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Pesticide Biochemistry and Physiology 160 (2019) 171–180

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Identification of a DnaJC3 gene in Apis cerana cerana and its involvement in various stress responses

T

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Xuemei Zhanga, Guilin Lia, Xinxin Yanga, Lijun Wanga, Ying Wangb, Xingqi Guoa, Han Lia, , ⁎ Baohua Xub, a b

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong 271018, PR China College of Animal Science and Technology, Shandong Agricultural University, Taian, Shandong 271018, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Apis cerana cerana AccDnaJC3 Stress response Expression analysis RNA interference

As molecular chaperones, DnaJs play critical roles in maintaining cytoplasmic structure and resisting various stresses. However, the functions of DnaJs in insects are poorly understood. In this study, we identified a DnaJC3 from Apis cerana cerana (AccDnaJC3) and investigated its roles in adverse conditions. Real-time quantitative PCR analysis showed that AccDnaJC3 was highly expressed in muscle and epidermis. In addition, AccDnaJC3 was induced by a variety of stresses, such as 4 °C, 24 °C, 44 °C, H2O2, HgCl2, VC, UV, cyhalothrin, abamectin and emamectin benzoate treatments, whereas it was inhibited by CdCl2 and paraquat treatments. Disc diffusion experiments indicated that overexpression of recombinant AccDnaJC3 enhanced Escherichia coli tolerance to some stress conditions. In contrast to the control group, when AccDnaJC3 was knocked down with RNAi technology, several other antioxidant genes were downregulated, suggesting that AccDnaJC3 may play important roles in stress response. Furthermore, we found that the enzyme activities of superoxide dismutase, peroxidase and catalase were lower in AccDnaJC3-knockdown bees than in control bees. Taken together, these results suggest that AccDnaJC3 may be involved in various stress responses in Apis cerana cerana.

1. Introduction Cells are inevitably subjected to different biotic and abiotic stresses, which results in unfolded or misfolded proteins in the cells (Vendruscolo, 2012; Meher et al., 2017). Misfolded proteins aggregate together, and this aggregation triggers protein toxicity, which in turn destroys protein homeostasis and hampers the normal cellular functions (Weids et al., 2016; Tamás et al., 2014). To mitigate this danger, cells have developed a limited number of molecular chaperones (Peralescalvo et al., 2018). Molecular chaperones are thought to be a fundamental defense mechanism; they can reestablish functional protein conformations and maintain protein homeostasis under different stress conditions (Rajan and D'Silva, 2009a). Most molecular chaperones are of a class known as heat shock proteins (HSPs), which can assist misfolded proteins to refold, degrade terminally misfolded proteins, maintain cytoplasmic structure and protect organisms from protein toxicity damage (Tamadaddi and Sahi, 2016; Pratt and Toft, 1997; Csermely et al., 1998). HSPs are a large family, and their expression can be induced by different environmental stresses. On the basis of molecular weight,

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HSPs can be divided into six major families (HSP20, HSP40, HSP60, HSP70, HSP90, and HSP100) (Ratheesh Kumar et al., 2012; Peng-Mian et al., 2013). Among these, HSP40s, also called DnaJs, are a group of highly conserved proteins (Pascarella et al., 2018; Goffin and Georgopoulos, 1998). They were originally identified in Escherichia coli (E. coli) and have since been found to be widespread in animals, plants and microbes (Goffin and Georgopoulos, 1998; Chen et al., 2010; Zhang et al., 2018). Six DnaJs occur in E. coli (Zhang et al., 2018), 22 in yeast (Walsh et al., 2004) and 41 in humans (Qiu et al., 2006). Based on their domain organization, DnaJs can be classified into three types, namely, DnaJA, DnaJB and DnaJC (Cheetham and Caplan, 1998). All types contain an approximately 70 amino acid sequence called the J domain, which is the reason for the name DnaJ (Greene et al., 1998; Kim et al., 2014; Misselwitz et al., 1998). It has been demonstrated that DnaJs are cochaperones of HSP70s and function by stimulating HSP70 ATPase activity and helping HSP70s accomplish cellular protein refolding and translocation (Rampuria et al., 2018). Moreover, DnaJs themselves can act as molecular chaperones, binding unfolded proteins to promote recovery of damaged proteins during various environmental stresses (Rampuria et al., 2018).

Corresponding authors. E-mail addresses: [email protected] (H. Li), [email protected] (B. Xu).

https://doi.org/10.1016/j.pestbp.2019.08.007 Received 7 October 2018; Received in revised form 23 August 2019; Accepted 29 August 2019 Available online 31 August 2019 0048-3575/ © 2019 Published by Elsevier Inc.

