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. 2019 Nov 26;25(1):119–131. doi: 10.1007/s12192-019-01053-4

Effects of abiotic stress on the expression of Hsp70 genes in Sogatella furcifera (Horváth)

Cao Zhou 1, Xi-bin Yang 1, Hong Yang 1,2,✉, Gui-yun Long 1, Zhao Wang 1,3, Dao-chao Jin 1
PMCID: PMC6985323  PMID: 31773487

Abstract

Sogatella furcifera (Horváth), a prominent rice pest in Asia, is a typical R-strategic and highly adaptable insect. Heat shock proteins (Hsps) are highly conserved molecular chaperones regulating responses to various abiotic stresses; however, limited information is available regarding their role in responding to abiotic stress in S. furcifera. This study aimed to investigate the effect of abiotic stresses on the expression of Hsp70 genes in the S. furcifera. Five Hsp70 genes were isolated from S. furcifera, and the expression patterns at different developmental stages and temperatures, upon treatment with different insecticides and ultraviolet A (UV-A) stress, were analyzed. Hsp70 genes were expressed at different developmental stages. Hsp70-2, Hsp70-5, and Hsp70-6 were significantly upregulated upon heat shock at 40 °C for 30 min. Hsp70-3 and Hsp70-4 were significantly upregulated upon heat shock at 30 °C for 30 min. Under UV-A stress, Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 were significantly upregulated. Conversely, Hsp70-2 was significantly downregulated under UV-A stress. The five Hsp70 genes were significantly downregulated in 3rd-instar nymphs on exposure to thiamethoxam, buprofezin, and avermectin at LC10 and LC25 concentrations. Hence, Hsp70 genes significantly contribute to the tolerance of S. furcifera to temperature and UV-A stress; however, they are not involved in the response to insecticides.

Electronic supplementary material

The online version of this article (10.1007/s12192-019-01053-4) contains supplementary material, which is available to authorized users.

Keywords: Sogatella furcifera, Temperature, Ultraviolet A radiation, Insecticides, Heat shock protein

Heat shock proteins (Hsps) serve as molecular chaperones in response to heat shock stress and can be rapidly synthesized to cope with various environmental stressors, including cold shock, insecticides, heavy metals, and diapause (Lu et al. 2017; Rinehart et al. 2007; Shu et al. 2011). Hsps can be divided into five families based on molecular weight and homology: Hsp90, Hsp70, Hsp60, Hsp40, and small Hsps (Feder and Hofmann 1999). The Hsp70 family is further divided into two subgroups based on their response patterns to various stimuli: stress-inducible (Hsp70) and constitutively expressed (Hsc70) proteins (Cheng et al. 2016; Shim and Lee 2015; Sun et al. 2016).

Recent studies have reported that Hsps are induced by various stresses including heat shock, cold shock, hypoxia, dryness, starvation, heavy metal exposure, and insecticide exposure (King and MacRae 2015; Koo et al. 2015; Lu et al. 2016b; Sun et al. 2016; Tang et al. 2015; Tungjitwitayakul et al. 2016; Yang et al. 2016). Hsp induction would help insects adapt to high-temperature environments owing to global warming. Hsp70 overexpression in numerous insects including Laodelphax striatellus (Wang et al. 2017), Nilaparvata lugens (Lu et al. 2017), and Rhopalosiphum padi (Li et al. 2017) leads to enhanced heat tolerance. In addition to environmental temperature, insecticide stress is another important response factor among insects. Sublethal concentrations of beta-cypermethrin (LC10 and LC30) can upregulate RpHsp70-1 and RpHsp70-2 in R. padi (Li et al. 2017). Cydia pomonella was exposed to acetamiprid, methomyl, carbaryl, and cypermethrin for 36 h, and CpHSP70-2 expression was induced (Yang et al. 2016). Furthermore, stress tolerance induced by Hsp70s in insects varies depending on the stage of insect development and the type of stress (Sharma et al. 2007).

