Characterization of two novel heat shock protein 70s and their transcriptional expression patterns in response to thermal stress in adult of Frankliniella occidentalis (Thysanoptera: Thripidae)

1. Introduction

Insects are poikilotherm, whose activities are affected by many biotic and abiotic factors, including temperature,humidity, light, radiation, heavy metals, food, parasitic,predation and so on. Temperature is one of the most common environmental factors which affects the insect ecology and evolution directly or indirectly within time and space (Hoffmann et al. 2003). Since global warming has led to more frequent fluctuations of the extreme high and low temperatures, insects have developed many behavioral and physiological abilities to adapt to various environmental temperatures by searching for ideal shelters, experiencing cold or heat hardening, and synthesizing heat shock proteins and antioxidant enzymes (Joanisse and Storey 1998; Lalouette et al. 2011; Li et al. 2011b; Lu et al. 2014).

Heat shock proteins (HSPs) are widely found in various organisms. They are special proteins that are highly conserved in evolution and have high homology in all organisms. HSPs can be synthesized rapidly to help organism adapt to adverse environments (Lindquist 1986;Sǿrensen et al. 2003; Zhao and Jones 2012; Sun et al.2014), such as functioning as molecular chaperones in organism and help denatured protein refolding (Hartl 1996).HSPs can be divided into three categories based on the molecular weight and homology, including high molecular weight HSPs (HMW HSPs, 90–110 kDa), medial molecular weight HSPs (MMWHSPs, 62–72 kDa), and low molecular weight HSPs (LMWHSPs, 15–30 kDa) (Lindquist and Craig 1988; Morimoto 1990). Among these, heat shock protein 70 (HSP70) is one of the most conservative and important family and is much more sensitive than other HSPs when exposed under various stress (Glick 1995; Kim et al. 1998). The HSP70 family consists of two proteins,the non-inducible heat shock cognate protein 70 (HSC70)which exists in almost all cells under normal conditions, and stress inducible HSP70 (Welch 1992). The classic structure of HSP70 contains 44 kDa fragment (amino acid residues 1–386) from the N-terminus, 18 kDa peptide-binding domain(amino acid residues 384–543), 10-kDa C-terminus (amino acids residues 542–646), to the highly conserved EEVD terminal sequence glycine/proline-rich aperiodic segment(Flaherty et al. 1990; Bork et al. 1992; Hightower et al.1994; Morshauser et al. 1995). These four terminal amino acids are ubiquitous in all eukaryotic HSP70 and affect the amount of mRNA translation during heat shock stress(Denisenko and Yarchuk 1990). In addition, HSP70 also plays important roles in anti-apoptotic function, immune response, antioxidant and stress tolerance of cells.

Western flower thrips, Frankliniella occidentalis, is a worldwide economically detrimental insect pest to a wide range of crops. F. occidentalis is native to western North America and damages plants by both direct feeding and transmitting plant viruses (Kirk 2002; Reitz et al. 2011). In 2003, F. occidentalis was recorded in a greenhouse on pepper plant in Beijing, China for the first time (Zhang et al.2003). Afterwards, F. occidentalis has been found in 14 provinces across China (Chen et al. 2011). The spread of F. occidentalis is in large part due to its ability in tolerance of temperature extremes. Previous studies have shown that the accumulation of inducible HSP70 determined the thermotolerance of cells (Parsell and Lindquist 1994).F. occidentalis did not diapause and tolerate extreme temperature through cold or heat hardening or by producing heat shock proteins (e.g., FoHSP90, FoHSP70, FoHSP60)and antioxidase (e.g., catalase) (Ishida et al. 2003; Li 2011b;Wang et al. 2014; Lu et al. 2016; Qin et al. 2017a, b). In this study, to evaluate the thermal response of F. occidentalis adults, we analyzed the sequence characteristics and expression patterns of two novel full-length HSP70 protein genes obtained from the male adults (AM) and female adults (AF) under various temperature treatments. The discussion of thermal tolerance related to the two novel genes in this study provided fundamentals for further study of F. occidentalis adaptation.

