Genes encoding heat shock proteins in the endoparasitoid wasp,Cotesia chilonis, and their expression in response to temperatures

PAN Dan-dan, CAO Shuang-shuang, LU Ming-xing, HANG San-bao, DU Yu-zhou,

1 School of Horticulture and Plant Protection & Institute of Applied Entomology, Yangzhou University, Yangzhou 225009, P.R.China

2 Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education/Yangzhou University,Yangzhou 225009, P.R.China

Correspondence DU Yu-zhou, E-mail: yzdu@yzu.edu.cn

? 2018 CAAS. Publishing services by Elsevier B.V. All rights reserved.

1. Introduction

Temperature is one of the most important environmental factors for all living organisms, which could affect nearly all biological processes, rates and functions (Willmeret al.2000). Insect tolerance to temperatures varies considerably and has significant behavioral and ecological implications(Hausmannet al. 2005; Samietzet al. 2005). When exposed to extreme temperatures, insects may rapidly synthesize a set of proteins called heat shock proteins (HSPs) that participate in the refolding and relocalization of proteins(Hoffmann and Parsons 1991; Sorensenet al.2003; Sonoda and Tsumuki 2007). The HSPs are ubiquitous proteins that have been extensively studied in different organisms(Kimet al. 1998; Feder and Hofmann 1999; Aevermann and Waters 2008; Waterset al. 2008). HSPs often exert protective functions in response to a range of environmental stresses, including heat, cold, oxidation, hypertonic stress,UV, heavy metals, organic pollutants, osmolarity, and even high population density of organisms (Feder and Hofmann 1999; Akerfeltet al. 2010; Padmini 2010).

HSPs can be divided into different gene families based on molecular weights, such as HSP100, HSP90, HSP70,HSP60, HSP40 and small HSPs (sHSPs) (Feder and Hofmann 1999; Li and Srivastava 2004). HSP90, HSP70,HSP60 and HSP40 are highly conserved families that exist across species from prokaryotes to eukaryotes protecting against protein aggregation, renaturing damaged proteins to restore their biological activity (Feder and Hofmann 1999; Li and Srivastava 2004). HSP40 was first known to stimulate the ATPase activity of DnaK, the bacterial HSP70 homologue(Yochemet al. 1978; Libereket al. 1988; Walshet al. 2014).The HSP40 family members contain the J domain, which is present at the N-terminal region of the proteins and binds to their partners HSP70s and HSP90s (Cajoet al. 2006; Qiuet al. 2006). HSP60 was initially discovered and cloned in mammalian mitochondria (Jindalet al. 1989). Most of the HSP60 sequences contain one or more repeat nucleotide sequences like GGM or GGGM at their C-terminus. As a molecular chaperone, HSP 60 has functions in protecting against protein aggregation, renaturing damaged proteins to restore their biological activity (Sanderset al. 1992;Slavotinek and Biesecker 2001), and in the transport of proteins from cytoplasm to organelles (Fink 1999). HSP70 family is the most conservative heat shock protein, and involved in various cellular processes including protein folding and degradation. There are three different groups of proteins in form of HSP70s (S?rensen 2010). The first one is called a solely constitutive group, heat shock cognate 70 (HSC70) that is defined by its constitutive expression and cytoplasmic localization and is distributed under normal cellular conditions in all living cells, such as nuclei, cytoplasm, endoplasmic reticulum, mitochondria and chloroplasts (Boutetet al. 2003). The second one is a solely inducible group, HSP70, which is induced by cellular stress such as temperature changes or exposure to toxic chemicals (Morimotoet al. 1990; McKayet al. 1994; Ravauxet al. 2007). The last one is a constitutive and inducible group: expressed during normal cell functioning and also up-regulated in response to stressful stimuli (Callahanet al.2002). HSP90 family is also highly conserved, and widely exists in various organisms. This family can be categorized into five sub-families: HSP90A, HSP90B, HSP90C, TRAP(tumor necrosis factor receptor-associated protein) and HTPG (High temperature protein G) (Chenet al. 2006).

