk and hemocyte (Fig. 2A). For the hsp70 transcript, it was significantly induced in most tissues, except in stomach and intestine (Fig. 2B). The hsp70 transcript was the most induced in thoracic ganglia with a 24-fold increase. Most strikingly, the hsp90 transcript was significantly up-regulated under the heat shock condition in all tissues with the most induction in hepatopancreas (12-fold; Fig. 2C). The expression levels of the internal control, ef1˛, were not significantly different among the nine examined tissues under untreated and heat shock conditions (Fig. 2D). 3.2. Time-course induction of the hsp genes in hepatopancreas and gill under heat shock stress Hepatopancreas has been previously reported to be a heatsensitive tissue in crustaceans (Bhavan and Geraldine, 2001) which was consistent with our results from tissue distribution analysis showing the significant inductions of all three hsp genes in this tissue (Fig. 2A–C). Therefore, in the time-course heat shock experiment, the hsp expression patterns were further analyzed in hepatopancreas (Fig. 3). To assess a time-dependent induction of the hsp gene expression, copy numbers of the hsp21, hsp70 and hsp90 transcripts were measured in P. monodon juveniles exposed to the heat shock stress before recovering back to a room temperature seawater tank (Fig. 1A). Under untreated conditions, the basal expression levels of hsp21 and hsp90 in hepatopancreas were lower than the hsp70 level, which were consistent to the observations in the tissue distribution analysis (untreated, Fig. 3A–C). When P. monodon was placed in the elevated temperature for 1 h (1 h HS), the transcripts of hsp21 hsp70 and hsp90 were increased by 3-, 2and 6-fold, respectively. When the heat shock exposure time was prolonged to 2.5 h (2.5 h HS), the levels of hsp21 and hsp70 transcripts remained similar to those of the 1-h exposure group (1 h HS) (Fig. 3A and B), but the hsp90 transcript level was significantly induced by 11-fold from its untreated level (Fig. 3C). Once shrimp were recovering from the heat shock treatment to the room temperature (RT), the hsp90 transcript level gradually declined to the similar level as in untreated control within 3 h. Interestingly, the transcript levels of hsp21 and hsp70 still remained high where that
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Fig. 2. Expression profiles of hsp21, hsp70 and hsp90 in nine different tissues of P. monodon juveniles with no heat shock (untreated, open bars) and under the heat shock condition for 2.5 h (solid bars). Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and the copy number of ef1˛ as the internal control (D) were examined. GL = gill, HT = heart, HP = hepatopancreas, ST = stomach, IN = intestine, ES = eyestalk, PL = pleopod, TH = thoracic ganglion and HC = hemocyte. The error bars represent the corresponding standard deviations from triplicate trials. Asterisks denote statistically significant differences between untreated control and heat shock groups (P < 0.05).
of the latter was further increased during the recovering phase at room temperature. In addition to hepatopancreas, expression profiles of the hsp genes were also determined in gill due to its important role in respiration and a direct contact to the aquatic environment (Fig. 4). In gill, the hsp21 transcript level was significantly increased when shrimp exposed to heat shock for 1 h (1 h HS) and further increased after 2.5 h of the heat shock treatment (2.5 h HS), then reached the highest level at the beginning of recovery phase at room temperature (0 h RT) before gradually declined after 1 and 3 h of the recovery phase (1 and 3 h RT), respectively (Fig. 4A). In contrast, the level of hsp70 transcript was already high in the untreated control, thus, its induction level after the heat shock was not significant until 2.5 h of the heat shock (2.5 h HS) (Fig. 4B). The transcript level of hsp70 was lowered during the recovery phase (0 h, 1 h, 3 h RT) after the heat shock treatment. The expression pattern of the hsp90 transcript in gill was similar to the pattern observed in hepatopancreas (Fig. 3C and 4C). Under the heat shock stress, the hsp90 expression level was induced 18-fold within 1 h (1 h HS) and 23-fold in 2.5 h (2.5 h HS). Once the shrimp were returned back to the room temperature for recovery, the hsp90 transcript level became gradually lowered until it reached the same level as in the untreated control after 3 h of the recovery time. The expression level of ef1˛ in gill did not show any significant change during the time-course experiment (Fig. 4D). 3.3. Expression profile analysis of the hsp genes in gill of P. monodon challenged with V. harveyi To further investigate roles of the hsp genes in the pathogenic stress, the expression profiles of these three hsp genes were determined in gill of P. monodon after injected with the pathogenic bacterium, V. harveyi (Fig. 1B). Gill tissue was chosen in this study
because it could be collected using a non-lethal procedure, while this procedure could not be done for hepatopancreas tissue. The gill tissue was sampled at 3, 12, 24, and 72 h after the V. harveyi exposure. In a parallel experiment, gill was also collected from the shrimp injected with a saline solution as a control group and the expression levels of the hsp transcripts in this group were used as the basal expression levels to compare to the V. harveyi treated group. Comparing to the control group, the expression level of hsp21 was slightly increased after 12 h of the V. harveyi injection and significantly induced at 24 h of the treatment, before returned to the similar level to the control after 72 h of the treatment (Fig. 5A). Meanwhile, the hsp70 and hsp90 transcripts showed significant increases at the first 3 h of the V. harveyi exposure (2.6 and 3.4fold comparing to the control at the same time point, respectively) (Fig. 5B and 5C). The hsp70 transcript level continued to significantly increase after 12 h of treatment, peaked at 24 h (4.8-fold and 4.4- fold of induction comparing to the control at the same time point, respectively) and still significant at 72 h in the presence of V. harveyi (Fig. 5B). On the other hand, the induction of the hsp90 transcript reached the highest level at 12-h of the V. harveyi exposure (5.7-fold comparing to the control at the same time point), began to decline at 24 h (3.6-fold comparing to the control of the same time point) and eventually returned to the similar level as the control at 72 h of post-treatment (Fig. 5C). When we compared the overall expression patterns of the three hsp genes over the timecourse of the V. harveyi exposure, hsp21, hsp70 and hsp90 exhibited increasing patterns until they reached the maximum induction and then became lower to the similar levels of the control group. The group of juveniles that were injected with the saline solution did not show significant difference in transcript levels of hsp21, hsp70 and hsp90 overtime. The expression of ef1˛ was not significantly altered in juveniles injected with the saline solution and heat-killed V. harveyi (Fig. 5D).
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Fig. 3. Time-course gene expression of hsp21, hsp70 and hsp90 under the heat shock condition in hepatopancreas of 4-month old P. monodon. Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and the copy number of ef1˛ as the internal control (D) were examined. The 1 h HS and 2.5 h HS denote the 1 and 2.5 h exposure to the heat shock condition where 0 h RT, 1 h RT and 3 h RT are 0, 1 and 3 h after recovering at the room temperature seawater tank. The error bars represent the corresponding standard deviations from at least triplicate trials. Different letters (a, b and c) denote statistically significant differences (P < 0.05).
