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

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

这款煮锅的手柄处设计有一个出水口，整个中空手柄可作为一个出水管，再配合一个过滤网，可将煮锅里的水从食物中过滤出来，很适合油炸食物将油与食物分离。不过，设计师可能忽略了中空手柄的清洁问题，里面日积月累的污垢如果不清洁会变得很不卫生。

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 (Willmer et al.2000). Insect tolerance to temperatures varies considerably and has significant behavioral and ecological implications(Hausmann et al. 2005; Samietz et 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; Sorensen et al. 2003; Sonoda and Tsumuki 2007). The HSPs are ubiquitous proteins that have been extensively studied in different organisms(Kim et al. 1998; Feder and Hofmann 1999; Aevermann and Waters 2008; Waters et 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; Akerfelt et 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(Yochem et al. 1978; Liberek et al. 1988; Walsh et 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 (Cajo et al. 2006; Qiu et al. 2006). HSP60 was initially discovered and cloned in mammalian mitochondria (Jindal et 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 (Sanders et 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 (Sørensen 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 (Boutet et 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 (Morimoto et al. 1990; McKay et al. 1994; Ravaux et 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 (Callahan et 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) (Chen et al. 2006).

Cotesia chilonis (Matsumura) (Hymenoptera: Braconidae)is a major endoparasitoid of Chilo suppressalis and primarily distributed in southeastern and eastern areas of Asia (Huang et al. 2011; Wu et al. 2013). It has been claimed that 10–30%overwintering larvae of C. suppressalis were parasitized by C. chilonis in Jiangsu, Anhui, Zhejiang and Hunan provinces in China (Chen et al. 2002; Pan et al. 2016). Similarly,approximate 8.1–20.3% C. suppressalis larvae were reported to be parasitized by C. chilonis in Japan (Hang and Lin 1989; Chen et al. 2002; Wu et al. 2013). Secondinstar larvae of C. chilonis had 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 of C. chilonis. For example, the parasitic rate of C. chilonis could be increased by the low temperature (Chen et al.2002). However, the molecular basis of thermotolerance in C. chilonis is still covered. Therefore, in order to firstly exploit the molecular mechanism of thermotolerance, the genomic and structures of five different HSP genes (Cchsp40,Cchsp60, Cchsp70, Cchsc70 and Cchsp90) in C. chilonis were 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

Cchsp90 The full-length cDNA of Cchsp90 was 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 of CcHSP90. 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 of Cchsp90 was deposited in GenBank with accession no. KM103517. Again, comparison between DNA and genomic sequences indicated that Cchsp90 lacked introns (Appendix B; Fig. 1-E).

2.2. Cloning and RACE

Total RNA was extracted from C. chilonis using the SV Total RNA Isolation System (Promega, USA) and treated with DNase I to eliminate DNA contamination. cDNA was synthesized using an oligo(dT)18 primer (TaKaRa, Japan).Based on five hsps (hsp40, hsp60, hsp70, hsc70 and hsp90) 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

The genomic DNA of C. chilonis was extracted according to the AxyprepTM Multisource Genomic DNA Kit (Axygen, USA).Based on the sequences of the full-length cDNAs of the five genes encoding HSPs, primers were designed to amplify Cchsps genomic 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 competent Escherichia coli DH5α cells for sequencing.

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

1) F, forward; R, reverse.

Primer name1) Primer sequences (5´→3´)For partial segments cloning hsp40-F ATGGGTAAAGACTACTATAAAA hsp40-R TGGTGGKTCTTGAGCGTGATCT hsp60-F ATCACCAAGGAYGGYGTBACTGT hsp60-R TCKCCDACCTGDCCCAARTCGCT hsp70-F ATGAARGARACNGCNGARGC hsp70-R GTCATNACDCCDCCDGCDGT hsc70-F ATGAAGGGARACMGCTGAGGC hsc70-R GCYTCRTCVGGRTTGATGGA hsp90-F GCKGAGATCGCYCAGCTKATGTC hsp90-R GCCTTCATGATRCGYTCCATGTTGGC For 5´-RACE hsp40 CTGAATGCTCCTTGCCCGCC hsp60 GGCGACATCCTGAACCAACTTAGC hsp70 ACGCCTTCGTAGAGCGCGTCTATTTC hsc70 GCCTCGGCGGTTTCCTTCATCTT hsp90 TGTTGAGTGGCAAGTCCTCGCTGTCG For 3´-RACE hsp40 TGTCTGGAGCACGACAGGGTGG hsp60 GTCAAAGCTCCAGGCTTCGGTG hsp70 TTTGGTGGCTTGACGCTGCGAAT hsc70 TTCCGCAGCACCCTGGAGCCCGT hsp90 GCTGCTCGCTCAAGGACTACGTTGGTCGT For genome amplification hsp40-F TTGGGGAAGAAGGATTGAAAGGC hsp40-R ATGGTTCTTTCGGGAATGGTAGT hsp60-F ACAGACTACCTGGACTGATGCGT hsp60-R CATACCACCCATACCTCCCATAC hsp70-F GACGTTAAGTGAGGAGTTA hsp70-R AGTCAACCTCTTCAACCG hsc70-F GCAGTTGGTATTGATTTGGGAA hsc70-R CTCCAGCAGTTGACTTGACCTC hsp90-F TTGAGACCTTCGCTTTCCA hsp90-R GCTGCGGTTTGAACTATCT