Cotesia chilonis(Matsumura) (Hymenoptera: Braconidae)is a major endoparasitoid ofChilo suppressalisand primarily distributed in southeastern and eastern areas of Asia (Huanget al. 2011; Wuet al. 2013). It has been claimed that 10–30%overwintering larvaeofC.suppressaliswere parasitized byC.chilonisin Jiangsu, Anhui, Zhejiang and Hunan provinces in China (Chenet al. 2002; Panet al. 2016). Similarly,approximate 8.1–20.3%C.suppressalislarvae were reported to be parasitized byC.chilonisin Japan (Hang and Lin 1989; Chenet al. 2002; Wuet al. 2013). Secondinstar larvae ofC.chilonishad the strongest resistance to low temperature. The mortality rate of second-instar larvae was 44% while the mortality rates of first- and third-instar larvae were 60 and 77% at 5°C for 30 days (Galichet 1979).The temperature also affects the parasitic and development ofC.chilonis. For example, the parasitic rate ofC.chiloniscould be increased by the low temperature (Chenet al.2002). However, the molecular basis of thermotolerance inC.chilonisis still covered. Therefore, in order to firstly exploit the molecular mechanism of thermotolerance, the genomic and structures of five different HSPgenes (Cchsp40,Cchsp60,Cchsp70,Cchsc70andCchsp90) inC.chiloniswere characterized. In addition, we also investigated the expression patterns of these genes in response to different temperatures in this study.

2. Materials and methods

2.1. Insects

Populations ofC.suppressalisandC.chiloniswere collected from a suburb (32.39°N, 119.42°E) in Yangzhou, Jiangsu,China. More than three generations of theC. suppressalislarvae are feeding as the host for multiple generations ofC.chilonisto reserve.Insects were reared in glass containers (10 cm×13 cm) containing rice plants and maintained in a growth chamber at (27±1)°C, 16 h L:8 h D photoperiod, 60–70% relative humidity as described in Hang(1993).C.suppressalislarvae were then parasitized by the adults ofC.chilonisemerged in 24 h.

2.2. Cloning and RACE

Total RNA was extracted fromC.chilonisusing the SV Total RNA Isolation System (Promega, USA) and treated with DNase I to eliminate DNA contamination. cDNA was synthesized using an oligo(dT)18primer (TaKaRa, Japan).Based on fivehsps(hsp40,hsp60,hsp70,hsc70andhsp90) sequences from other species, degenerate primers of the five genes were designed and synthesized to amplify internal fragments (Table 1). The full-length cDNAs of the genes encoding HSPs were determined using 5′- and 3′-RACE, according to the sequence information obtained from internal fragment.

2.3. Characterization of the genomic DNA

Thegenomic DNA ofC.chiloniswas extracted according to the AxyprepTMMultisource Genomic DNA Kit (Axygen, USA).Based on the sequences of the full-length cDNAs of the five genes encoding HSPs, primers were designed to amplifyCchspsgenomic fragments (Table 1). DNA products were first purified using Gel Extraction Kit (Axygen, USA), then cloned into pGEM-T Easy vector (Promega, USA), and transformed into competentEscherichia coliDH5α cells for sequencing.

Table 1 The primer sequences used in the gene cloning of five hsps

2.4. Temperature treatment

One-day-old adults (both sexes) were exposed to –13, –12,–9, –6, –3, 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36°C for 1 h in a constant-temperature incubator. Adults were allowed to recover at 27°C for 1 h, and then number of survivors was recorded. The surviving adults were frozen in liquid nitrogen and stored at –80°C. The insects maintained at 27°C were used as controls. Each experimental treatment contained 20 adults, and each treatment was repeated three times.

2.5. qRT-PCR analysis

Total RNA of insects with different temperature treatments was extracted as mentioned above. The integrity of RNA was verified by comparing the ribosomal RNA bands in ethidium bromide-stained gels. RNA sample purity was examined using spectrophotometric measurements at 260 and 280 nm. Real-time PCR reactions were performed in a 20-μL total reaction volume consisting of 10 mL 2× iTaqTMUniversal SYBR?Green Supermix (Bio-Rad, USA), 6 mL sterilized H2O, 1 mL of each gene specific primer, and 2 mL cDNA templates. Reactions were carried out on a CFX-96 Real-time PCR System (Bio-Rad, USA). The homogeneity of the PCR products was con firmed by melting curve analysis,which was evaluated every 5 s per 0.5°C increment from 65 to 95°C. The quantity ofCchspsmRNAs was calculated using the 2–ΔΔCTmethod and normalized to the abundance of theC.chilonisribosomal protein L10 (CcRPL10) gene(Schmittgen and Livak 2008), and each reaction was executed in triplicate (Table 2).