4. Discussion The organisms respond to a sudden temperature elevation by synthesizing a set of proteins including heat shock proteins (HSPs) (Ritossa, 1996). HSPs are known to play protective roles as molecular chaperones to assist in protein folding, protein assembly and damaged proteins degradation (Kregel, 2002). While most of the hsp genes are well characterized in model organisms such as D. melanogaster (Loeschcke et al., 1997; Vabulas et al., 2002; Morrow and Tanguay, 2003), a little information is known in P. monodon. Although several families of the hsp genes have been identified, only full-length cDNAs of hsp21, hsp70 and hsp90 were characterized in P. monodon (Huang et al., 2008; Lo et al., 2004; Jiang et al., 2009). The extensive studies of the three major families of the heat shock proteins in model organisms demonstrate that HSP70 plays the primary role in assisting folding of nascent polypeptide chains, facilitate in repairing or degrading of damaged proteins (Feder and Hofmann, 1999). The HSP90 has been shown to involve in various cell components such as protein kinases in signal transduction pathways, transcriptional factors, cell cycle regulators, and steroid hormone receptors (Xu et al., 2002; Nathan et al., 1997; Wu and Chu, 2008). The HSP21 acts as a chaperone by binding to an unfolded protein and mediating in protein folding (Jaya et al., 2009; Lee et al., 1997). To gain more insight on the hsp genes in P. monodon, the expression profiles of hsp21, hsp70 and hsp90 in different tissues and the time-course induction patterns of these genes under
heat shock in gill and hepatopancreas of juvenile shrimp were assessed. The expression and distribution patterns of hsp21, hsp70, and hsp90 were quantified in nine tissues of P. monodon juveniles. Under no heat shock condition, the three hsp genes were commonly expressed in all examined tissues suggesting that all three forms of hsp gene products were required to maintain cellular homeostasis. However, their expression levels varied in each tissue. The hsp21 transcript was the most abundance in heart whereas the transcript of hsp70 was mostly expressed in hepatopancreas and hsp90 showed no significant difference in its basal expression level. The differential expression levels in different organs are observed in other model organisms. In flesh fly (Sarcophaga crassipalpis), the expression levels of hsp23, hsp70, and hsp90 are highest in brain, followed by epidermis and mid-gut. Among the three genes, the hsp70 transcript was expressed at a higher level suggesting that hsp70 was a dominant form of heat shock response proteins. On the other hand, the hsp21 and hsp90 levels were less abundance in comparison to that of the hsp70. The more abundance of the hsp70 than hsp21 and hsp90 from our results was also consistent with the higher frequency of hsp70 clones found in the P. monodon EST libraries than the hsp21 or hsp90 (Tassanakajon et al., 2006). In addition, our results were consistent with a previous report that the P. monodon hsp70 is the heat shock cognate isoform. Therefore, hsp70 in P. monodon is constitutively and highly expressed acting as a housekeeping gene whose protein plays a
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Fig. 4. Time-course expression of the hsp21, hsp70 and hsp90 under the heat shock condition in gill of 4-month old P. monodon. Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and (D) the copy number of ef1˛ as the internal control were examined. The 1 h HS and 2.5 h HS denote the 1 and 2.5 h exposure to the heat shock condition where 0 h RT, 1 h RT and 3 h RT are 0, 1 and 3 h after recovering at the room temperature. The error bars represent the corresponding standard deviations from at least triplicate trials. Different letters (a, b and c) denote statistically significant differences (P < 0.05).
putative role as defense mechanism against cell damages (Lo et al., 2004). When P. monodon juveniles exposed to the heat shock stress, the transcripts of hsp21, hsp70 and hsp90 were significantly upregulated in gill, heart, hepatopancreas, pleopod and thoracic ganglia. Among these organs, a time-course induction of the hsp21, hsp70 and hsp90 transcripts was further examined in hepatopancreas and gill. The expression levels of hsp21, hsp70 and hsp90 were significantly induced in hepatopancreas within the first hour after the heat exposure. Our results suggest that hepatopancreas in P. monodon was a heat-sensitive organ. Hepatopancreas is a major organ in crustaceans that plays important roles in digestion, absorption and secretory of nutrients (Brunet et al., 1994). Due to its metabolically active, hepatopancreas can be sensitive to environmental changes. Moreover, it also has been reported to be heat-sensitive in Monsoon river prawn (Macrobrachium malcolmsonii) (Bhavan and Geraldine, 2001). When we examined time-course expression patterns of hsp21, hsp70 and hsp90 in gill, the transcripts of hsp21 and hsp90 were up-regulated after 1 h of the heat shock exposure whereas the transcript of hsp70 became significantly induced after 2.5 h of the treatment. Strikingly, the transcripts of hsp21 and hsp70 still remained at high levels during the recovery phase in hepatopancreas but gradually declined to a similar level to the untreated control in gill within a 3-h period. The faster response observed in gill than in hepatopancreas is logical because gill directly contacts and constantly exchanges to the
environment. Thus, it may sense environmental changes, such as a shift in temperature, faster than hepatopancreas. To further investigate involvement of the hsp genes in immune response, we determined the expression profiles of hsp21, hsp70 and hsp90 in gill of P. monodon exposed to V. harveyi. Upon pathogen invasion, P. monodon triggers innate immune responses as their defense mechanisms and the well-known mechanism of shrimp innate immune system against bacterial infection is phagocytosis and dominantly takes place in hemocytes (Jiravanichpaisal et al., 2006). The phagocytosis process involves in the release of reactive oxygen species (ROS) to kill pathogenic bacteria. In consequence, the accumulation of ROS is harmful to host cell proteins, resulting in a similar protein damage to heat shock stress (Parsell and Lindquist, 1993; Imlay, 2003). Since HSPs have been well characterized as molecular chaperones, which assist in protein folding and repairing along with the significant induction of the hsp70 and hsp90 transcripts in hemocytes under heat shock stress also suggests for possible involvement of hsp in shrimp immune response. Thus, we investigated if the hsp genes were inducible upon the presence of bacterial antigens in P. monodon. Although hemocyte is a primary organ for immune response against pathogens, there has been a report that gill tissue is also one of sites for bacteria accumulation in pacific white shrimp (Burgents et al., 2005). Therefore, we chose to determine the hsp gene expression in gill upon V. harveyi infection. To reduce other complications that may cause by disease progression in host shrimp, we opted to inject juveniles
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Fig. 5. Expression profiles of hsp21, hsp70 and hsp90 in gill of P. monodon injected with the saline solution as the control (open bars) or heat-killed V. harveyi (solid bars). Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and (D) the copy number of ef1˛ as the internal control were examined. The 3, 12, 24 and 72 h refer to number of hours after the injection. The error bars represent the corresponding standard deviations from at least triplicate trials. Asterisks denote statistically significant differences between control and V. harveyi injection groups (P < 0.05).