该方法以两个节点间相似度的拓扑或语义度量评估连接形成的概率进行预测.Wang等人[11]利用马尔科夫随机场和无向图,设计局部概率模型对两跳邻居进行局部建模,可预测大型图中两节点间发生连接的概率;Lü等人[12]提出随机关系模型,通过最大化边缘概率将链路间的结构依赖看作矩阵中的实体依赖,并映射为自适应高斯核函数进行链路预测.该方法要求给出符合目标网络特点的概率模型,但大多数实际环境中,这一要求往往难以现实.

2. Materials and methods

2.1. Insects

The F. occidentalis adults were originally collected in Hangzhou, China in 2008 and reared in the laboratory according to Li et al. (2011a). The climate chamber conditions were maintained at (25±0.5)°C, with a photoperiod of 16 h L:8 h D for F. occidentalis colony. Newly emerged AF and AM were used in our experiment.

2.2. Thermal treatments

Two hundred newly emerged adults (1-day-old) ( and )were collected and placed in a glass tube and exposed to certain temperature treatments for 1 h. Temperature treatments included exposure to cold from –14 to –6°C at 2°C intervals, and heat from 33 to 41°C at 2°C intervals using a temperature controller (DC-3010, Jiangnan Instrument Factory, Ningbo, China). After treatments, F. occidentalis adults were allowed to recover at (25±0.5)°C for 1 h and then were frozen in liquid nitrogen and stored at 70°C.F. occidentalis maintained at 26°C was treated as control.Each treatment was replicated four times.

2.3. RNA extraction and cDNA synthesis

Total RNA was extracted from F. occidentalis adults using the SV Total RNA Isolation System (Promega, USA).The concentration and quality of RNA were analyzed by spectrophotometry (Eppendorf Bio Photometer Plus,Germany) and agarose gel electrophoresis. A total of 1 μg of total RNA was used as template and the oligo(dT)18 primer was also used to generate the first strand complementary DNA (cDNA) according to the protocol of First Strain cDNA Synthesis Kit (Clontech, USA).

2.4. Cloning of HSP transcripts

Gene-specific primers (Table 1) were designed based on sequences from de novo transcriptome in F. occidentalis to amplify the partial cDNA fragments of HSP70. The PCR reaction conditions were set as follows: 94°C for 3 min,19 cycles of 94°C for 30 s, 65–45°C (decreasing by 1°C/cycle) for 30 s, 72°C for 1 min, and then 25 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min, with extension at 72°C for 10 min. Purified products were cloned into the pGEM-T Easy vector (Promega, USA) and transformed into competent Escherichia coli DH5α cells for sequencing.

Gene-specific primers (Table 1) were designed to obtain 5´ and 3´ regions using the SMART RACE cDNA Amplification Kit (Clontech, USA) based on the sequence of partial fragments. PCR parameters were set as follows:94°C for 3 min, 35 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min, followed by extension at 72°C for 10 min.Bands of the expected size were cloned and sequenced as described above.

2.5. Bioinformatic analysis

To determine the expression of Fohsc704 and Fohsc705 in AF and AM at different heat and cold temperatures, the relative mRNA levels of the two genes were quantified by using real-time quantitative PCR. GAPDH and 18S were used as candidate reference genes in heat and cold stress,respectively (Zheng et al. 2014).