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group for the 4 °C, 24 °C, 44 °C, UV, abamectin, paraquat, emamectin benzoate and cyhalothrin treatments. Bees injected with 0.5 μL of double distilled water in the chest were used as the control group for the H2O2, CdCl2, HgCl2 and VC treatments. The control groups were raised in normal living conditions at 34 °C with 70% relative humidity and fed a diet of pollen dough and 30% sucrose solution, and the living conditions of the experimental groups were consistent with the control groups except the temperatures used in groups 1–3. Each treatment was performed for three times. All the bees that were treated were immediately stored in liquid nitrogen at the appropriate time point (Table S1) and then stored at −80 °C until use.

Previous studies have shown that DnaJs have functions in suppressing the aggregation and toxicity of misfolded proteins in Drosophila models (Kuo et al., 2013). In addition, Hageman et al. demonstrated that DnaJs can protect cells from damage due to misfolded protein toxicity in a Xenopus laevis model (Hageman et al., 2010). Although the roles of DnaJs have been studied extensively in plants and microbes, they have rarely been reported in insects (Li et al., 2016a), including Apis cerana cerana (A. cerana cerana). A. cerana cerana is uniquely valuable indigenous species in China; this bee can utilize sporadic nectar supplies under extreme weather conditions and in mountain or forest regions (Han et al., 2018; Nie et al., 2018). In addition, A. cerana cerana has anti-pest and cold tolerance traits and plays an important role in the balance between agricultural economic development and regional ecology (Han et al., 2018). Nevertheless, the population of bees has declined in recent decades owing to the loss of habitat, the continued accumulation of chemical residues and other environmental threats, such as extremes of cold and heat, excessive use of pesticides, and unjustified use of heavy metals (Goulson et al., 2015; Gallai et al., 2009). Therefore, it is essential to research the stress responses of bees to adverse environments. To explore the possible roles of AccDnaJC3 in various stress conditions, a DnaJC3 gene, namely, AccDnaJC3, was isolated from A. cerana cerana in this study. We investigated the expression pattern of AccDnaJC3 in different tissues and under various stresses. At the same time, a disk diffusion experiment displayed a variety of stress tolerance responses due to recombinant AccDnaJC3. In addition, to further confirm our results, we knocked down AccDnaJC3 using RNA interference technology and evaluated enzyme activities and the expression of other antioxidant genes in A. cerana cerana. Collectively, our data reveal that AccDnaJC3 may play an important role in response to different environmental stresses.

2.2. Total RNA extraction and cDNA synthesis Total RNA was extracted from the bees using RNAiso Plus reagent (Takara, Dalian, China) according to the reagent instructions. Then, the extracted RNA was used for reverse transcription to first-strand cDNA by using the HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer's directions. 2.3. Real-time quantitative PCR Real-time quantitative PCR (RT-qPCR) was carried out using TB Green™ Premix Ex Taq™ (Takara, Dalian, China) on the CFX96™ RealTime System (Bio-Rad, Hercules, CA, USA). The RT-qPCR was carried out in a total volume of 25 μL, including 12.5 μL of TB Green Premix Ex Taq (Tli RNase H Plus) (2×), 0.5 μL of PCR Forward Primer (10 μM), 0.5 μL of PCR Reverse Primer (10 μM), 2 μL of cDNA template (< 100 ng) and 9.5 μL of sterile water. The two-step PCR amplification standard procedure is as follows: step 1 is 95 °C for 30 s, step 2 is 40 cycles of PCR that consist of 95 °C for 5 s and 60 °C for 30 s, and step 3 is a melt curve. The housekeeping gene β-actin (GenBank accession number XM-640276), which is expressed stably in all cells of bees, was used as an endogenous control (Cunha et al., 2005). All experiments were carried out with at least three separate biological replicates, and all RT-qPCRs were also repeated in triplicate. The Bio-Rad CFX Manager 3.1 was used to analyze the RT-qPCR data, and the 2-ΔΔCT method was applied to calculate the relative expression level of the target gene (Yu et al., 2007). Data are presented as the means ± standard error (SE) of triplicate replicates (n = 3). The statistical analyses were performed using SPSS Statistics. Statistical significance was set at P < 0.01.