with the heat-killed V. harveyi instead of challenging them with a live bacterial culture. Our study revealed that the hsp21, hsp70 and hsp90 transcripts were inducible upon the presence of the pathogen where the transcripts of hsp70 and hsp90 were highly induced during the first 3 h of the treatment. Meanwhile, the expression level of hsp21 became significantly induced later at 24 h after the treatment. This result provides the evidence for putative roles of protein products of hsp21, hsp70 and hsp90 in an immune response, especially in the case of the highly induced hsp90 expression level during the V. harveyi challenge. In mammalian systems, heat shock proteins have been proposed to act as signaling molecules to activate the host innate immune response. Heat shock proteins such as HSP70 and HSP90 are released from damaged cells to interact with host immune cells (Srivastava, 2002; Basu et al., 2001). Although the stimulation of innate immune responses by heat shock proteins in hosts has not yet been extensively characterized in invertebrates, many reports have shown the correlation between the increased levels of heat shock proteins and the reduction of pathogens such as the induction of HSP70 by a short exposure to heat shock results in the reduction of Gill-Associated Virus (GAV) in infected P. monodon (Vega et al., 2006). Additionally, we hypothesize that the bacterial infection such as V. harveyi may cause similar tissue and protein damages to heat shock stress. Therefore, upon the presence of pathogens, shrimp induced the expression of the hsp genes, which may act as chaperone proteins to repair protein damages and may play additional role as signaling molecules to modulate innate immune response in host shrimp. The induction of hsp70 from our study was consistent with the previous expressed sequence tag (EST) analysis of cDNA library from lym-
phoid organ of the V. harveyi-infected P. monodon (Pongsomboon et al., 2008). They have reported two clones with hsp70 sequence (out of the total of 625 clones) as the evidence for an involvement of hsp70 in immune response. However, clones with hsp21 and hsp90 sequences were not reported in this EST library analysis (625 clones) and may be due to an insufficient number of clones sequenced to identify genes expressed at lower copy numbers such as hsp21 and hsp90. These were concordant with the finding on lower expression levels of hsp21 and hsp90 than that of hsp70 in our study. Among the three hsp genes, the hsp90 of P. monodon showed a distinct, consistent and rapidly responsive expression pattern under the heat shock stress and V. harveyi challenge. In heat shock stress, the hsp90 transcript was expressed at a relatively low level during the untreated condition but sharply induced after the exposure to 1 h of heat shock and became higher with a longer exposure time. This expression profile of the hsp90 transcript was obviously time-dependent. During the recovery phase after the heat shock stress, the hsp90 expression level was gradually declined until it reached a similar level as seen in the untreated condition. A similar expression pattern of hsp90 was also observed when P. monodon exposed to V. harveyi. This similar response suggests that hsp90 was an acute and common response to the heat shock treatment and bacterial infection. The HSP90, encoded by hsp90, has been reported as a dimer protein and its activity required ATP (Buchner, 1999). In both eukaryotes and prokaryotes, HSP90 plays a crucial role in protein folding as one of protective mechanisms against heat shock stress (Freeman and Morimoto, 1996; Wandinger et al., 2008). Interestingly, unlike other molecular chap-
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erones, HSP90 also involves in several signal transduction systems such as protein kinases in MAP kinase family and nuclear receptors for steroid hormones (Scherrer et al., 1993; Pratt, 1997). Moreover, HSP90 also plays an important role in bacterial DNA and LPS-mediated macrophage activation in the host (Byrd et al., 1999; Zhu and Pisetsky, 2001). In other aquatic animal model such as a disk abalone (Haliotis discus), the hsp90 transcript is inducible after the bacterial LPS challenge (Wang et al., 2010). In this work, we provide an evidence for the involvement of hsp90 in P. monodon under both heat shock and bacterial stresses. The transient up-regulation of hsp90 in gill of P. monodon under stress conditions provides an advantage for a non-lethal screening assay and hsp90 may be a good candidate gene as the biomarker to assess level of environmental and disease stresses in P. monodon farming. In conclusion, this work examined the expression patterns of hsp21, hsp70, and hsp90 under in nine tissues of P. monodon, and hsp70 was the most dominant form. The time-course induction patterns of the three hsp transcripts were determined in gill and hepatopancreas where all three genes were significantly induced under heat shock stress. In addition to heat shock stress, the transcripts of hsp21, hsp70 and hsp90 were significantly up-regulated when P. monodon exposed to the presence of V. harveyi. This work provides the evidence to support the putative roles of hsp21, hsp70 and hsp90 as a part of the host immune response. The further studies on the involvement of heat shock proteins under environmental and pathogenic stresses will be essential to elucidate the protective roles of heat shock proteins in host shrimp. Acknowledgements We thank Assoc. Prof. Somkiat Piyatiratitivorakul, Center of Excellence for Marine Biotechnology, Chulalongkorn University for advices and facility. We thank Umaporn Uawisetwathana, Thidathip Wongsurawat, Pacharaporn Angthong and Ittipon Suriyachay for their assistance to this project. This project was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. References Parsell, D.A., Lindquist, S., 1993. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437–496. Kregel, K.C., 2002. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol. 92 (5), 2177–2186. Morimoto, R.I., 1998. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 12 (24), 3788–3796. Sharma, S.K., Christen, P., Goloubinoff, P., 2009. Disaggregating chaperones: an unfolding story. Curr. Protein Pept. Sci. 10 (5), 432–446. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282. Moseley, P., 2000. Stress proteins and the immune response. Immunopharmacology 48 (3), 299–302. Nover, L., Scharf, K.D., 1997. Heat stress proteins and transcription factors. Cell Mol. Life Sci. 53 (1), 80–103. Cotto, J.J., Morimoto, R.I., 1999. Stress-induced activation of the heat-shock response: cell and molecular biology of heat-shock factors. Biochem. Society Symposium 64, 105–118. Huang, P., Kang, S., Chen, W., Hsu, T., Lo, C., Liu, K., et al., 2008. Identification of the small heat shock proteins HSP21, of shrimp Penaeus monodon and the gene expression of HSP21 is inactivated after white spot syndrome virus (WSSV) infection. Fish Shellfish Immunol. 25, 250–257. Lo, W., Liu, K., Song, Y., 2004. Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon). Cell Stress Chaperones 9 (4), 332–343.