当前国内各地区基础规划方面，存在着旅游资源发展不均衡、休闲场所较少等情况，尤其是在现代城镇居民生活水平的不断提升下，居民对于休闲生活的关注越来越高，但相应的基础设施建设显然还不能够满足居民的此种需求，各地区都呈现出小假期旅游没有地方可去的情况。另外，普遍地区特色小镇自身具有着一定的自然与人文优势，结合国家政府方面近几年对绿色发展、生态环保事业、保护古镇风貌的政策指导来看，引入资本创建特色小镇具有其必要性，能够迎合现代政策引导的需求以及城镇居民对休闲生活的需求。

Table 1 The primers used in the study

Primer name Primer sequence (5´→3´)For RT-PCR Fohsc704 GCTTGATTGGCAGACGATTTGAG CAAGTGAAAGAGGGGTAACATCC Fohsc705 GGAAAGAACAGAGTTATCGGTG TCAACTTCACCCAGTAAGCGTA For RACE PCR Fohsc704-5´ CAACCTCCAGCAGGCATCACTTA Fohsc704-3´ CAGCATCCTCAAATCGTCTGCCAATC Fohsc705-5´ TTGTGGGTGGCTCCTCAAGAGTTCC Fohsc705-3´ TGTGAGGGTTCGTGATTTCAGCGT For genomic ampli fication Fohsc704 TAGTTTCTGTCCTACCGTTTCCAG TGACATTTGGTATGAAGGACACAC Fohsc705 GTGCAATTTCTTCGTGCCCTTTC CCTTATGGTTGAGCTCGGATGTA For qRT-PCR Fohsc704 CTCACCCAGTCAAATAGGAGCAT CAGCATCTTTTGTAGCCTGTCTC Fohsc705 AGACTTTGATGTCCTTTTAGCCG TCACCCAGTAAGCGTAAATAAGC 18S AACACGGGAAACCTCACCA CAGACAAATCGCTCCACCAA GAPDH AAGGGTGCTCAGGTTGTTGCT CGACCGTGGGTGGAGTCATAT

2.6. Ampli fication of genomic DNA

The deduced amino acid sequences of FoHSC704 and FoHSC705 were aligned with other insect species. A high degree of conservation was observed among these sequences (Appendices C and D). FoHSC704 displayed 85, 86, 84, 84, and 85% identity with the Zootermopsis nevadensis (KDR08641.1), Athalia rosae(XP_012264348.1), Lasius niger (KMQ94354.1), Eufriesea mexicana (OAD62568.1), and Epicauta chinensis(AHK26790.1), respectively. But there were some highly variable regions, like the region (1–51 aa) located at N-terminal, 613–638 aa and 659–688 aa regions located at C-terminal. We found a putative ATP/GTP-binding site,AESYLNT (Saraste et al. 1990) and a EF-hand (EFh) domain(EIQKGVFEVKSTNGDTFLGGEDFDNTLVNF, 255–284 aa)in these sequences. FoHSC705 displayed 74, 74, 73,and 73% identity with Myzus persicae (XP_022174838.1),A. rosae (XP_020709814.1), Melipona quadrifasciata(KOX73871.1), and Liposcelis bostrychophila (AKP54258.1)respectively. There was a thymopoietin domain (271–303 aa)in these sequences.

2.7. Quantitative real-time reverse transcriptase PCR(qRT-PCR) analysis

The complete cDNA of the Fohsc704 and Fohsc705 was submitted to GenBank (accession no. KY914547 and MF377626, respectively). The full-length cDNA of Fohsc704 and Fohsc705 consisted of 2 073 and 1 476 bp ORF which encoded 690 and 491 aa with calculated molecular weights of 75 and 54.5 kDa, respectively. The polyadenylation signal(AATAAA) was present at 13 bp upstream from the ployA in Fohsc704 (Tabaska and Zhang 1999). Three canonical signatures that defined the HSC70 family were found in FoHSC704, including IDLGTTnS (aa residues 56–63),VYDLGGGTfdiSIL (241–254), and VlLvGGmSRMPkVqQ(382–396). The FoHSC705 protein contained typical feature of the HSP70 family at aa residues 341–355(VeIvGGsSRVPaIkQ). The conserved motif EKKN and GIFL was found at the C-terminal in Fohsc704 and Fohsc705,respectively (Appendices A and B).