2.6. Bioinformatic analysis

ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to identify the open reading frames (ORFs). Deduced amino acid sequences were aligned using Clustal X ver. 2.0 Software. Sequence analysis tools of the ExPASy Molecular Biology Server (Swiss Institute of Bioinformatics) including Translate, Compute pI/MW, and Blast were used to analyze the deducedhspsequences. Amino acid sequences ofhspsinC.chilonis, together with publishedhspssequences of other insectswere used to conduct phylogeny with the neighborjoining methods. Phylogenetic trees were constructed with 1 000 bootstrap replicates in MEGA 7.0 (Kumaret al. 2016).

2.7. Statistical analysis

Homogeneity of variances among different treatments was evaluated by Levene’s test. Differences between treatments were identified using Tukey’s test (homogeneity of variances). Statistical analysis was performed using SPSS16.0 software, and presented as means±SE (standard error) (Pallant 2007).

3. Results

3.1. Sequence analysis of C. chilonis hsps

Cchsp40 The full-length cDNA ofCchsp40was 1 265 bp(GenBank accession no. MF377628), which included a 123-bp 5′-untranslated region (UTR), a 74-bp 3′-UTR, and a 1 068-bp ORF. The deduced protein consisted of 355 amino acids with a molecular weight of 39.1 kDa and an isoelectric point of 9.09 (Table 3). MotifScan analysis (http://www.ca.expasy.org) revealed three conserved signature sequences, including J-domain (aa 3–57), G/F domain (aa 70–125) and C domain (aa 176–341), respectively (Appendix A). The genomic DNA sequence ofCchsp40was deposited in GenBank (accession no. MF377630). Comparison between the cDNA and genomic sequences indicated thatCchsp40lacked introns (Appendix B; Fig. 1-A).

Cchsp60 The full-length cDNA ofCchsp60was 2 551 bp(GenBank accession no. MF377629), which contained a 128-bp 5′-UTR, a 707-bp 3′-UTR, and a 1 716-bp ORF. The deduced protein possessed 571 amino acids with a molecular weight of 60.6 kDa and an isoelectric point of 5.29 (Table 3). MotifScan analysis showed that this protein contained 25 amino acid sequence(MYRLPGLMRGVASRQMQLQARSYK, 1–25) at its N-terminus. In addition, AAVEEGIVPGGG (427–438), a classical family pro file of HSP60, was also observed in theCcHSP60 sequence.. A (GGM)nrepeat motifwas also detected inthe C-terminal ofCcHSP60, suggesting thatCcHSP60 is a member of the mitochondrial Hsp60 family.Furthermore, various binding sites were also observed in theCcHSP60 sequence (Appendix A). The genomic DNA sequence ofCchsp60was deposited in GenBank (accession no. MF377631). Similarly, comparison between the cDNA and genomic sequences indicated thatCchsp60lacked introns (Appendix B; Fig. 1-B).

Cchsp70 The cDNA ofCchsp70was 2 094 bp in length(GenBank accession no. KM077498) that contained an 89-bp 5′-UTR, a 53-bp 3′-UTR, and a 1 953-bp ORF.The deduced protein contained 650 amino acids with a molecular weight of 71.45 kDa and an isoelectric point of 5.46 (Table 3). MotifScan analysis indicated that this protein had three conserved signature sequences that were commonly present in other HSP70. These three conserved signature sequences were IGIDLGTTYS (aa 3–13),IFDLGGGTFDVSIL (aa 194–207), and VVLVGGSTRIP KIQS(aa 332–346), respectively. Moreover, four motifs were also detected in this protein that included an ATP-GTP binding site AEAYLGQK (aa 128–135), a bipartite nuclear localization signal sequence (NLS) ERKYRKNLKTNPRALRRL(aa 244–261), a non-organellar consensus motif RARFEEL(aa 297–303) as well as the cytoplasmic motif EEVD(aa 647–650) (Appendix A). The genomic DNA sequence ofCchsp70was deposited in GenBank with accession no.KM103515. Likewise, comparison between the cDNA and genomic sequences indicated thatCchsp70lacked introns(Appendix B; Fig. 1-C).