2. Materials and methods 2.1. Biomaterials and treatments The A. cerana cerana tested in this article was collected from honeycomb at the experimental apiary of Shandong Agricultural University (Taian, China). We used back labels to identify the ages of the bees; A thousand newly emerged one-day-old adult worker bees (A1) were marked with paint, of which 700 fifteen-day postemergence worker bees (A15) were collected at the hive after 15 days. Afterward, the bees were fed a diet of pollen dough and 30% sucrose solution and were kept in a constant temperature incubator (34 °C) with 70% relative humidity under a 24 h dark regimen for 1 day. For the analysis of gene expression in different tissues, 100 fifteen-day postemergence bees were specifically dissected on ice to obtain nine tissues, namely, the brain, muscle, leg, epidermis, antennae, poison gland, honey sac, midgut and wing tissue. The fifteen-day postemergence adult bees were divided into 14 groups of 40 individuals each, and each group was subjected to different stress conditions. Groups 1–3 were maintained in constant temperature incubators at 4 °C, 24 °C and 44 °C, respectively. Groups 4–7 were injected with H2O2 (0.5 μL, final concentration of 2 mM), CdCl2, HgCl2 (0.5 μL, final concentration of 1 mM) and Vitamin C (VC) (0.5 μL, final concentration of 10 mM) solutions, respectively, in the chest of each worker bee using a microsyringe. Specifically, for the injection, the wings of each bee were grasped using one hand to stabilize the bee, and the reagent was then injected into the chest of the stabilized bee using a microsyringe. Group 8 was exposed to UV (254 nm, 30 mj/cm2). Groups 9–12 were treated with pesticides (abamectin, paraquat, emamectin benzoate, cyhalothrin) by using fumigation. The specific pesticide was added to 0.5 g of cotton. The cotton containing the specific pesticide was held in a cage, to avoid bees accessing to the pesticide, and then the cage was placed the middle of artificial wooden box (5 cm × 5 cm × 2 cm), so that the bees could were fumigated by the volatile pesticide. In addition, untreated bees constituted the control

2.4. Isolation of the open reading frame of AccDnaJC3 A pair of specific primers was designed to obtain the internal gene fragments of AccDnaJC3. The primer sequence and PCR amplification procedures are shown in Tables S2 and S3, respectively. The recovered PCR products were ligated to the pEASY-T1 vectors, and then transformed into E. coli competent cells. Finally, the positive clones were screened and sequenced by the Sangon Biotech Company (Shanghai, China). 2.5. Bioinformatic analysis and phylogenetic tree construction of AccDnaJC3 Several homologous amino acid sequences were filtered using the BLAST tool from NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and DNAMAN version 5.22 was used to compare homologous amino acid sequences (Lynnon Biosoft, Pointe-Claire, QC, Canada). Theoretical isoelectric point and protein molecular weight were predicted using the Compute pI/Mw tool (https://web.expasy.org/compute_pi/). Conserved domains of AccDnaJC3 were identified by conserved domain search services in NCBI sequence analysis (https://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi). The three-dimensional structure of AccDnaJC3 was constructed by SWISS-MODEL (https://swissmodel. expasy.org/interactive). A phylogenetic tree was established based on 172

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groups were kept in an incubator at 34 °C with 70% relative humidity. Two days after injection of dsRNA, the live bees were frozen in liquid nitrogen and stored at −80 °C. The effects of AccDnaJC3 knockdown on the expression of other DnaJ genes in A. cerana cerana were analyzed by RT-qPCR. We also used RT-qPCR to test the expression levels of some antioxidant genes, including AccCAT, AccSOD1, AccSOD2, AccTpx1, AccTpx5, AccGrx1, AccGrx2, AccGSTD, AccGSTS4 and AccCYP4G11, in conjunction with AccDnaJC3 knockdown. Three biological replicates were performed in all the above experiments.

species kinship by using Molecular Evolutionary Genetics Analysis version 4.1. 2.6. Prokaryotic expression and antibody preparation of recombinant AccDnaJC3 A pair of specific primers was designed to amplify the full-length open reading frame (ORF) sequence of AccDnaJC3. The amplified product was digested with restriction endonucleases and ligated into the expression vector pET-30a (+). Next, the recombinant plasmid pET-30a (+)-AccDnaJC3 was transformed into E. coli Transetta (DE3) competent cells (TransGen Biotech, Beijing, China). The bacteria were cultured at 37 °C to an OD600 nm of 0.3–0.5. The expression of AccDnaJC3 was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG, Promega) at a 0.1 mM final concentration and the cells were further incubated at 28 °C for 8 h. The induced protein was identified by 12% SDS-PAGE with the correct molecular weight. The SDS-PAGE gel containing the target protein was excised and added to the appropriate amount of normal saline solution, and the recombinant AccDnaJC3 protein was used to immunize mice to obtain antibody. The specific procedures involving the immunizing of mice to obtain antibody were performed as described in previous studies (Li et al., 2016b; Yan et al., 2013).

2.10. Activities of SOD, POD and CAT after AccDnaJC3 knockdown After AccDnaJC3 knockdown, the bees were ground under liquid nitrogen and transferred to physiological saline to obtain a 10% tissue homogenate (tissue weight (g): volume of physiological saline (mL) = 1:9). A 1% total protein concentration was measured using the total protein assay kit (with the standard: BCA method; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the kit instructions. The Total Superoxide Dismutase (T-SOD) assay kit (Hydroxylamine method), Peroxidase (POD) assay kit and Catalase (CAT) assay kit (Visible light) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to assay the activities of SOD, POD and CAT (U/mgprot), respectively, according to the requirements of the kit. Three biological replicates were performed for each experiment.