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Loeschcke, V., Krebs, R.A., Dahlgaard, J., Michalak, P., 1997. High-temperature stress and the evolution of thermal resistance in Drosophila. Exs 83, 175–190. Vabulas, R.M., Wagner, H., Schild, H., 2002. Heat shock proteins as ligands of toll-like receptors. Curr. Top. Microbiol. Immunol. 270, 169–184. Morrow, G., Tanguay, R.M., 2003. Heat shock proteins and aging in Drosophila melanogaster. Semin. Cell Dev. Biol. 14 (5), 291–299. Xu, W., Mimnaugh, E.G., Kim, J.S., Trepel, J.B., Neckers, L.M., 2002. Hsp90, not Grp94, regulates the intracellular trafficking and stability of nascent ErbB2. Cell Stress Chaperones 7 (1), 91–96. Nathan, D.F., Vos, M.H., Lindquist, S., 1997. In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone. Proc. Natl. Acad. Sci. U. S. A. 94 (24), 12949–12956. Wu, L., Chu, K., 2008. Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: evidence for its role in the regulation of vitellogenin synthesis. Mol. Reprod. Dev. 75, 952–959. Jaya, N., Garcia, V., Vierling, E., 2009. Substrate binding site flexibility of the small heat shock protein molecular chaperones. Proc. Natl. Acad. Sci. U. S. A. 106 (37), 15604–15609. Lee, G.J., Roseman, A.M., Saibil, H.R., Vierling, E., 1997. A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state. EMBO J. 16 (3), 659–671. Tassanakajon, A., Klinbunga, S., Paunglarp, N., Rimphanitchayakit, V., Udomkit, A., Jitrapakdee, S., et al., 2006. Penaeus monodon gene discovery project: the generation of an EST collection and establishment of a database. Gene 384, 104–112. Brunet, M., Arnaud, J., Mazza, J., 1994. Gut structure and digestive cellular process in marine crustacea. Oceanogr. Mar. Biol. 32, 335–367. Jiravanichpaisal, P., Lee, B.L., Soderhall, K., 2006. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology 211 (4), 213–236. Imlay, J.A., 2003. Pathways of oxidative damage. Annu. Rev. Microbiol. 57, 395–418. Burgents, J.E., Burnett, L.E., Stabb, E.V., Burnett, K.G., 2005. Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp Litopenaeus vannamei. Dev. Comp. Immunol. 29 (8), 681–691. Srivastava, P., 2002. Roles of heat-shock proteins in innate and adaptive immunity. Nat. Rev. Immunol. 2 (3), 185–194. Basu, S., Binder, R.J., Ramalingam, T., Srivastava, P.K., 2001. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 14 (3), 303–313. Pongsomboon, S., Wongpanya, R., Tang, S., Chalorsrikul, A., Tassanakajon, A., 2008. Abundantly expressed transcripts in the lymphoid organ of the black tiger shrimp Penaeus monodon, and their implication in immune function. Fish Shellfish Immunol. 25 (5), 485–493. Buchner, J., 1999. Hsp90 & Co – a holding for folding. Trends Biochem Sci. 24 (4), 136–141. Freeman, B.C., Morimoto, R.I., 1996. The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding. EMBO J. 15 (12), 2969–2979. Wandinger, S.K., Richter, K., Buchner, J., 2008. The Hsp90 chaperone machinery. J. Biol. Chem. 283 (27), 18473–18477. Scherrer, L.C., Picard, D., Massa, E., Harmon, J.M., Simons Jr., S.S., Yamamoto, K.R., et al., 1993. Evidence that the hormone binding domain of steroid receptors confers hormonal control on chimeric proteins by determining their hormone-regulated binding to heat-shock protein 90. Biochemistry 32 (20), 5381–5386. Pratt, W.B., 1997. The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptors signaling via MAP kinase. Annu. Rev. Pharmacol. Toxicol. 37, 297–326. Byrd, C.A., Bornmann, W., Erdjument-Bromage, H., Tempst, P., Pavletich, N., Rosen, N., et al., 1999. 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Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci
Expression and distribution of three heat shock protein genes under heat shock stress and under exposure to Vibrio harveyi in Penaeus monodon Wanilada Rungrassamee a,∗ , Rungnapa Leelatanawit a , Pikul Jiravanichpaisal b , Sirawut Klinbunga b , Nitsara Karoonuthaisiri a a Microarray Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Pathumthani, 12120, Thailand b Aquatic Molecular Genetics and Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Road, Pathumthani, 12120, Thailand
a r t i c l e
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Article history: Received 12 April 2010 Received in revised form 24 May 2010 Accepted 24 May 2010 Available online 8 June 2010 Keywords: Heat shock response Heat shock protein Gene expression Black tiger shrimp Penaeus monodon Vibrio harveyi
a b s t r a c t A sudden increase in temperature results in heat shock stress of the cultured shrimp. To cope with the stress, shrimp has to overcome by triggering a response known as heat shock response. To understand the heat shock response in the black tiger shrimp (Penaeus monodon), we examined expression patterns and distribution of three heat shock protein (hsp) genes in P. monodon juveniles. The expression levels of hsp21, hsp70 and hsp90 were determined by quantitative real-time PCR in nine tissues (gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocyte) under untreated and heat shock conditions. Under untreated condition, all three hsp genes were differentially expressed in all examined tissues where the hsp70 transcript showed the highest basal level. Under heat shock condition, only hsp90 was inducible in all nine tissues when comparing to its untreated level. The timecourse induction experiment in gill and hepatopancreas revealed that the transcriptional levels of hsp21, hsp70 and hsp90 were inducible under the heat shock condition and in time-dependent manner. To determine the response of the hsp genes upon bacterial exposure, we further determined transcript levels of the hsp genes in gill of P. monodon after Vibrio harveyi injection. The expression levels of hsp70 and hsp90 were significantly increased after a 3-h exposure to V. harveyi where the hsp21 transcript was induced later after a 24-h exposure. This evidence suggests for putative roles and involvement of the hsp genes as a part of immunity response against V. harveyi in P. monodon. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Natural and farming environments can be variable and unpredictable. An organism needs mechanisms to adapt to a wide range of the stressful conditions. In some habitats, a stress from daily or seasonal fluctuation of environmental temperature causes an organism to respond by inducing sets of proteins including heat shock proteins (HSPs) and this process is known as a heat shock stress response (Parsell and Lindquist, 1993). The HSPs are ubiquitous, highly conserved and found in all organisms (Kregel, 2002). They play critical roles as molecular chaperones in heat tolerance by repairing and refolding denatured proteins (Morimoto, 1998; Sharma et al., 2009). In addition to heat shock stress, HSPs respond to other factors such as pathogen infection, oxidative stress, heavy metals and xenobiotics stresses (Feder and Hofmann, 1999; Moseley, 2000). The HSPs are found in various forms and categorized into different families based on their molecular weights
∗ Corresponding author. Tel.: +66 2 564 6700x3255; fax: +66 2 564 6707. E-mail address: [email protected] (W. Rungrassamee). 0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2010.05.012
(kDa) such as HSP110, HSP100, HSP90, HSP70, HSP60 and small HSPs (Parsell and Lindquist, 1993; Feder and Hofmann, 1999). The HSP70 family has been extensively characterized as a primary family of heat shock proteins, and the members in HSP70 family can be constitutive or inducible expressed under heat shock stress (Nover and Scharf, 1997). The heat shock responses are well characterized in many model organisms such as Drosophila melanogaster, mouse and Arabidopsis; however, the responses can vary in different organisms (Feder and Hofmann, 1999; Cotto and Morimoto, 1999). In black tiger shrimp (Penaeus monodon), one of the important aquaculture species, a complete understanding of how the animal responds to heat shock stress will help improving farming condition to boost the shrimp immunity and protecting them from a risk of disease outbreak. In an attempt to understand heat shock stress response in P. monodon, heat shock proteins have previously been characterized by identifying full-length cDNA sequences of hsp21, hsp70 and hsp90 (Huang et al., 2008; Lo et al., 2004; Jiang et al., 2009). The hsp21 transcript has been shown to be heat inducible in pleopods and was down-regulated during the infection by White Spot Syndrome Virus (Huang et al., 2008). The hsp70 was inducible in hemocytes
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Fig. 1. Schematic representation of experiments performed in this study: (A) heat shock and (B) V. harveyi experiments. (A) In heat shock (HS) experiment, a group of 4-month old P. monodon juveniles was maintained at the room temperature (RT) where tissues were collected as the untreated control. The juvenile shrimp were then placed to a 35 ◦ C seawater tank and tissues were collected at 1 and 2.5 h of exposure time (1 h HS and 2.5 h HS, respectively). For the recovery phase, the juvenile shrimp were returned to the room temperature seawater tank, and tissues were collected immediately after 0 h RT, while the remainders were allowed to recover in the seawater tank for 1 and 3 h (1 h RT and 3 h RT, respectively) before tissue collection. Gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocyte were collected at the sampling time with asterisk (*) (N = 5). Otherwise, only gill and hepatopancreas were collected (N = 5). (B) In V. harveyi experiment, a group of 4-month old juveniles was injected with 100 l of heat-killed V. harveyi 1526 in the saline solution (∼2.0 OD600 ) at the lateral side of abdominal muscles, where a control group was injected with 100 l of the saline solution but without bacterial cells (control). The gill tissues were dissected out from shrimp at 3, 12, 24 and 72 h after the injection (N = 3 for each time point).