2.4. Temperature treatment

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× iTaqTM Universal 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 of Cchsps mRNAs was calculated using the 2–ΔΔCT method and normalized to the abundance of the C. chilonis ribosomal protein L10 (CcRPL10) gene(Schmittgen and Livak 2008), and each reaction was executed in triplicate (Table 2).

2.5. qRT-PCR analysis

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.

2.6. Bioinformatic analysis

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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 of Cchsp40 was 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 of Cchsp40 was deposited in GenBank (accession no. MF377630). Comparison between the cDNA and genomic sequences indicated that Cchsp40 lacked introns (Appendix B; Fig. 1-A).

Cchsp60 The full-length cDNA of Cchsp60 was 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 the CcHSP60 sequence.. A (GGM)n repeat motif was also detected in the C-terminal of CcHSP60, suggesting that CcHSP60 is a member of the mitochondrial Hsp60 family.Furthermore, various binding sites were also observed in the CcHSP60 sequence (Appendix A). The genomic DNA sequence of Cchsp60 was deposited in GenBank (accession no. MF377631). Similarly, comparison between the cDNA and genomic sequences indicated that Cchsp60 lacked introns (Appendix B; Fig. 1-B).

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 deduced hsp sequences. Amino acid sequences of hsps in C. chilonis, together with published hsps sequences of other insects were used to conduct phylogeny with the neighborjoining methods. Phylogenetic trees were constructed with 1 000 bootstrap replicates in MEGA 7.0 (Kumar et al. 2016).

以财税制度和法律为准绳划清分配过程中的合法收入和非法收入，坚决取缔非法收入。对诚实劳动和合法经营的高收入者，要合理调节；少数垄断性行业的过高收入，政府要进行宏观调控，以抑制高收入者收入的过快增长；对于非法获得高额收入的行为依法予以打击和取缔。只有这样，才能依法保护合法的劳动收入和非劳动收入，进一步提高社会主义市场经济的效率和公平。

Cchsp70 The cDNA of Cchsp70 was 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 of Cchsp70 was deposited in GenBank with accession no.KM103515. Likewise, comparison between the cDNA and genomic sequences indicated that Cchsp70 lacked introns(Appendix B; Fig. 1-C).

Table 2 Primers used for qRT-PCR analysis1)

1) The qRT-PCR primers used in this study were validated.

Gene Primer sequences (5´→3´) Tm (°C) Length (bp) Ampli fiction efficiency (%) R2 hsp40 F: TGGGGAAGAAGGATTGAAAG 53.1 120 92.9 0.983 R: GGTGATGATGAGCCGAAAAA hsp60 F: CGAAGACATTGATGGCGGG 57.1 116 109.9 0.981 R: GAAGAGTGGCTTTGCGGTTG hsp70 F: CACCCAGCTATGTTGCCTTTA 54.6 107 94.8 0.998 R: GAGTCGTTTCGCGTCGAAT hsc70 F: GGAAACAAAATGACGAAAGCAC 55.0 76 92.6 0.984 R: GTTGAAAAACTCCAACACAGGAG hsp90 F: TCCCAACAAAAATGACCGT 57.1 256 109.9 0.989 R: GGCAGCTGATTCCCACAAG RPL10 F: CCCAAAATCCCGTTTCTGT 56.7 121 99.9 0.976 R: CTGGCTTTCTTCTTTCCCAA

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

Gene cDNA size(bp)GenBank accession no.(DNA)hsp40 1 265 1 068 39.1 9.09 MF377628 MF377630 hsp60 2 551 1 716 60.6 5.29 MF377629 MF377631 hsp70 2 094 1 953 71.45 5.46 KM077498 KM103515 hsc70 2 297 1 956 71.19 5.42 KM077499 KM103516 hsp90 2 635 2 166 82.92 4.96 KM077500 KM103517 ORF size(bp)(kDa) pI GenBank accession no.(cDNA)MW

Cchsc70 The cDNA of Cchsc70 was 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). Cchsc70 contained a 461-bp intron and was deposited in GenBank under accession no.KM103516 (Appendix B; Fig. 1-D).