2.8. Statistical analysis

The expression of Fohsc705 in AF and AM was found inducible by heat and cold stress. At high temperatures,this gene expression level reached its peak in AF at 33°C and decreased significantly with the temperature increasing.However, no significant difference was found in expression level of AM (Fig. 4-A). In the cold treatment, the relative expression level of Fohsc705 was induced significantly to increase in both AF (1.83-fold) and AM (4.18-fold) at–8°C (Fig. 4-B). However, Fohsc705 exhibited different expression patterns in different genders of F. occidentalis under the same cold stress. For example, the expression level of AM is higher than that of AF at –8 and –12°C,respectively (heat temperature: F=3.217, P=0.007; cold temperature: F=16.875, P＜0.001).

3. Results

3.1. Cloning and sequence analysis of HSPs

The mRNA level of the two hsc70s was measured by comparative quantitative real-time PCR amplification. The 18S rRNA and GAPDH were used as internal standard for cold and heat treatments respectively (Zheng et al. 2014).PCR reactions were performed in 20-μL reaction volumes that included iTaq Universal SYBR Green Supermix (2×) (Bio-Rad,USA), 1 μL of each forward and reverse primer (10 μmol L–1)(Table 1), 2 μL of cDNA template (2.5×10–4 μg μL–1), and 6 μL of ddH2O. The PCR reaction was set as follows: 95°C for 30 s,40 cycles of 95°C for 30 s, and incubation at the Tm value of primer pairs (Table 1, see qRT-PCR) for 15 s, and then the melting curve analysis was carried out to determine the specificity of PCR products. Reactions were conducted using a CFX-96 Real-Time PCR System (Bio-Rad, Berkeley, USA).Each PCR reaction included three replicates.

由于血液透析过程中会导致部分血液的流失,若透析脱水速度过快,人体组织液渗透作用缓慢来不及补充失去的血液,导致有效循环血量减少,机体为保证心脑等重要脏器的供血供氧,进一步加重肾功能损害,严重影响患者预后,护理人员应尽早发现及处理。同时,患者出现低血压反应时,护理人员应警惕患者在透析结束起床时出现一过性脑供血不足引起的头晕、跌倒等安全问题。

3.2. Sequence alignment and phylogenetic analysis

Genomic DNA was extracted from adults of F. occidentalis by using AxyprepTM multisource Genomic DNA Kit (Axygen,USA). Specific primers (Table 1) flanking the ORF were designed to amplify genomic sequences of the three genes.Touch-down PCR was performed as follows: 94°C for 5 min,14 cycles of 94°C for 30 s, 60–45°C (decreasing by 1°C/cycle) for 30 s, 72°C for 2 min 30 s, and then 25 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 2 min 30 s, with a final extension of 72°C for 10 min. PCR products were cloned and sequenced as described above.

Pairs of Fohsc704 and Fohsc705 cDNA-specific primers were designed to amplify 3 114 and 1 870 bp sequence from F. occidentalis genome (accession no. MF377632 and MF377627, respectively). Comparison of the genomic and cDNA sequence revealed the presence of four introns in Fohsc704, including 87- and 71-bp introns in the coding region, a 962-bp intron across coding region and 3´-UTR,and a 70-bp intron in the 3´-UTR. Fohsc705 contained six introns in the coding region and the lengths were 633,200, 145, 95, 696 and 73 bp, respectively. Fig. 2 showed the difference of introns of HSP70 family among the same insect species. In F. occidentalis, no introns were found in Fohsp703, and four, seven, four and six intorns were found in Fohsc701, Fohsc702, Fohsc704 and Fohsc705,respectively. The size of introns ranged from 76 to 962 bp,and all locations were almost not conservative, there were only two similar locations in Fohsc702 and Fohsc704.