under heat shock conditions and the increase in the hsp70 expression was correlated with the reduction of Gill-Associated Virus (GAV) replication (Vega et al., 2006). Similarly, hsp90 was heat inducible in brain, stomach and heart and may play a role in ovary maturation (Jiang et al., 2009). Nonetheless, the gene expression patterns for hsp21, hsp70 and hsp90 in P. monodon were investigated under different heat shock treatment conditions in their studies. In this study, we aimed to characterize gene expression patterns of all three hsp21, hsp70, and hsp90 transcripts under the same heat shock treatment conditions to give a better understanding of the response to the stress. We also investigated if hsp genes were inducible upon the presence of a heat-killed shrimp pathogen. The expression patterns of the three major hsp genes (hsp21, hsp70 and hsp90) in nine tissues of P. monodon under both untreated and heat shock conditions were examined to validate for inducibility of hsp genes under the stress condition. We provided the first report on the time dependency of expression patterns of hsp21, hsp70 and hsp90 in hepatopancreas and gill tissues during and after heat shock conditions. Moreover, expression patterns of these genes were examined in P. monodon upon an exposure to the heat-killed shrimp pathogen, Vibrio harveyi, to determine the possible roles of the hsp genes as part of the immune response in shrimp.
in Chachoengsao province (Eastern Thailand) were transferred to seawater tanks with aeration and maintained at room temperature (27 ± 2 ◦ C, RT) with salinity at 20 parts per thousand (ppt). In the heat shock experiment, a group of 50 juvenile shrimp was transferred from the RT seawater tank to a 35 ± 2 ◦ C seawater tank. Tissue samples were collected from t = 0 (untreated at room temperature (RT), prior to heat shock treatment), heat shock (HS) = 1 and 2.5 h for heat shock treatment duration (N = 5 for each time point). Then, the group of remaining juveniles were returned to the RT seawater tank, the tissue samples (N = 5 for each time point) were collected at RT = 0 (immediate after placing back to room temperature), RT = 1 and RT = 3 h (1 and 3 h, respectively, after placing back to room temperature). The collected tissues were quickly placed in liquid nitrogen (−80 ◦ C). For V. harveyi infection (Fig. 1B), a group of 25 juvenile shrimp was injected with 100 l of heat-killed V. harveyi 1526 (Rengpipat et al., 2003) (∼2 × 108 cells/ml) at the lateral side of abdominal muscles, where the control group of 25 juvenile shrimp was injected with the same diluent but without bacterial cells (100 l of saline solution). The gill tissues were dissected out from shrimp at 3, 12, 24 and 72 h after injection (N = 3 for each time point). 2.2. RNA extraction and cDNA synthesis
2. Materials and methods 2.1. Experimental animals, heat shock treatment and Vibrio harveyi infection The heat shock treatment was depicted in Fig. 1A. Sixty of 4month-old P. monodon juveniles (13.4 ± 2.0 g) obtained from a farm
Each tissue sample was ground using a mortar in liquid nitrogen and transferred to TriReagent® (Molecular Research Center). RNA extraction was performed according to supplier’s instruction. The RNA pellets were resuspended in 50 l of RNase-free water and treated with DNaseI (0.5 unit/g, Promega) for 30 min at 37 ◦ C to remove DNA contamination. The DNA-free RNA was purified by phenol: chloroform extraction and precipitated with 1/10 volume
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Table 1 Oligonucleotides used in this study.
3. Results
Primer
Sequence 5 –3
Amplicon size (bp)
hsp21 F hsp21 R hsp70 F hsp70 R hsp90 F hsp90 R ef1˛ F ef1˛ R
AAT TCA TTG CGG AAG CGA GCC A ACT TCA GCG TGA TCG ACC AGG AAT AGA AGT CAC TCC GTG ATG CCA AGA ACT CCT TGC CGT TGA AGA AGT CCT GCA TGA AGG AGA ACC AGA AGC ACA TGA ACG CAG TAT TCG TCG ATG GGT GGC GTA CTG GTA AGG AAC TGG AA GAG GAG CAT ACT GTT GGA AGG TCT C
100 123 145 123
of 3 M sodium acetate and 1 volume of isopropanol. The concentration of each RNA sample was measured by using a NanoDrop UV–vis Spectrophotometer (ND-8000). Treated RNA samples were further confirmed for an absence of DNA contamination by PCR. A RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas) was used to synthesize the first strand cDNA using total RNA (1.5 g) as a template in a total reaction volume of 20 l. Aliquots of 0.5 l of cDNA preparation were used for quantitative real-time PCR reactions. 2.3. Quantification of hsp21, hsp70 and hsp90 expression by real-time qRT-PCR The abundance of the hsp21, hsp70 and hsp90 transcripts was measured by real-time quantitative reverse transcription PCR (real-time qRT-PCR). The reactions were carried out in an iCycler (BIORAD) using IQTM SYBR® Green Supermix (BIORAD) with primer pairs for hsp21 (the amplicon size = 100 bp), hsp70 (123 bp), and hsp90 (145 bp), respectively (Table 1). All reactions were carried out according to the supplier’s instructions. The housekeeping gene, ef1˛, was used as an internal control for all real-time PCR experiments (primers ef1˛ F + R, 123 bp; Table 1). The cycling parameters used were as follows: an initial denaturation at 95 ◦ C for 3 min, 40 cycles at 95 ◦ C for 20 s, 57 ◦ C for 30 s and 72 ◦ C for 30 s. The fluorescent signal intensities were recorded at the end of each cycle. Melting curve analysis was performed from 55 to 95 ◦ C with continuous fluorescence reading every 0.5 ◦ C increment. Standard curves for the quantification of number of mRNA transcripts per g of total RNA in the quantitative real-time PCR were plotted from the result of amplification reactions using each primer pair and plasmid templates containing the partial sequences of the hsp21, hsp70 or hsp90 genes. In order to construct these templates, a DNA fragment containing hsp21 (100 bp), hsp70 (123 bp) or hsp90 (145 bp) partial sequence was amplified by PCR using the same primer pairs in the real-time PCR reaction. The PCR products were gel-purified (Qiagen Gel Purification Kit) and subsequently cloned into a plasmid vector pGEM-T (Promega). Each resulting vector was transformed into E. coli DH5␣ and the transformants were selected on LB plates containing ampicillin (50 g/ml). Similarly, a plasmid containing the ef1˛ segment was constructed using a TOPO-TA cloning kit (Invitrogen) and the TOPO cloning reaction and transformation were done according to supplier’s instruction. All plasmids were extracted from the 4 ml overnight cultures using a plasmid miniprep kit (Favorgen). Plasmid concentrations were quantified using a Nanodrop (ND-8100) at a wavelength of 260 nm and the copy numbers were calculated. Five concentrations of 10-fold serial dilutions (106 to 102 copies) of each plasmid were used as templates to construct standard curves for hsp21, hsp70, hsp90 and ef1˛. Real-time PCR samples were set up in SYBR green reactions with primers for hsp21, hsp70, hsp90 and ef1˛ (Table 1). The cycling parameters used were the same as described earlier. The standard curve allowed a quantification of mRNA in samples. Data was analyzed by iCycler software. All real-time PCR experiments were performed in five independent trials.