2.7. Disk diffusion experiment 3. Results The recombinant AccDnaJC3 was expressed in E. coli cells. LB-kanamycin agar was seeded at 5 × 108 cells/ml E. coli and incubated at 37 °C. Cells including the pET-30a (+) empty vector were used as the control. After 1 h, filter disks soaked with 2 μL of different concentrations of cumene hydroperoxide (0, 50, 100, 200, and 400 mM), HgCl2 (0, 75, 100, 150, and 200 mM) and paraquat (0, 50, 100, 200, and 400 mM) were placed on the agar surface. After the cells were cultured at 37 °C for 10 h, the killing ranges around the disks were measured.

3.1. Presence of DnaJ genes in the A. cerana cerana genome To obtain complete information of the DnaJ genes in A. cerana cerana, we manually identified the DnaJ genes of A. cerana cerana using the sequences published in the NCBI database. After deduplication of the data, a total of 24 DnaJ family members, including 1 DnaJA member, 5 DnaJB members, 15 DnaJC members, and 3 unclassified DnaJ members in the A. cerana cerana genome, were identified (Table S4).

2.8. Western blot analysis of AccDnaJC3 Protein extraction was performed using the Tissue Protein Extraction Kit (ComWin Biotech, Beijing, China) according to instructions, and the total protein assay kit (with standard: BCA method; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to quantify the protein. After the protein was separated by a 12% SDSPAGE gel, the protein was transferred to a PVDF membrane (ComWin Biotech, Beijing, China) by the semidry transfer method with the TransBlot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% Difco™ Skim Milk (Solarbio, Beijing, China) for 1 h at 4 °C and immunoblotted with anti-AccDnaJC3 antibody at 1:500 overnight. α-Tubulin (Beyotime, Jiangsu, China) was diluted to 1:1000 as a protein control. Next, the membranes were washed with TBST (200 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20) 3 times and then incubated at room temperature in peroxidase-conjugated goat anti-mouse immunoglobulin G (1:2000, Jingguo Changsheng Biotechnology, Beijing, China) antibody for 2 h. After the membranes were washed with TBST three times, the corresponding bands were visualized using a ChampChemi (Sage Creation, Beijing, China).

3.2. Sequence analysis of AccDnaJC3

2.9. Silencing of AccDnaJC3 by RNA interference and transcript levels of some antioxidant genes after AccDnaJC3 knockdown

3.3. Putative cis-acting elements of AccDnaJC3

The ORF of AccDnaJC3 was obtained by PCR and found to consist of 1449 bp; it encoded a polypeptide of 482 amino acids with a predicted molecular weight of 55.446 kDa and a theoretical pI of 5.08. Comparison between AccDnaJC3 and DnaJC3s of other species, including Apis mellifera, Habropoda laboriosa, Linepithema humile and Bombyx mori, showed that DnaJC3s were highly conserved in these species, and the common DnaJC3 characteristics comprised a conserved J domain consisting of a His-Pro-Asp (HPD) motif (Fig. S1A). To investigate the evolutionary relationships between DnaJC3s of different species, we constructed a phylogenetic tree and found that AccDnaJC3 was classified with DnaJC3s from the order Hymenoptera and that DnaJC3s from the same insect orders were phylogenetically closer together than they were to DnaJC3s from other orders (Fig. S1B). As shown in the predicted tertiary structure diagram of the J domain of AccDnaJC3 (Fig. S1C), we found that the J domain consisted of four α helices with an HPD motif between the second and third α helices, which is consistent with previous reports (Zarouchlioti et al., 2018).

To study the transcriptional regulatory regions of AccDnaJC3, we used TFBIND software to search for cis-acting elements in a 1543 bp promoter sequence. The result showed many putative binding sites for transcription factors, including heat shock factors (HSF), nuclear factorerythroid 2-related factor 2 (Nrf2), activating protein-1 (AP-1), cAMP response element binding protein (CREB), X-box binding protein-1 (XBP-1), p53 and caudal-related homeobox (CdxA), which is associated

The dsRNA-AccDnaJC3 and dsRNA-GFP RNAs were produced using the RiboMAX™ Large Scale RNA Production System-T7 (Promega, USA). Then, the concentration was measured and adjusted to 16 μg/μL. The fifteen-day postemergence worker bees were randomly divided into three groups (n = 50) and injected with 0.5 μL (8 μg) dsRNAAccDnaJC3, dsRNA-GFP and water using microsyringes. The three 173