Table 2 Primers used for qRT-PCR analysis1)

Table 3 A summary of the five HSP genes from Cotesia chilonis

Cchsc70 The cDNA ofCchsc70was 2 297 bp in length(GenBank accession no. KM077499) that included a 137-bp 5′-UTR, a 206-bp 3′-UTR, and a 1 956-bp ORF.The deduced protein consisted of 651 amino acids with a predicted molecular weight of 71.19 kDa and a theoretical pI of 5.42 (Table 3). Similarly, MotifScan analysis showed that there were three conserved signature sequences in this protein, which included IDLGTTYS (aa 9–16),IFDLGGGTFDVSIL (aa 197–210) and IVLVGGSTRIPKIQK(aa 334–348). Additionally, five motifs including an ATPGTP binding site AEAYLGQT (aa 131–138), a bipartite NLS(KRKYKKDLT SNKRALRRL, aa 246–263), a non-organellar consensus motif RARFEEL (aa 299–305), a four-fold repeat of the tetrapeptide ‘GGM(I)P’ (aa 615–630) and the cytoplasmic motif EEVD (aa 648–651) were observed in the protein (Appendix A).Cchsc70contained a 461-bp intron and was deposited in GenBank under accession no.KM103516 (Appendix B; Fig. 1-D).

Cchsp90 The full-length cDNA ofCchsp90was 2 635 bp(GenBank accession no. KM077500), which contained a 112-bp 5′-UTR, a 358-bp 3′-UTR and a 2 166-bp ORF. The predicted protein had 721 amino acids with a molecular weight of 82.92 kDa and a theoretical pI of 4.96 (Table 3).MotifScan analysis indicated that five conserved signature sequences were observed in this protein, which included NKEIFLRELISNSSDALDKIR (aa 35–55), LGTIAKSGT(aa 102–110), IGQFGVGFYSAYLVAD (aa 126–141),IKLYVRRVfi(aa 351–360), and GVVDSEDLPLNISRE(aa 377–391). In addition, a consensus sequence MEEVD was found at the C-terminus ofCcHSP90. Except for that, this protein also contained several other features,such as (1) a typical histidine kinase-like ATPase domain(aa 12–221), which is ubiquitous in all HSP90 family members; (2) two highly charged domains, including a hinge-domain (aa 225–258) and a C-terminal domain(aa 560–682); (3) a NLS (KKKKKK, aa 263–268);and (4) a basic helix-loop-helix (bHLH) protein-folding domain EADKNDKSVKDLVVLLFETALLSSGFSLD DPQVHAARIYRMIKLGLGI (aa 641–688) (Appendix A).The genomic DNA sequence ofCchsp90was deposited in GenBank with accession no. KM103517. Again, comparison between DNA and genomic sequences indicated thatCchsp90lacked introns (Appendix B; Fig. 1-E).

3.2. Phylogenetic analysis

Fig. 1 Position and numbers of introns in hsp40 (A), hsp60 (B),hsp70 (C), hsc70 (D) and hsp90 (E) in different insect species.GenBank accession numbers of corresponding insect HSP genes are shown in the brackets.

Fig. 2 Neighbor-joining phylogenetic trees of CcHSP40 (A),CcHSP60 (B), CcHSP70 (C), CcHSC70 (D) and CcHSP90(E). The location of Cotesia chilonis HSPs were marked with rectangles. Numbers on the branches indicated bootstrap values obtained from 1 000 replicates (only bootstrap values＞50 are shown).

The deduced amino acid sequences of the fiveCchspsinC.chilonisdisplayed high degrees of homology compared with that of other insects. The overall results of phylogenetic analysis were consistent among the five proteins (Fig. 2).Phylogenetic trees of HSP40, HSP60, HSP90and HSC70 could be categorized into three clusters, Braconidae,Apoidea, and Formicidae. Similarity betweenCcHSP60 ofC.chilonisandMdHSP60 ofMicroplitis demolitorreached to 80%. Moreover, similarity ofCcHSP40,CcHSP90 andCcHSC70 inC.chiloniswas almost identical to that inCotesia vestalis, which showed a shared amino acid identity with 97, 96 and 98%, respectively. The phylogenetic tree of HSP70 in the Hymenoptera could be divided into three clusters, Ichneumonoidea, Apoidea, and Formicidae. In addition,CvHSP70 inC.vestaliswas the most closely related ortholog relative toCcHSP70 inC.chilonis; which shared amino acid similarity of 96% (Fig. 2).