3.1. Tissue distribution of hsp21, hsp70 and hsp90 in P. monodon To validate the inducibility of hsp genes in heat shock condition, the expression levels of the hsp21, hsp70, and hsp90 transcripts were measured by quantitative real-time PCR in nine tissues (gill, heart, hepatopancreas, stomach, intestine, eyestalk, pleopod, thoracic ganglia and hemocytes) from P. monodon juveniles under untreated and heat shock conditions (Fig. 2). The elevated temperature at 35 ◦ C for 2.5 h was chosen for the heat shock condition because at this temperature the heat shock stress response can be induced with a minor effect on shrimp survival rates (data not shown). The transcripts of hsp21, hsp70 and hsp90 transcripts were detected with varied expression levels in all examined tissues. Under untreated condition, the basal expression level of the hsp21 transcript was highest in heart but lowest in hemocytes (Fig. 2A), whereas the hsp70 basal expression level was highest in hepatopancreas and lowest in thoracic ganglia (Fig. 2B). On the other hand, the basal levels of the hsp90 transcript were detected with marginal differences in all tissues (Fig. 2C). Among the three hsp transcripts, the hsp70 transcript was expressed at the highest level under untreated condition in all tissues except in thoracic ganglia. Under the heat shock condition, hsp21 was significantly induced in gill, heart, hepatopancreas, pleopod and thoracic ganglia (1.9, 2.0-, 1.8-, 2.5- and 2.4-fold of induction, respectively) while its expression levels remained relatively the same in stomach, intestine, eyestalk and hemocyte (Fig. 2A). For the hsp70 transcript, it was significantly induced in most tissues, except in stomach and intestine (Fig. 2B). The hsp70 transcript was the most induced in thoracic ganglia with a 24-fold increase. Most strikingly, the hsp90 transcript was significantly up-regulated under the heat shock condition in all tissues with the most induction in hepatopancreas (12-fold; Fig. 2C). The expression levels of the internal control, ef1˛, were not significantly different among the nine examined tissues under untreated and heat shock conditions (Fig. 2D). 3.2. Time-course induction of the hsp genes in hepatopancreas and gill under heat shock stress Hepatopancreas has been previously reported to be a heatsensitive tissue in crustaceans (Bhavan and Geraldine, 2001) which was consistent with our results from tissue distribution analysis showing the significant inductions of all three hsp genes in this tissue (Fig. 2A–C). Therefore, in the time-course heat shock experiment, the hsp expression patterns were further analyzed in hepatopancreas (Fig. 3). To assess a time-dependent induction of the hsp gene expression, copy numbers of the hsp21, hsp70 and hsp90 transcripts were measured in P. monodon juveniles exposed to the heat shock stress before recovering back to a room temperature seawater tank (Fig. 1A). Under untreated conditions, the basal expression levels of hsp21 and hsp90 in hepatopancreas were lower than the hsp70 level, which were consistent to the observations in the tissue distribution analysis (untreated, Fig. 3A–C). When P. monodon was placed in the elevated temperature for 1 h (1 h HS), the transcripts of hsp21 hsp70 and hsp90 were increased by 3-, 2and 6-fold, respectively. When the heat shock exposure time was prolonged to 2.5 h (2.5 h HS), the levels of hsp21 and hsp70 transcripts remained similar to those of the 1-h exposure group (1 h HS) (Fig. 3A and B), but the hsp90 transcript level was significantly induced by 11-fold from its untreated level (Fig. 3C). Once shrimp were recovering from the heat shock treatment to the room temperature (RT), the hsp90 transcript level gradually declined to the similar level as in untreated control within 3 h. Interestingly, the transcript levels of hsp21 and hsp70 still remained high where that
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Fig. 2. Expression profiles of hsp21, hsp70 and hsp90 in nine different tissues of P. monodon juveniles with no heat shock (untreated, open bars) and under the heat shock condition for 2.5 h (solid bars). Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and the copy number of ef1˛ as the internal control (D) were examined. GL = gill, HT = heart, HP = hepatopancreas, ST = stomach, IN = intestine, ES = eyestalk, PL = pleopod, TH = thoracic ganglion and HC = hemocyte. The error bars represent the corresponding standard deviations from triplicate trials. Asterisks denote statistically significant differences between untreated control and heat shock groups (P < 0.05).
of the latter was further increased during the recovering phase at room temperature. In addition to hepatopancreas, expression profiles of the hsp genes were also determined in gill due to its important role in respiration and a direct contact to the aquatic environment (Fig. 4). In gill, the hsp21 transcript level was significantly increased when shrimp exposed to heat shock for 1 h (1 h HS) and further increased after 2.5 h of the heat shock treatment (2.5 h HS), then reached the highest level at the beginning of recovery phase at room temperature (0 h RT) before gradually declined after 1 and 3 h of the recovery phase (1 and 3 h RT), respectively (Fig. 4A). In contrast, the level of hsp70 transcript was already high in the untreated control, thus, its induction level after the heat shock was not significant until 2.5 h of the heat shock (2.5 h HS) (Fig. 4B). The transcript level of hsp70 was lowered during the recovery phase (0 h, 1 h, 3 h RT) after the heat shock treatment. The expression pattern of the hsp90 transcript in gill was similar to the pattern observed in hepatopancreas (Fig. 3C and 4C). Under the heat shock stress, the hsp90 expression level was induced 18-fold within 1 h (1 h HS) and 23-fold in 2.5 h (2.5 h HS). Once the shrimp were returned back to the room temperature for recovery, the hsp90 transcript level became gradually lowered until it reached the same level as in the untreated control after 3 h of the recovery time. The expression level of ef1˛ in gill did not show any significant change during the time-course experiment (Fig. 4D). 3.3. Expression profile analysis of the hsp genes in gill of P. monodon challenged with V. harveyi To further investigate roles of the hsp genes in the pathogenic stress, the expression profiles of these three hsp genes were determined in gill of P. monodon after injected with the pathogenic bacterium, V. harveyi (Fig. 1B). Gill tissue was chosen in this study
because it could be collected using a non-lethal procedure, while this procedure could not be done for hepatopancreas tissue. The gill tissue was sampled at 3, 12, 24, and 72 h after the V. harveyi exposure. In a parallel experiment, gill was also collected from the shrimp injected with a saline solution as a control group and the expression levels of the hsp transcripts in this group were used as the basal expression levels to compare to the V. harveyi treated group. Comparing to the control group, the expression level of hsp21 was slightly increased after 12 h of the V. harveyi injection and significantly induced at 24 h of the treatment, before returned to the similar level to the control after 72 h of the treatment (Fig. 5A). Meanwhile, the hsp70 and hsp90 transcripts showed significant increases at the first 3 h of the V. harveyi exposure (2.6 and 3.4fold comparing to the control at the same time point, respectively) (Fig. 5B and 5C). The hsp70 transcript level continued to significantly increase after 12 h of treatment, peaked at 24 h (4.8-fold and 4.4- fold of induction comparing to the control at the same time point, respectively) and still significant at 72 h in the presence of V. harveyi (Fig. 5B). On the other hand, the induction of the hsp90 transcript reached the highest level at 12-h of the V. harveyi exposure (5.7-fold comparing to the control at the same time point), began to decline at 24 h (3.6-fold comparing to the control of the same time point) and eventually returned to the similar level as the control at 72 h of post-treatment (Fig. 5C). When we compared the overall expression patterns of the three hsp genes over the timecourse of the V. harveyi exposure, hsp21, hsp70 and hsp90 exhibited increasing patterns until they reached the maximum induction and then became lower to the similar levels of the control group. The group of juveniles that were injected with the saline solution did not show significant difference in transcript levels of hsp21, hsp70 and hsp90 overtime. The expression of ef1˛ was not significantly altered in juveniles injected with the saline solution and heat-killed V. harveyi (Fig. 5D).