Sogatella furcifera (Horváth) is a prominent rice pest in Asia. Adult S. furcifera and nymphs can puncture the phloem of the rice plant, thus absorbing sap and causing serious damage to rice plants; simultaneously, S. furcifera propagates the southern rice black-streaked dwarf virus (Zhou et al. 2008), causing severe agricultural losses. In rice fields, S. furcifera are often affected by various abiotic stresses, including temperature, insecticides, and ultraviolet (UV) radiation. Insect growth, development, survival, and geographic distribution are altered by environmental factors (high temperature/low temperature, insecticides, and UV radiation) (Bale et al. 2002; Wang et al. 2014). Similar to most insects, S. furcifera is extremely resistant and adaptable to these abiotic stresses (Kisimoto 1976; Mu et al. 2016). However, limited information is available regarding the mechanism underlying the adaptation to abiotic stresses in S. furcifera. In this study, we cloned full-length complementary DNA (cDNA) of five Hsp70 genes of S. furcifera. We assessed the expression of these five genes at different developmental stages and at different temperatures, and their role in the response to insecticide stress in S. furcifera was analyzed. Furthermore, their expression profiles were assessed under UV stress. Our findings may further the current understanding of the molecular mechanism underlying the adaptation to abiotic stresses in S. furcifera.

Materials and methods

Insects and insecticides

As described previously (Zhou et al. 2019), S. furcifera were collected from a rice field in Huaxi, Guiyang, Guizhou, China, in 2013 and maintained in the laboratory on rice seedlings at 25 ± 1 °C and 70 ± 10% relative humidity with a 16:8 h (L:D) photoperiod without insecticides. Third-instar nymphs were used in the study. Thiamethoxam (96%, technical formulation) was obtained from PFchem Co., Ltd. (Nanjing, China); abamectin (96.4%, technical formulation) was obtained from Shandong Qilu King-Phar Pharmaceutical Co., Ltd. (Shandong, China); and buprofezin (97%, technical formulation) was obtained from Guangxi Pingle Pesticide Factory (Guangxi, China).

Cloning of Hsp70 genes

Using the Hsp70 gene annotated in the transcriptome as a query sequence (Zhou et al. 2018), Geneious 9.0 (Kearse et al. 2012) software was used to assemble the transcriptome database of S. furcifera, using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA) software–designed gene-specific primers (Table S1) to verify the sequence assembled via RT-PCR. Total RNA was extracted from ten 5th-instar nymphs with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using Thermo Scientific NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The total cDNA was synthesized using the PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, China) in accordance with the manufacturer’s instructions.

Sequence and phylogenetic analysis

The gene and protein sequences of all five Hsp70 members were subjected to a homology search using NCBI-BLAST (http://www.ncbi.nih.gov/BLAST/). The deduced protein sequences were analyzed using DNAMAN software (version 6.0; Lynnon Biosoft, Quebec, Canada). ORFs were identified using the ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Molecular weights and theoretical isoelectric points were calculated using the SWISS-PROT (ExPASy server) tool “Compute pI / Mw” (http://au.expasy.org/tools/pi_tool.html). Conserved domains were analyzed using PFAM (http://pfam.xfam.org). Thereafter, phylogenetic analysis was carried out using Hsp70 gene sequences, using the neighbor-joining (NJ) method with 1000 bootstrap replicates, using the MEGA program package, v. 6.0 (Tamura et al. 2013).

Collection of samples at different developmental stages

To analyze the expression pattern of Hsp70 genes, we harvested eggs, 1st instar, 2nd instar, 3rd instar, 4th-instar first day, 4th-instar second day, 5th-instar first day, 5th-instar second day, 5th-instar third day, 5th-instar fourth day, and female insects emerging at 12 h, 1 day, 2 days, 3 days, 4 days, and 5 days, amounting to 16 developmental stages in total and subjected to qRT-PCR analysis. Each sample was biologically repeated three times.