Populations of C. suppressalis and C. chilonis were collected from a suburb (32.39°N, 119.42°E) in Yangzhou, Jiangsu,China. More than three generations of the C. suppressalis larvae are feeding as the host for multiple generations of C. chilonis to 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. suppressalis larvae were then parasitized by the adults of C. chilonis emerged in 24 h.

4.在廉政建设方面，加强对党员干部党风党纪、廉洁从业和艰苦奋斗的教育。积极开展党风廉政教育活动，过党员大会、党小组会议认真组织党员干部观看反腐倡廉警示教育片。通过一个个鲜活的案例，让大家更加清楚的认识到贪污腐败对国家、社会、家庭、个人带来的危害，从而增强党员干部的法制观念和廉洁自律意识，永葆共产党员的先进性和纯洁性，起到表率带头作用。

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 five Cchsps in C. chilonis displayed 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, HSP90 and HSC70 could be categorized into three clusters, Braconidae,Apoidea, and Formicidae. Similarity between CcHSP60 of C. chilonis and MdHSP60 of Microplitis demolitor reached to 80%. Moreover, similarity of CcHSP40, CcHSP90 and CcHSC70 in C. chilonis was almost identical to that in Cotesia 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 in C. vestalis was the most closely related ortholog relative to CcHSP70 in C. chilonis; which shared amino acid similarity of 96% (Fig. 2).

目前，临床主要依靠术前应用胰岛素合理控制血糖，同时预防性应用抗生素等方式来预防术后切口感染，但临床实践表明，预防性应用抗生素并不能获得理想的切口感染预防效果。临床在长期探索的过程中发现，在患者实施手术治疗操作的整个过程中，如果能够配合科学化的手术室护理干预，规范这个手术流程，从术前检查、手术室及手术用品消毒、术中护理、缩短手术时间和控制手术室人员流动等全方位进行管理和干预，则能够从根本上降低患者术后切口的感染发生率[5]。