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abamectin and emamectin benzoate. In contrast, the paraquat treatment reduced the mRNA expression level of AccDnaJC3, which reached its minimum at 3 h (Fig. 2L). 3.7. Prokaryotic expression of recombinant AccDnaJC3 protein and Western blot analyses AccDnaJC3 was overexpressed in E. coli Transetta (DE3) competent cells. The recombinant protein was separated by SDS-PAGE gel after induction with IPTG. An SDS-PAGE analysis revealed that the target protein had a molecular weight of approximately 62.446 kDa (containing cleavable N- and C-terminal His-tags of approximately 7 kDa) (Fig. S4). Since the recombinant protein was mainly present in inclusion bodies, protein purification using a HisTrap™ FF column was difficult. To further study the response of AccDnaJC3 to different adverse environments, we performed Western blot analyses using antibodies prepared from the above-mentioned recombinant protein. We examined AccDnaJC3 protein levels from bees treated with 44 °C, VC and abamectin using anti-AccDnaJC3 antibody. As shown in Fig. 3A, AccDnaJC3 protein expression was induced significantly after 0.5 h of 44 °C treatment. Following VC injection, the level of AccDnaJC3 protein expression was maximally induced at 2 h (Fig. 3B). As shown in Fig. 3C, the AccDnaJC3 protein level was increased to the maximum at 1.5 h after abamectin treatment.

Fig. 1. Expression profile of AccDnaJC3 in different tissues. The different tissues include the brain (BR), muscle (MS), leg (LE), epidermis (EP), antennae (AN), poison gland (PG), honey sac (HS), midgut (MG) and wing (WI) tissue. The βactin gene was employed as an internal control.

with tissue development and environmental stress. In addition, a TATA box, which is a component of the eukaryotic core promoter, was found (Fig. S2). 3.4. Genomic structure of AccDnaJC3

3.8. Assessment of AccDnaJC3 disk diffusion under different stress conditions

To study the genomic structure of AccDnaJC3, we analyzed its fulllength DNA fragment (2906 bp). As shown in Fig. S3, AccDnaJC3 contains six exons and five introns, implying that the regulation of AccDnaJC3 is diverse. In addition, the number of exons in AccDnaJC3 is highly conserved compared to those of its homologs in the same insect taxonomic order, while the size of the exons varies greatly among different insect taxonomic orders; these results indicate both conservation and variability of this gene during evolution.

To investigate the protective abilities of recombinant AccDnaJC3 in cells, disc diffusion assays were performed under the various stresses. Compared with those of cells carrying pET-30a (+) empty vectors, as shown in Fig. 4, the halo diameters around the filter disks of cells containing recombinant AccDnaJC3 were smaller after the cumene hydroperoxide, HgCl2 and paraquat treatments.

3.5. mRNA levels of AccDnaJC3 in different tissues

3.9. Knockdown of AccDnaJC3 and expression profiles of other antioxidant genes after AccDnaJC3 knockdown

Different mRNA levels of AccDnaJC3 in different tissues may indicate the specific needs of those tissues. To explore the mRNA levels of AccDnaJC3 in different tissues, we used RT-qPCR. As shown in Fig. 1, the mRNA levels of AccDnaJC3 differed constitutionally in different tissues, and the highest expression of AccDnaJC3 was found in muscle (MS), followed by epidermis (EP) and wings (WI).

RNA interference (RNAi) causes posttranscriptional gene silencing and is commonly used to study gene function (Huvenne and Smagghe, 2010). Therefore, we used the RNAi technology to verify the functions of AccDnaJC3 in A. cerana cerana. Fifteen-day postemergence bees were injected with water, dsRNA-GFP and dsRNA-AccDnaJC3 to cause RNAimediated silencing. As shown in Fig. S5A, compared with the control groups (water injection and dsRNA-GFP injection), the transcript level of AccDnaJC3 was lower at 2 days after the dsRNA-AccDnaJC3 microinjection, indicating that AccDnaJC3 had been knocked down successfully. Additionally, AccDnaJC3 knockdown had little effect on the expression of other DnaJ genes in A. cerana cerana (Fig. S5B). After dsRNA injection, the bees showed significant mortality on day 1 and then almost no mortality on days 2–5. To eliminate interference from the injection stimulus, the survival rate of the bees was analyzed 2–5 days after dsRNA injection. As shown in Fig. S5C, the survival rate of AccDnaJC3-knockdown bees was not significantly different compared with that of the control groups, which suggested that AccDnaJC3 knockdown might not result in obvious signs of sickness. To elucidate the potential functions of AccDnaJC3 in response to environmental stresses, we examined the transcript levels of other antioxidant genes after AccDnaJC3-knockdown. The RT-qPCR analyses show that, compared those in with the control group, the expression profiles of AccCAT, AccSOD1, AccSOD2, AccTpx1, AccTpx3, AccTpx5, AccGrx2, AccGSTD and AccCYP4G11 were all downregulated (Fig. 5).