3.3. Genomic structure analysis

Twenty-seven full-length HSP70 proteins from Lepidoptera, Hymenoptera, Thysanoptera, Hemiptera,Coleoptera, and Psocoptera were used for phylogenetic analysis and tree construction using the NJ method (Fig. 1).The tree was divided into four branches, and FoHSP70 was distributed in all branches. The FoHSC704 and FoHSC705 in this study were found at the top and bottom of the phylogenetic tree, respectively. The FoHSC704 has a close evolutionary relationship with HSP70s of Laodelphax striatella and Nilaparvata lugens, which belong to Hemiptera.FoHSC705 was the closest to Lygus hesperus, which also belongs to Hemiptera. The other two FoHSP70s were closed toTribolium castaneum.

3.4. Expression of Fohsc70s at different temperatures

Nucleotide and amino acid sequence similarities were evaluated using the BLAST Program available at the NCBI website (http://www.blast.ncbi.nlm.nih.gov/Blast).Open reading frames (ORFs) were identified using ORF Finder Software (http://www.ncbi.nlm.nih.gov/projects/gorf/). The molecular weights of predicated proteins were calculated by the SWISS-PROT (ExPASy server)“Compute pI/Mw” Program (http://au.expasy.org/tools/pi_tool.html). The online tool ScanProsite was used to identify the features of HSP families (http://www.expasy.org/). The sequence alignment and identity analysis were carried out using the DNAMAN Software Package (Lynnon Corporation, Canada). Phylogenetic trees were constructed with the neighbor joining (NJ) method using MEGA 7(Kumar 2016).

孙悟空三调芭蕉扇！全部疑点都集中到这辆花车上。根据在场目击者的回忆，德公公还原出这样的场景：每当有人走过那辆花车，铁扇公主都会笑着朝经过的人扇扇子，好像是在调戏路人。孙悟空呢，每次都调皮地拿金箍棒戳向围观的人，当然不是真戳。

对水体中COD、氮、磷的净化量和净化效率进行主要估算。河道是个半开放体系，其水体净化效益较难定量评估，故通常以单项工程来实施对进出水水质指标控制。

企业信息化的手段在帮助企业建立科学的决策机制中可以发挥的作用就是把决策的程序和方法固化下来，同时把决策的过程记录在案，再配合后续的责任追究机制，来迫使决策者必须按照既定程序，获取足够的决策信息后来做出决策，且谁决策谁负责，谁参与决策谁根据自己的角色职责承担责任。

For Fohsc704, when exposed to heat stress, the expression level in F. occidentalis adults was low and similar, except for those of AF at 39°C, whose level was significantly decreased (Fig. 3-A). When exposed to cold stress, the expression of Fohsc704 reached to the highest level (2.28-fold) in AM at –12°C. No significant difference of the expression level was found in AF (Fig. 3-B).When exposed to high temperature, the expression level of AF is slightly higher than that of AM at 41°C (heat temperature: F=4.732, P＜0.001, cold temperature: F=2.930,P=0.01).

Fig. 1 Phylogenetic tree of FoHSC704 and FoHSC705 in Frankliniella occidentalis and other insect species based on neighborjoining method using Mega 7.0. The percentage bootstrap values obtained from 1 000 re-samplings were shown at the nodes and percentages lower than 50 were deleted. The FoHSP70 in our study was marked with black triangles, and FoHSP70s in previous studies were marked with black square.

Fig. 2 Comparison of genomic structure of Fohsp70s in Frankliniella occidentalis. The lines represented the cDNA sequences of hsp70s and GenBank accession numbers of species were given on the left of the line. Intron positions were indicated by black rectangles, and the number above the boxes represented the length of introns (bp).