3.3. Expression of Cchsps genes in response to temperature

The relative mRNA levels of the fiveCchspsgenes were monitored at temperature gradients ranging from –13 to 36°C. The results showed that the expression patterns varied in the fiveCchspsgenes in response to temperature(Fig. 3).Cchsp40andCchsp60responded similarly to the thermal stress. The expression of these two genes were up-regulated by cold, which were significantly different at–6 and –3°C (Cchsp40:F17,36=53.109,P＜0.001;Cchsp60:F17,36=43.535,P＜0.001), but they showed no response to heat. The mRNA expression level ofCchsp40increased by 653.83-fold at –3°C and 616.2-fold at –6°C compared with the control (Fig. 3-A). Similarly, relative to the control,the mRNA expression level ofCchsp60increased by 647.85-fold and 573.6-fold at –3 and –6°C, respectively(Fig. 3-B).Cchsp70andCchsc70also showed similar response to the thermal stress, which could be induced by both cold and heat stress (Cchsp70:F17,36=8.638,P＜0.001;Cchsc70:F17,36=7.731,P＜0.001). The results showed that as temperatures increased, transcription ofCchsp70andCchsc70enhanced. The highest expression level forCchsp70was at –3°C and increased by 6.84-fold relative to the control, while these values forCchsc70were –6°C and 6.77-fold, respectively (Fig. 3-C and D).Cchsp90could only be induced by heat and mild cold stress (Cchsp90:F17,36=10.151,P＜0.001), but not by cold stress (Fig. 3-E).

4. Discussion

Heat shock proteins usually contain stress-inducible and constitutively-expressed genes. They function as chaperones of other proteins (Boorsteinet al. 1994;Daugaardet al. 2007) and play an important role in the thermotolerance of the organisms (Rinehartet al. 2007; Luet al. 2013, 2016; Panet al. 2017; Wanget al. 2017). In this study, five genes encodinghsps(Cchsp40,Cchsp60,Cchsp70,Cchsc70,andCchsp90) were identified inC.chilonis. The results showed that the predicted amino acid sequences of these proteins shared high similarities with published HSPs of other insects in Hymenoptera.For example, amino acid ofCchsp40,Cchsp60,Cchsp70,Cchsc70,andCchsp90inC.chilonishad a high similarity of 97, 80, 96, 98, and 96%, respectively, compared with that of the closed-related speciesC.vestalis. All core signature sequences and motifs were conserved in the fivehspsbetweenC.chilonisandC.vestalisorM.demolitor.These high similarities con firmed the identification of thesegenes as functional HSPs inC.chilonis. The conserved motifs EEVD and MEEVD were consistently located at the C-terminal in the cytoplasm and were supposed to facilitate the interactions of HSPs with other proteins. The results implied that these conserved motifs could enableCcHSP70,CcHSC70 andCcHSP90 to bind other proteins or cochaperones (Zhang and Denlinger 2010) in the cytoplasm ofC.chilonis(Gupta 1995). The amino acid sequence ofCcHSP40 showed that none of the three conserved repeats was displayed as the consensus sequence CxxCxGxG(cysteine-rich region or zinc finger motif), which suggested thatCcHSP40 inC.chiloniswas the Type II HSP40s having lower efficiency in forming chaperone pairs with cytosolic HSP70 and folding proteins (Caplanet al. 1993; Fanet al.2004). A classical family pro file (AAVEEGIVPGGG) and one (GGM)nrepeat motifwere found inCcHSP60. These results indicated thatCcHSP60 is a member of the typical mitochondrial HSP60 family.

Fig. 3 Relative mRNA expression levels of five hsps of Cotesia chilonis adults exposed to different temperatures. A, Cchsp40.B, Cchsp60. C, Cchsp70. D, Cchsc70. E, Cchsp90. Data are means±SE. Different letters indicate significant differences(P＜0.01).

This study found thatCchsp40,Cchsp60,Cchsp70andCchsp90lacked introns, butCchsc70had a single intron.The nucleotide sequences at the intron splice junctions inCchsc70were consistent with the canonical GT-AG rule.The position and numbers of introns inhsc70homologs are variable. For example, although sequence ofCchsc70andCvhsc70were very similar, they differed in the number and sizes of introns.Cchsc70had a single 461 bp intron at the 5′ end, whileCvhsc70had two introns at the 5′ end(119 and 460 bp; Fig. 1-D). Similarly, as observed in other species, no intron was found in the genome ofCchsp70(Fig. 1-C).Cchsp40(Fig. 1-A),Cchsp60(Fig. 1-B)andCchsp90(Fig. 1-E)inC.chilonis, which was also true for other insects within the same genus. However, these threehspshad variable introns for insects in another genus.The absence of introns favors the expression of stressresponsive genes because no mRNA processing could slow transcript accumulation and/or be disrupted by stress(King and MacRae 2014). Previous studies have suggested that there was a negative correlation between intron size and gene expression level, which was con firmed by our study that expression ofCchsc70was less than that ofCchsp40,Cchsp60andCchsp70(Fig. 3) (Comeron 2004).In summary,Cchsc70might be constitutive and inducible protein expressed during normal cell functioning and also up-regulated in response to stressful stimuli (Callahanet al.2002).