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Fig. 3. Time-course gene expression of hsp21, hsp70 and hsp90 under the heat shock condition in hepatopancreas of 4-month old P. monodon. Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and the copy number of ef1˛ as the internal control (D) were examined. The 1 h HS and 2.5 h HS denote the 1 and 2.5 h exposure to the heat shock condition where 0 h RT, 1 h RT and 3 h RT are 0, 1 and 3 h after recovering at the room temperature seawater tank. The error bars represent the corresponding standard deviations from at least triplicate trials. Different letters (a, b and c) denote statistically significant differences (P < 0.05).
4. Discussion The organisms respond to a sudden temperature elevation by synthesizing a set of proteins including heat shock proteins (HSPs) (Ritossa, 1996). HSPs are known to play protective roles as molecular chaperones to assist in protein folding, protein assembly and damaged proteins degradation (Kregel, 2002). While most of the hsp genes are well characterized in model organisms such as D. melanogaster (Loeschcke et al., 1997; Vabulas et al., 2002; Morrow and Tanguay, 2003), a little information is known in P. monodon. Although several families of the hsp genes have been identified, only full-length cDNAs of hsp21, hsp70 and hsp90 were characterized in P. monodon (Huang et al., 2008; Lo et al., 2004; Jiang et al., 2009). The extensive studies of the three major families of the heat shock proteins in model organisms demonstrate that HSP70 plays the primary role in assisting folding of nascent polypeptide chains, facilitate in repairing or degrading of damaged proteins (Feder and Hofmann, 1999). The HSP90 has been shown to involve in various cell components such as protein kinases in signal transduction pathways, transcriptional factors, cell cycle regulators, and steroid hormone receptors (Xu et al., 2002; Nathan et al., 1997; Wu and Chu, 2008). The HSP21 acts as a chaperone by binding to an unfolded protein and mediating in protein folding (Jaya et al., 2009; Lee et al., 1997). To gain more insight on the hsp genes in P. monodon, the expression profiles of hsp21, hsp70 and hsp90 in different tissues and the time-course induction patterns of these genes under
heat shock in gill and hepatopancreas of juvenile shrimp were assessed. The expression and distribution patterns of hsp21, hsp70, and hsp90 were quantified in nine tissues of P. monodon juveniles. Under no heat shock condition, the three hsp genes were commonly expressed in all examined tissues suggesting that all three forms of hsp gene products were required to maintain cellular homeostasis. However, their expression levels varied in each tissue. The hsp21 transcript was the most abundance in heart whereas the transcript of hsp70 was mostly expressed in hepatopancreas and hsp90 showed no significant difference in its basal expression level. The differential expression levels in different organs are observed in other model organisms. In flesh fly (Sarcophaga crassipalpis), the expression levels of hsp23, hsp70, and hsp90 are highest in brain, followed by epidermis and mid-gut. Among the three genes, the hsp70 transcript was expressed at a higher level suggesting that hsp70 was a dominant form of heat shock response proteins. On the other hand, the hsp21 and hsp90 levels were less abundance in comparison to that of the hsp70. The more abundance of the hsp70 than hsp21 and hsp90 from our results was also consistent with the higher frequency of hsp70 clones found in the P. monodon EST libraries than the hsp21 or hsp90 (Tassanakajon et al., 2006). In addition, our results were consistent with a previous report that the P. monodon hsp70 is the heat shock cognate isoform. Therefore, hsp70 in P. monodon is constitutively and highly expressed acting as a housekeeping gene whose protein plays a
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Fig. 4. Time-course expression of the hsp21, hsp70 and hsp90 under the heat shock condition in gill of 4-month old P. monodon. Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and (D) the copy number of ef1˛ as the internal control were examined. The 1 h HS and 2.5 h HS denote the 1 and 2.5 h exposure to the heat shock condition where 0 h RT, 1 h RT and 3 h RT are 0, 1 and 3 h after recovering at the room temperature. The error bars represent the corresponding standard deviations from at least triplicate trials. Different letters (a, b and c) denote statistically significant differences (P < 0.05).
putative role as defense mechanism against cell damages (Lo et al., 2004). When P. monodon juveniles exposed to the heat shock stress, the transcripts of hsp21, hsp70 and hsp90 were significantly upregulated in gill, heart, hepatopancreas, pleopod and thoracic ganglia. Among these organs, a time-course induction of the hsp21, hsp70 and hsp90 transcripts was further examined in hepatopancreas and gill. The expression levels of hsp21, hsp70 and hsp90 were significantly induced in hepatopancreas within the first hour after the heat exposure. Our results suggest that hepatopancreas in P. monodon was a heat-sensitive organ. Hepatopancreas is a major organ in crustaceans that plays important roles in digestion, absorption and secretory of nutrients (Brunet et al., 1994). Due to its metabolically active, hepatopancreas can be sensitive to environmental changes. Moreover, it also has been reported to be heat-sensitive in Monsoon river prawn (Macrobrachium malcolmsonii) (Bhavan and Geraldine, 2001). When we examined time-course expression patterns of hsp21, hsp70 and hsp90 in gill, the transcripts of hsp21 and hsp90 were up-regulated after 1 h of the heat shock exposure whereas the transcript of hsp70 became significantly induced after 2.5 h of the treatment. Strikingly, the transcripts of hsp21 and hsp70 still remained at high levels during the recovery phase in hepatopancreas but gradually declined to a similar level to the untreated control in gill within a 3-h period. The faster response observed in gill than in hepatopancreas is logical because gill directly contacts and constantly exchanges to the
environment. Thus, it may sense environmental changes, such as a shift in temperature, faster than hepatopancreas. To further investigate involvement of the hsp genes in immune response, we determined the expression profiles of hsp21, hsp70 and hsp90 in gill of P. monodon exposed to V. harveyi. Upon pathogen invasion, P. monodon triggers innate immune responses as their defense mechanisms and the well-known mechanism of shrimp innate immune system against bacterial infection is phagocytosis and dominantly takes place in hemocytes (Jiravanichpaisal et al., 2006). The phagocytosis process involves in the release of reactive oxygen species (ROS) to kill pathogenic bacteria. In consequence, the accumulation of ROS is harmful to host cell proteins, resulting in a similar protein damage to heat shock stress (Parsell and Lindquist, 1993; Imlay, 2003). Since HSPs have been well characterized as molecular chaperones, which assist in protein folding and repairing along with the significant induction of the hsp70 and hsp90 transcripts in hemocytes under heat shock stress also suggests for possible involvement of hsp in shrimp immune response. Thus, we investigated if the hsp genes were inducible upon the presence of bacterial antigens in P. monodon. Although hemocyte is a primary organ for immune response against pathogens, there has been a report that gill tissue is also one of sites for bacteria accumulation in pacific white shrimp (Burgents et al., 2005). Therefore, we chose to determine the hsp gene expression in gill upon V. harveyi infection. To reduce other complications that may cause by disease progression in host shrimp, we opted to inject juveniles
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Fig. 5. Expression profiles of hsp21, hsp70 and hsp90 in gill of P. monodon injected with the saline solution as the control (open bars) or heat-killed V. harveyi (solid bars). Gene expression patterns of hsp21 (A), hsp70, (B) and hsp90 (C), and (D) the copy number of ef1˛ as the internal control were examined. The 3, 12, 24 and 72 h refer to number of hours after the injection. The error bars represent the corresponding standard deviations from at least triplicate trials. Asterisks denote statistically significant differences between control and V. harveyi injection groups (P < 0.05).