The results of qPCR were analyzed by using the 2–ΔΔCt method (Livak and Schmittgen 2001). The statistical significance of difference between treatments was analyzed by either t-test or one-way analysis of variance (ANOVA)followed by a Tukey’s honesty significant difference test for multiple comparisons. For the ANOVA, hsp expression levels were log- and cosine-transformed according to Tomanek and Somero (1999) to assure homogeneity of variances among different groups. One-way ANOVA was used to detect significant differences in mRNA levels among treatments,followed by Tukey’s honesty significant difference test with P＜0.05. All statistical analysis were performed using SPSS ver. 16.0 (SPSS, Chicago, USA)

点绛唇，词牌名，又名“点樱桃”“十八香”“南浦月”“沙头雨”“寻瑶草”等。以冯延巳词《点绛唇·荫绿围红》为正体，双调四十一字，前段四句三仄韵，后段五句四仄韵；另有四十一字前后段各五句四仄韵，四十三字前段四句三仄韵，后段五句四仄韵的变体。代表作有苏轼的《点绛唇·红杏飘香》等。姜夔的这首《点绛唇》属于正体。

Fig. 3 Relative mRNA expression level of Fohsc704 in Frankliniella occidentalis adults under heat (A) and cold (B) stress. All statistics indicate means±SE. Columns labeled with different letters indicate significant differences using one-way ANOVA followed by Tukey’s multiple comparison analysis.

4. Discussion

Fig. 4 Relative mRNA expression level of Fohsc705 in Frankliniella occidentalis adults under heat (A) and cold (B) stress. All statistics indicate means±SE. Columns labeled with different letters indicate significant differences using one-way ANOVA followed by Tukey’s multiple comparison analysis.

In this study, we identified new Fohsc704 and Fohsc705 genes, members of HSC70s in F. occidentalis adults.We found a putative ATP/GTP-binding site, AESYLNT(Saraste et al. 1990) and a EF-hand (EFh) domain(EIQKGVFEVKSTNGDTFLG GEDFDNTLVNF, 255-284 aa)in FoHSC704. In normal situations, a balance exists between the generation of reactive oxygen species (ROS)and antioxidant defenses (Poljsak et al. 2013). When cells exposed to extreme temperatures, the balance will be broken and surplus ROS can trigger severe damage to biological molecules, including lipids, proteins, and nucleic acids (Martindale and Holbrook 2002), resulting in damage or genomic instability (Dizdaroglu and Jaruga 2012). EFh can help prevent cell from oxidative damaging (Chen 2007).Thymopoietin domain in FoHSC705 is related to cellular immunity. The motifs at the C-terminus were EKKN and GIFL for FoHSC704 and FoHSC705, respectively. The motif at C-terminus of HSP70 members in insect is specific in which the sequence ended with EEVD located in the cytoplasm,KDEL in endoplasmic reticulum, and PEAEYEEAKK in mitochondrion (Guy and Li 1998). EKKN and GIFL found in our study were different from the description as above,and the motif at C-terminus of FoHSP70s in previous studies was EEVD or KDEL (Wang et al. 2014; Lu et al. 2016). In addition, the full length of Fohsp705 was only 1 476 bp which encoded 491 aa, and its molecular weight was only 54.5 kDa which was less than HSPs in previous studies. Thus,we summarized that the FoHSC704 and FoHSC705 found in our study were two novel members in HSP70 family. The specific locations need to be certified in the future study.

The phylogenetic tree showed the decentralized distribution of FoHSP70s, indicating that HSP70s in insecta are diverse and mutated. The FoHSC704 and FoHSC705 in this study were not located in the same branch with other FoHSP70s, but they were close to Hemiptera. The result suguested that the evolutionary history of the two Fohsc70s be parallel to insects in Hemiptera. Meanwhile,the two FoHSP70s shared high sequence similarity with other insects. These results suggested that the two FoHSP70s may have other functions. Wang and Lei(2005) reported that the HSP70 proteins from the same cell compartment among species were more similar than those from different organs of one species, due to the specialized function of members in HSP70 family. The analysis of genomic sequence suggested that four of the five Fohsp70s contained introns, and their number and size differ from each other. Since the introns were the remnants of a process that speeded up evolution, the number and location of introns reflect the evolutionary history to a certain extent (Gilbert 1985). The genomic structure analysis result was consistent with the phylogenetic analysis that the FoHSP70s were different in evolutionary process. Compared to HSP70, HSC70 existed in normal cells and tissues and could not be induced easily. We hereby speculated that the presence of introns improved the stability of HSP70 and have affected the expression of hsp70 genes. Previous studies have shown that the presence of introns offset the efficiency of transcription and affected other molecular biological processes. It can also influence the gene expression in mRNA transport, stability,and translation efficiency (Nott et al. 2003). Comeron(2004) also reported that there was negative correlation between intron size and gene expression level, and the shorter or no intron genes had high expression level.