3.6. Expression patterns of AccDnaJC3 under various stresses Previous studies have proven that DnaJ is involved in resistance to various stresses (Rampuria et al., 2018). To evaluate the responses of AccDnaJC3 to different stress conditions, we detected the expression patterns of AccDnaJC3 when A. cerana cerana were subjected to diverse stresses, and the housekeeping gene β-actin was used as an internal control. As shown in Fig. 2A-C, the expression levels of AccDnaJC3 were upregulated in 4 °C, 24 °C and 44 °C treatments and reached their peak levels at 5 h, 0.5 h and 5 h, respectively. After H2O2 treatment, AccDnaJC3 was induced to the highest level at 0.5 h (Fig. 2D). The AccDnaJC3 expression level was significantly increased by HgCl2 injection and reached its maximum at 3 h, whereas expression was constantly suppressed and reached its minimum at 1 h after CdCl2 injection (Fig. 2E-F). Under UV treatment, the expression of AccDnaJC3 was increased slightly at 1 h (Fig. 2G). In addition, the expression of AccDnaJC3 increased to a maximum level at 0.5 h after VC injection (Fig. 2H). Interestingly, the mRNA levels of AccDnaJC3 were either upregulated or downregulated under different pesticide treatments. As shown in Fig. 2I-K, the mRNA expression levels of AccDnaJC3 were upregulated to the maximum at 0.5 h, 1.5 h and 1 h, respectively, and then restored to the basic level, after treated with cyhalothrin,

3.10. Determination of enzyme activity after AccDnaJC3 silencing To further examine the antioxidant properties of AccDnaJC3 against 174

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Fig. 2. Expression profile of AccDnaJC3 under different environmental stress conditions. The environmental stresses were 4 °C (A), 24 °C (B), 44 °C (C), H2O2 (D), HgCl2 (E), CdCl2 (F), UV (G), VC (H), abamectin (I), emamectin benzoate (J), cyhalothrin (K) and paraquat (L). The β-actin gene was employed as an internal control. The data represent the means ± SE of three biological replicates. The letters above the columns represent significant differences (P < 0.01) on the basis of Duncan's multiple range tests.

a large protein family (Pulido and Leister, 2018). DnaJs are reported to participate in many cellular processes, including growth and development (Bekhochir et al., 2013), signal transduction (Rajan and D'Silva, 2009b), and various stress responses (Rajan and D'Silva, 2009b; Zhou et al., 2012). However, the functions of DnaJs in stress response have seldom been reported in A. cerana cerana. Here, we conducted a series of experiments to explore the responses of AccDnaJC3 to various stresses. Our study indicated that AccDnaJC3 might be involved in various stress responses and is likely to play an important role in

oxidative stress, we determined its possible effects on enzyme activity, including those of SOD, POD and CAT. As shown in Fig. 6, the enzyme activities of SOD, POD and CAT were reduced in bees with AccDnaJC3knockdown compared to those of bees in the control groups, whereas the enzyme activities in the control groups showed little change. 4. Discussion DnaJs exist in prokaryotes and eukaryotes and are considered to be 175