Temperature is one of the most important factors in determining the distribution and abundance of insects (Jing and Kang 2004). When exposed to extreme temperatures,insects may respond in different ways, such as adopting behaviors to avoid or escape extreme temperatures and/or alter their physiology to withstand the temperature(Hoffmann and Parsons 1991). Various studies have indicated that thermotolerance is largely due to the regulation and expression levels of genes encoding HSPs(S?rensenet al. 2003; Luet al. 2015). In this study, we found that the fivehspsinC.chiloniscan be significantly induced by thermal stress, which was consistent with previous results (Mahroofet al. 2005; Sonodaet al. 2006;Zhanget al. 2010; Wanget al. 2017). In addition, we found that the responses of the fiveCchspsto temperature were different inC.chilonis. The expression ofCchsp40,Cchsp60,Cchsp70andCchsc70could be induced by the cold stress. The highest expression levels ofCchsp40,Cchsp60andCchsp70were at –3°C after 1 h, while it wasat –6°C forCchsc70(Fig. 3-A–D). Compared withCchsp70andCchsc70,Cchsp40andCchsp60were more sensitive to low temperature but showed no response to heat. However,hsp40inAphaenogaster piceaandA.rudis(Cahanet al. 2017), andhsp60inC.suppressalisandSiniperca chuatsicould response to high temperature treatments (Cuiet al. 2014; Wanget al. 2017).Cchsp70andCchsc70showed similar responses to thermal stress and could be induced by both cold and heat. Combining with genomic structures, the results indicatedCchsp70was solely inducible protein induced by temperature changes(Morimotoet al. 1990; McKayet al. 1994; Ravauxet al.2007), butCchsc70was constitutive and inducible protein expressed during normal cell functioning, which also upregulated in response to stressful stimuli (Callahanet al.2002). The expression levels ofhsp70inGrapholita molestaandMusca domesticawere up-regulated with the rise of temperature (Tanget al. 2012; Chenet al. 2014).Fohsc701inFrankliniella occidentalislarvae plays an important role in resisting low temperature stress (Li and Du 2013). Similar induced expression pattern was also reported in other species, such asPlutella xylostella,Locusta migratoriaandMacrocentrus cingulum(Sonodaet al. 2006; Wanget al.2007; Xuet al. 2010). In many organisms, HSP70 are the main family of heat shock proteins that are considered to be a class of thermally induced proteins. However, in this study, we found thathsp70 showed a low level of expression at different temperatures. It is possible that there exist other heat-induced HSP70s inC.chilonis. In this study,we also found thatCchsp90could be induced by heat and mild cold stress, but not cold shock (Fig. 3-E), which was also observed inSehsp90ofSpodoptera exigua(Xuet al.2011) andSihsp90ofSesamia inferens(Sunet al. 2014;Tanget al. 2015).

5. Conclusion

We obtained five genes encoding heat shock proteins(HSPs) forCotesia chilonis,Cchsp40,Cchsp60,Cchsp70,Cchsc70andCchsp90and their predicted amino acid sequences showed high similarities with published HSPs of other insects in Hymenoptera. Expression patterns varied in the fiveCchsps in response to temperature. The expression of heat shock protein is related to the thermotolerance of the organism as a physiological mechanism. Physiological acclimation to temperature variation appears to involve modulation of the heat shock response. Our results would be useful in exploring the thermotolerance ofC.chilonisat the molecular level. Further researches are needed to evaluate the expression levels of the other HSPs ofC.chilonisunder different temperatures to gain a deep understanding of various roles of all heat shock proteins.

Acknowledgements

This research was funded by the National Key R&D Program of China (2017YFD0200400) and the National Basic Research Program of China (973 Program, 2013CB127604).We sincerely thank Dr. Yang Fei from Texas A&M University,USA for editing and providing comments on the manuscript.We also express our deep gratitude to the Testing Center of Yangzhou University, China.

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