The induction of Fohsc704 and Fohsc705 by heat and cold treatments in our study indicated the important roles they played in thermotolerance adaptation. There were differences in gene expression in genders of F. occidentalis in our study, for instance, the Fohsc704 expression level of AF was significantly higher than that of AM at 41°C,which was consistent with the previous reports that AF was more thermotolerant than AM (Li et al. 2011a). Similarly,Grapholita molesta (Busck) adult genders were found to be correlated with the expression of hsp70 gene (Chen et al. 2014). However, the Fohsc705 expression in AM at–8 and –12°C was significantly higher than AF. This was inconsistent with previous reports in Li et al. (2011a, b).The difference in thermotolerance may be influenced by various factors including water and ion homeostasis, levels of proline, asparagine and glutamic acid, etc. HSPs only contributed partially to thermotolerance (Fields et al. 1998;MacMillan et al. 2015). For Fohsc704, the expression difference was only found in AF under the heat stress,and this was opposite when exposed to cold stress. This finding indicated that the sensitivity of different genders to temperature was variable. In F. occidentalis, we found that the relative expression of Fohsc704 was lower than Fohsc705 in adults in same treatments, particularly under heat stress. We surmised that the polyadenylation signal(AATAAA) in 3´ nontranslated regions in Fohsc704 resulted in the situation. The signal had greater mRNA stability at normal temperatures (Colgan and Manley 1997; Rubenstein and Lyons 2001), and therefore it contributed to the maintenance and re-establishment of basal levels of gene expression (Lindquist and Petersen 1990).

The expression level of Fohsc705 with six introns was induced by temperature, which was similar to the expression level of Fohsp703 with no intron in Qin et al. (2017b).Therefore, the situation was consistent with the study that the presence or absence of introns cannot be used to distinguish HSC70 from HSP70, and genes with introns can encode stress-inducible HSP70 (Qin et al. 2003). The highest expression of Fohsc705 in adults was 4.18-fold of those in the control, indicating that there might be other members of HSP70 family contributing to temperature response. In addition, thymopoietin domain in the gene is related to cellular immunity, not cell stress. What’s more,development of insects will affect the gene expression level,Lu et al. (2016) reported that Fohspc701 and Fohspc702 expression levels were significantly higher in pupae than that in adults. Expression levels of hsps were also altered by treatment and recovery time. For example, the expression of hsp90 and hsp70 in Grapholita molesta increased with the treatment time (Zhang and Denlinger 2010), and the expression of Fohsp60 and Fohsp90 was the highest at 2-h treatment (Lu et al. 2016). Thus, the developmental stages of F. occidentalis status and treatment pattern played important roles in expression of hsp70s.

5. Conclusion

We obtained two novel Fohsc70s with introns and found that there was difference in expression patterns of the two hsc70s in genders of F. occidentalis. When exposed to heat stress, the two genes in AF were induced whereas not in AM. However, when exposed to cold stress, the expression differences of two genes were found in both AF and AM.Our results indicated that the two FoHSP70s are associated with thermal adaption and provided useful information in understanding the thermotolerance of F. occidentalis at the molecular basis.

Acknowledgements

This research was funded by the Special Fund for Agro-Scientific Research in the Public Interest of China(201103026, 200803025) and the Science and Technology Innovation Project of Student in Yangzhou University, China(X20160637). The authors would like to thank the Testing Center of Yangzhou University, China for assistance.

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

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