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stress can induce heat shock gene expression (Ahn and Thiele, 2003). In addition, pesticides are common environmental pollutants that can lead to impaired cell function and threaten the lives of bees (Narendra et al., 2007). Previous studies have shown that HSP is induced by pesticides (Lu et al., 2017; Chen and Zhang, 2015), which is consistent with our results showing that abamectin, emamectin benzoate and cyhalothrin induce AccDnaJC3 expression (Fig. 2H-K). Our results showed that the expression levels of AccDnaJC3 were upregulated by various stresses (Fig. 2A-E, G-K), which suggests that this upregulation was the result of generalized stress. Thus, we speculate that the protective machinery is rapidly activated, and DnaJs may need to be induced to avoid protein aggregation and inappropriate folding of proteins in response to different stresses. Moreover, the data showed that the mRNA levels of AccDnaJC3 were reduced under CdCl2 and paraquat treatments (Fig. 2F, L). We speculated that low-dose treatments are less toxic to bees, leading to DnaJs being recruited to protect cells from toxicity at the initial stages of stress. These data collectively demonstrate that AccDnaJC3 may be involved in a variety of stress responses in A. cerana cerana. Additionally, we used a disc diffusion assay to explore the function of AccDnaJC3 under different adverse conditions by overexpressing the gene in E. coli. Previous studies have proven that overexpression of DnaJ can reduce ROS accumulation and maintain cell homeostasis under different environmental threats (Rampuria et al., 2018). In addition, H2O2, HgCl2 and paraquat treatments can induce increases in intracellular ROS (Colle et al., 2018; Li et al., 2018). Our results show that, compared with cells carrying the pET-30a (+) empty vector, cells including recombinant AccDnaJC3 have increased tolerance against cumene hydroperoxide and HgCl2 (Fig. 4A, B). In addition, although the transcription of AccDnaJC3 was downregulated (Fig. 2L), smaller killing zones appeared around paraquat-soaked filters on cells including recombinant AccDnaJC3 than on cells carrying the pET-30a (+) empty vector (Fig. 4C), which shows that AccDnaJC3 enhances the resistance of bacteria cells to paraquat stress. To clarify this result, low-dose treatments lead to downregulation of the expression level of AccDnaJC3 potentially due to the recruitment of DnaJs for protecting cells against toxicity. However, high-dose treatments resulted in the induction of AccDnaJC3 expression to produce more DnaJ protein for cellular protection. Studies have shown that heterologous expression of the DnaJ protein AdDjSKI enhances the stress tolerance of E. coli (Rampuria et al., 2018), which is consistent with our results. We inferred that AccDnaJC3 appears to protect proteins and reduce the ROS levels in cells presumably by aiding the folding of ROS-scavenging enzymes, which are considered to be essential for reducing the overproduction of ROS under stress conditions. Therefore, these findings further prove that the AccDnaJC3 protein can ensure the normal function of cells by reducing their ROS levels under different stress conditions. RNAi technology is a powerful reverse genetic tool, which can help researchers use functional genetics to study gene function in insects without genetically modified resources (Scott et al., 2013), and RNAi technology has been applied in bees. Previous studies have shown that molecular chaperones play established posttranslational roles in protein folding and degradation pathways (Csermely et al., 1998). However, a few studies have suggested that the roles of molecular chaperones have extended to transcriptional regulation (Floer et al., 2008). Previous studies have reported that genes including AccCAT, AccSOD1, AccSOD2, AccTpx1, AccTpx3, AccTpx5, AccGrx2, AccGSTD and AccCYP4G11 are involved in different stress responses (Li et al., 2016b; Yan et al., 2013; Yao et al., 2014; Ming et al., 2016). In addition, enzymatic antioxidants such as SOD, POD, and CAT could serve antioxidant functions and protect cells from oxidative damage (Corona and Robinson, 2006). Here, we successfully knocked down AccDnaJC3 by RNAi (Fig. S5A). The subsequent analysis revealed that the expression levels of other antioxidant genes were reduced to some extent in the AccDnaJC3knockdown bees the compared with those in the controls (Fig. 5), and this effect was accompanied by reduced SOD, POD and CAT activities

Fig. 3. Western blot analysis of AccDnaJC3. Fifteen-day-old adult bees were treated with 44 °C (A), VC (B), and abamectin (C). Total protein was extracted from the bee samples at specific times, and specific anti-AccDnaJC3 antibodies were then used to detect the levels of AccDnaJC3 protein in bees. Tubulin was used as an internal control.

resisting a variety of environmental stresses in A. cerana cerana. cis-Acting elements are involved in gene expression regulation. To better understand the functions of AccDnaJC3, we predicted cis-acting elements in the 5′-flanking region of AccDnaJC3 (Fig. S2). CdxA is known to be involved in embryo and tissue development, and other cisacting elements, including p53, HSF, AP-1, CREB and XBP-1, have been linked to environmental threats (Zhang et al., 2014). In addition, the 5′flanking region also contained a binding site for Nrf2, which is thought to be a major switch that regulates the expression of genes involved in stress response (Zhang et al., 2014). The tissue-specific expression of genes may reveal some of their biological and physiological functions. Tissue-specific transcriptional analysis of AccDnaJC3 showed that the maximum expression of AccDnaJC3 was detected in muscle, followed by the epidermis (Fig. 1). Previous studies have shown that muscle is not present in bee larvae without wings but is found during pupation and metamorphosis, suggesting that AccDnaJC3 may participate in the differentiation of muscle tissue (Fernandez et al., 2012). Additionally, the epidermis acts as the exoskeleton, which provides physical stability and resists various adverse environments (Marionnet et al., 2003). These results all suggest that AccDnaJC3 might be involved in development and stress response, and further studies are needed to confirm the functions of AccDnaJC3 in stress response. The expression of stress response genes is one of the main molecular mechanisms to maintain cell homeostasis and protect cells from protein toxicity under a variety of adverse conditions (Silvia et al., 2016), and these stress response genes include molecular chaperones such as DnaJs. Temperature is one of the most common factors inflicting stress on organisms (Wojda, 2017). High temperature can cause oxidative stress and cell damage (Murata et al., 2011). As an HSP40, DnaJs have been demonstrated to be induced by heat and by cold (Silvia et al., 2016), and similar results were observed in our study. The exposure of bees to 4 °C, 24 °C and 44 °C upregulated the expression levels of AccDnaJC3 (Fig. 2A-C), which suggested the potential involvement of AccDnaJC3 in the protection of A. cerana cerana against temperature stresses. Heavy metals, H2O2 and UV can also cause the accumulation of ROS, resulting in oxidative stress (Emamverdian et al., 2015; Goldshmit et al., 2001; Cadet et al., 2005). VC is a typical antioxidant and can scavenge ROS. However, high-dose VC, acting as a pro-oxidant, can also cause oxidative damage and DNA lesions due to its ability to mediate the formation of H2O2 and decompose lipid hydroperoxide to endogenous genotoxins (Lee et al., 2001; Ma et al., 2014; Maria et al., 2017). Our data also revealed that the expression of AccDnaJC3 was induced by H2O2, HgCl2, UV and VC in A. cerana cerana (Fig. 2D, E, G and H), which is consistent with previous observations that oxidative