with the heat-killed V. harveyi instead of challenging them with a live bacterial culture. Our study revealed that the hsp21, hsp70 and hsp90 transcripts were inducible upon the presence of the pathogen where the transcripts of hsp70 and hsp90 were highly induced during the first 3 h of the treatment. Meanwhile, the expression level of hsp21 became significantly induced later at 24 h after the treatment. This result provides the evidence for putative roles of protein products of hsp21, hsp70 and hsp90 in an immune response, especially in the case of the highly induced hsp90 expression level during the V. harveyi challenge. In mammalian systems, heat shock proteins have been proposed to act as signaling molecules to activate the host innate immune response. Heat shock proteins such as HSP70 and HSP90 are released from damaged cells to interact with host immune cells (Srivastava, 2002; Basu et al., 2001). Although the stimulation of innate immune responses by heat shock proteins in hosts has not yet been extensively characterized in invertebrates, many reports have shown the correlation between the increased levels of heat shock proteins and the reduction of pathogens such as the induction of HSP70 by a short exposure to heat shock results in the reduction of Gill-Associated Virus (GAV) in infected P. monodon (Vega et al., 2006). Additionally, we hypothesize that the bacterial infection such as V. harveyi may cause similar tissue and protein damages to heat shock stress. Therefore, upon the presence of pathogens, shrimp induced the expression of the hsp genes, which may act as chaperone proteins to repair protein damages and may play additional role as signaling molecules to modulate innate immune response in host shrimp. The induction of hsp70 from our study was consistent with the previous expressed sequence tag (EST) analysis of cDNA library from lym-
phoid organ of the V. harveyi-infected P. monodon (Pongsomboon et al., 2008). They have reported two clones with hsp70 sequence (out of the total of 625 clones) as the evidence for an involvement of hsp70 in immune response. However, clones with hsp21 and hsp90 sequences were not reported in this EST library analysis (625 clones) and may be due to an insufficient number of clones sequenced to identify genes expressed at lower copy numbers such as hsp21 and hsp90. These were concordant with the finding on lower expression levels of hsp21 and hsp90 than that of hsp70 in our study. Among the three hsp genes, the hsp90 of P. monodon showed a distinct, consistent and rapidly responsive expression pattern under the heat shock stress and V. harveyi challenge. In heat shock stress, the hsp90 transcript was expressed at a relatively low level during the untreated condition but sharply induced after the exposure to 1 h of heat shock and became higher with a longer exposure time. This expression profile of the hsp90 transcript was obviously time-dependent. During the recovery phase after the heat shock stress, the hsp90 expression level was gradually declined until it reached a similar level as seen in the untreated condition. A similar expression pattern of hsp90 was also observed when P. monodon exposed to V. harveyi. This similar response suggests that hsp90 was an acute and common response to the heat shock treatment and bacterial infection. The HSP90, encoded by hsp90, has been reported as a dimer protein and its activity required ATP (Buchner, 1999). In both eukaryotes and prokaryotes, HSP90 plays a crucial role in protein folding as one of protective mechanisms against heat shock stress (Freeman and Morimoto, 1996; Wandinger et al., 2008). Interestingly, unlike other molecular chap-
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erones, HSP90 also involves in several signal transduction systems such as protein kinases in MAP kinase family and nuclear receptors for steroid hormones (Scherrer et al., 1993; Pratt, 1997). Moreover, HSP90 also plays an important role in bacterial DNA and LPS-mediated macrophage activation in the host (Byrd et al., 1999; Zhu and Pisetsky, 2001). In other aquatic animal model such as a disk abalone (Haliotis discus), the hsp90 transcript is inducible after the bacterial LPS challenge (Wang et al., 2010). In this work, we provide an evidence for the involvement of hsp90 in P. monodon under both heat shock and bacterial stresses. The transient up-regulation of hsp90 in gill of P. monodon under stress conditions provides an advantage for a non-lethal screening assay and hsp90 may be a good candidate gene as the biomarker to assess level of environmental and disease stresses in P. monodon farming. In conclusion, this work examined the expression patterns of hsp21, hsp70, and hsp90 under in nine tissues of P. monodon, and hsp70 was the most dominant form. The time-course induction patterns of the three hsp transcripts were determined in gill and hepatopancreas where all three genes were significantly induced under heat shock stress. In addition to heat shock stress, the transcripts of hsp21, hsp70 and hsp90 were significantly up-regulated when P. monodon exposed to the presence of V. harveyi. This work provides the evidence to support the putative roles of hsp21, hsp70 and hsp90 as a part of the host immune response. The further studies on the involvement of heat shock proteins under environmental and pathogenic stresses will be essential to elucidate the protective roles of heat shock proteins in host shrimp. Acknowledgements We thank Assoc. Prof. Somkiat Piyatiratitivorakul, Center of Excellence for Marine Biotechnology, Chulalongkorn University for advices and facility. We thank Umaporn Uawisetwathana, Thidathip Wongsurawat, Pacharaporn Angthong and Ittipon Suriyachay for their assistance to this project. This project was supported by National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand. References Parsell, D.A., Lindquist, S., 1993. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437–496. Kregel, K.C., 2002. Heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol. 92 (5), 2177–2186. Morimoto, R.I., 1998. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 12 (24), 3788–3796. Sharma, S.K., Christen, P., Goloubinoff, P., 2009. Disaggregating chaperones: an unfolding story. Curr. Protein Pept. Sci. 10 (5), 432–446. Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282. Moseley, P., 2000. Stress proteins and the immune response. Immunopharmacology 48 (3), 299–302. Nover, L., Scharf, K.D., 1997. Heat stress proteins and transcription factors. Cell Mol. Life Sci. 53 (1), 80–103. Cotto, J.J., Morimoto, R.I., 1999. Stress-induced activation of the heat-shock response: cell and molecular biology of heat-shock factors. Biochem. Society Symposium 64, 105–118. Huang, P., Kang, S., Chen, W., Hsu, T., Lo, C., Liu, K., et al., 2008. Identification of the small heat shock proteins HSP21, of shrimp Penaeus monodon and the gene expression of HSP21 is inactivated after white spot syndrome virus (WSSV) infection. Fish Shellfish Immunol. 25, 250–257. Lo, W., Liu, K., Song, Y., 2004. Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon). Cell Stress Chaperones 9 (4), 332–343.
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