3.3. Expression of Cchsps genes in response to temperature

The relative mRNA levels of the five Cchsps genes were monitored at temperature gradients ranging from –13 to 36°C. The results showed that the expression patterns varied in the five Cchsps genes in response to temperature(Fig. 3). Cchsp40 and Cchsp60 responded 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 of Cchsp40 increased 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 of Cchsp60 increased by 647.85-fold and 573.6-fold at –3 and –6°C, respectively(Fig. 3-B). Cchsp70 and Cchsc70 also 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 of Cchsp70 and Cchsc70 enhanced. The highest expression level for Cchsp70 was at –3°C and increased by 6.84-fold relative to the control, while these values for Cchsc70 were –6°C and 6.77-fold, respectively (Fig. 3-C and D). Cchsp90 could 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 (Boorstein et al. 1994;Daugaard et al. 2007) and play an important role in the thermotolerance of the organisms (Rinehart et al. 2007; Lu et al. 2013, 2016; Pan et al. 2017; Wang et al. 2017). In this study, five genes encoding hsps (Cchsp40, Cchsp60,Cchsp70, Cchsc70, and Cchsp90) were identified in C. 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 of Cchsp40, Cchsp60, Cchsp70,Cchsc70, and Cchsp90 in C. chilonis had a high similarity of 97, 80, 96, 98, and 96%, respectively, compared with that of the closed-related species C. vestalis. All core signature sequences and motifs were conserved in the five hsps between C. chilonis and C. vestalis or M. demolitor.These high similarities con firmed the identification of thesegenes as functional HSPs in C. 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 enable CcHSP70,CcHSC70 and CcHSP90 to bind other proteins or cochaperones (Zhang and Denlinger 2010) in the cytoplasm of C. chilonis (Gupta 1995). The amino acid sequence of CcHSP40 showed that none of the three conserved repeats was displayed as the consensus sequence CxxCxGxG(cysteine-rich region or zinc finger motif), which suggested that CcHSP40 in C. chilonis was the Type II HSP40s having lower efficiency in forming chaperone pairs with cytosolic HSP70 and folding proteins (Caplan et al. 1993; Fan et al.2004). A classical family pro file (AAVEEGIVPGGG) and one (GGM)n repeat motif were found in CcHSP60. These results indicated that CcHSP60 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 that Cchsp40, Cchsp60, Cchsp70 and Cchsp90 lacked introns, but Cchsc70 had a single intron.The nucleotide sequences at the intron splice junctions in Cchsc70 were consistent with the canonical GT-AG rule.The position and numbers of introns in hsc70 homologs are variable. For example, although sequence of Cchsc70 and Cvhsc70 were very similar, they differed in the number and sizes of introns. Cchsc70 had a single 461 bp intron at the 5´ end, while Cvhsc70 had 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 of Cchsp70(Fig. 1-C). Cchsp40 (Fig. 1-A), Cchsp60 (Fig. 1-B) and Cchsp90 (Fig. 1-E) in C. chilonis, which was also true for other insects within the same genus. However, these three hsps had 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 of Cchsc70 was less than that of Cchsp40, Cchsp60 and Cchsp70 (Fig. 3) (Comeron 2004).In summary, Cchsc70 might be constitutive and inducible protein expressed during normal cell functioning and also up-regulated in response to stressful stimuli (Callahan et 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(Sørensen et al. 2003; Lu et al. 2015). In this study, we found that the five hsps in C. chilonis can be significantly induced by thermal stress, which was consistent with previous results (Mahroof et al. 2005; Sonoda et al. 2006;Zhang et al. 2010; Wang et al. 2017). In addition, we found that the responses of the five Cchsps to temperature were different in C. chilonis. The expression of Cchsp40,Cchsp60, Cchsp70 and Cchsc70 could be induced by the cold stress. The highest expression levels of Cchsp40,Cchsp60 and Cchsp70 were at –3°C after 1 h, while it was at –6°C for Cchsc70 (Fig. 3-A–D). Compared with Cchsp70 and Cchsc70, Cchsp40 and Cchsp60 were more sensitive to low temperature but showed no response to heat. However, hsp40 in Aphaenogaster picea and A. rudis (Cahan et al. 2017), and hsp60 in C. suppressalis and Siniperca chuatsi could response to high temperature treatments (Cui et al. 2014; Wang et al. 2017). Cchsp70 and Cchsc70 showed similar responses to thermal stress and could be induced by both cold and heat. Combining with genomic structures, the results indicated Cchsp70 was solely inducible protein induced by temperature changes(Morimoto et al. 1990; McKay et al. 1994; Ravaux et al.2007), but Cchsc70 was constitutive and inducible protein expressed during normal cell functioning, which also upregulated in response to stressful stimuli (Callahan et al.2002). The expression levels of hsp70 in Grapholita molesta and Musca domestica were up-regulated with the rise of temperature (Tang et al. 2012; Chen et al. 2014). Fohsc701 in Frankliniella occidentalis larvae plays an important role in resisting low temperature stress (Li and Du 2013). Similar induced expression pattern was also reported in other species, such as Plutella xylostella, Locusta migratoria and Macrocentrus cingulum (Sonoda et al. 2006; Wang et al.2007; Xu et 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 that hsp70 showed a low level of expression at different temperatures. It is possible that there exist other heat-induced HSP70s in C. chilonis. In this study,we also found that Cchsp90 could be induced by heat and mild cold stress, but not cold shock (Fig. 3-E), which was also observed in Sehsp90 of Spodoptera exigua (Xu et al.2011) and Sihsp90 of Sesamia inferens (Sun et al. 2014;Tang et al. 2015).

5. Conclusion

We obtained five genes encoding heat shock proteins(HSPs) for Cotesia chilonis, Cchsp40, Cchsp60, Cchsp70,Cchsc70 and Cchsp90 and their predicted amino acid sequences showed high similarities with published HSPs of other insects in Hymenoptera. Expression patterns varied in the five Cchsps 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 of C. chilonis at the molecular level. Further researches are needed to evaluate the expression levels of the other HSPs of C. chilonis under 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.

Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

选取2015年10月至2016年3月期间,我院收治的肝胆手术患者22例,其中男性11例,女性11例,患者年龄37岁至77岁,平均(48.96±7.51)岁,22例患者中肝胆结石14例,胆囊息肉3例,食道癌3例,喷门癌2例。所有患者均行手术治疗。

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