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Fig. 4. Disc diffusion assays of E. coli cells overexpressing AccDnaJC3. AccDnaJC3 was overexpressed in E. coli, and bacteria containing the pET-30a (+) empty vector were used as a control. Filter discs soaked with different concentrations of cumene hydroperoxide, HgCl2 and paraquat were placed on agar plates. (A), Cumene hydroperoxide concentrations of 0, 50, 100, 200, and 400 mM were tested. (B), The concentrations of HgCl2 included in the analysis were 0, 75, 100, 150, and 200 mM. (C), The paraquat concentrations tested were 0, 50, 100, 200, and 400 mM. The data presented are the means ± SE of three replicates.

and CAT were substantially reduced and that a large portion of the cellular tolerance to stresses was also decreased in DnaJ-deleted Beauveria bassiana (Wang et al., 2017). Therefore, the above results further indicate that AccDnaJC3 may play an essential role during the various stress responses of A. cerana cerana. In summary, we isolated an AccDnaJC3 from A. cerana cerana and studied its possible functions. The results revealed that AccDnaJC3 might be involved in response to various stresses and might play important roles in a variety of adverse environments. Our results provide basic knowledge of how AccDnaJC3 is involved in resisting adverse conditions in A. cerana cerana and may contribute to the functional study of DnaJC3s in other species.

(Fig. 6). Our results suggested that AccDnaJC3-knockdown might decrease the tolerance against various stresses by reducing the expression level of stress response genes and the activities of antioxidant enzymes in A. cerana cerana. Previous studies have shown that DnaJ-1 regulates transcription, and DnaJ-1, heat shock cognate 70–4 (Hsc70–4), BAG2 and myeloid leukemia factor (MLF) form a complex that regulates gene expression by modulating the stability of transcription factors (Dyer et al., 2016). We hypothesized that AccDnaJC3 silencing might lead to the inability to maintain the stability of transcription factors and transcription complexes that produce detox mRNAs, which results in the down-regulation of antioxidant gene expression and decreased synthesis of related antioxidant enzymes. In addition, AccDnaJC3 silencing might also lead to the inability to aid the folding of an enzyme to maintain its activity. However, further research is needed to identify the specific mechanism. Our results are supported by Wang's findings, which showed that the transcript levels and enzyme activities of SOD

Funding information This work was supported by the National Natural Science 177

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Fig. 5. Expression levels of other antioxidant genes after AccDnaJC3 knockdown in adult bees. Antioxidant genes included (A), AccCAT, (B), AccSOD1, (C), AccSOD2, (D), AccTpx1, (E), AccTpx3, (F), AccTpx5, (G), AccGrx2, (H), AccGSTD and (I), AccCYP4G11. The β-actin gene was used as the internal control. The data represent the means ± SE of three biological replicates. The letters above the columns represent significant differences (P < 0.01) on the basis of Duncan's multiple range tests.

Declaration of Competing Interest

Foundation of China (No. 31572470), Shandong Province Agricultural Fine Varieties Breeding Projects (2017LZN006) and the earmarked fund for the China Agriculture Research System (No. CARS-44), the Shandong Provincial Natural Science Foundation (ZR2017MC064).

The authors declare that they have no conflict of interest.

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Fig. 6. Enzyme activities of (A) SOD, (B) POD and (C) CAT after AccDnaJC3 knockdown. Water and dsRNA-GFP were injected into the control groups. The dsRNAGFP and dsRNA-AccDnaJC3 groups are abbreviated GFP and AccDnaJC3. The data represent the means ± SE of three biological replicates. The letters above the columns represent significant differences (P < 0.01) on the basis of Duncan's multiple range tests.

Appendix A. Supplementary data

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