Insecticide stress treatment

The rice stem dipping method (Zhou et al. 2017) was used in the study. One-hundred 3rd-instar nymphs were transferred to and reared separately in glass tubes (300 mm high × 30 mm diameter) containing rice seedlings dipped in a sublethal concentration (LC10 and LC25) of thiamethoxam, abamectin, and buprofezin, and rice stems treated with distilled water constituted the control. The treated insects were maintained at 25 ± 1 °C and 70 ± 10% relative humidity with a 16:8 h (L:D) photoperiod in an artificial climate box. After 48 h, 15 surviving insects were randomly selected for qRT-PCR analysis. Each sample was biologically repeated three times. The LC10 and LC25 values (Table S2) of thiamethoxam, abamectin, and buprofezin against S. furcifera were in accordance with previously reported values (Liu et al. 2016).

Temperature stress treatment

One hundred 3rd-instar nymphs were placed in a 10-ml plastic centrifuge tube and reared in an artificial climate box at 10 °C, 20 °C, 25 °C, 30 °C, and 40 °C. Fifteen nymphs were harvested for qRT-PCR analysis at 0 min, 10 min, 30 min, and 60 min, and each treatment was administered in triplicate. Each sample was biologically repeated three times.

Induction of UV-A stress

One-hundred 3rd instar nymphs were placed in a 10-ml plastic centrifuge tube. After dark adaptation for 2 h, they were irradiated with a UV-A ultraviolet lamp (model 58B-01; wavelength 315–400 nm; Nanjing Huaqiang Electronics Co., Ltd., Nanjing, China) with a radiation intensity of 300 μW/cm2 (Huang and Toledo 1982). Fifteen nymphs were harvested for RT-qPCR analysis at 0 min (control group CK), 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, and 120 min. Each sample was biologically repeated three times.

qRT-PCR analysis

As reported previously (Zhou et al. 2019), the Hsp70 messenger RNA (mRNA) levels under different treatment conditions were measured via qRT-PCR, using FastStart Essential DNA Green Master (Indianapolis, IN, USA) in a CFX96™ real-time quantitative PCR system (Bio-Rad, Hercules, CA, USA). The ribosomal protein L9 (RPL9) (GenBank accession number: KM885285) was considered the internal control. Specific primer pairs for each gene were designed using Primer Premier 6.0 (Premier Biosoft International, Palo Alto, CA, USA; Table S1). Each 20-μl reaction mixture comprised 1 μl of sample cDNA, 1 μl of each primer (10 μM), 7 μl of DEPC H2O, and 10 μl of FastStart Essential DNA Green Master. The qPCR cycling parameters were as follows: 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 30 s, and melting curve generation was carried out from 65 to 95 °C. To assess reproducibility, qPCR for each sample was performed in three technical and three biological replicates. The comparative 2−ΔΔCT method (Livak and Schmittgen 2001) was used to normalize relative mRNA expression levels.

Statistical analysis

Statistical analyses were performed using SPSS (version 17.0) (SPSS, Chicago, IL, USA). All data are presented as mean ± standard error (SE) values for relative expression levels. Differences among relative expression levels of each target gene were analyzed using one-way nested analysis of variance (ANOVA) and Bonferroni test (P < 0.05 indicating statistical significance). Furthermore, a heatmap figure was generated using the pheatmap (Kolde 2012) R packages (v.1.0.8).

Results

Cloning and characterization of Hsp70 genes

Using a previously described method (Zhou et al. 2019), five Hsp70 genes were identified in accordance with the transcriptome data of the S. furcifera and further verified via RT-PCR (Table 1), and their designations, accession numbers, lengths, ORF sizes, theoretical isoelectric points, and molecular weights are summarized in Table 1. Structural analysis revealed three characteristic domains in all five Hsp70 proteins (Figs. 1, 2, and 3). Meanwhile, the conserved C-terminal motif EEVD of cytoplasmic Hsp70 genes was observed. N-terminal sequences were more conserved than C-terminal sequences on the alignment of the Hsp70-2, Hsp70-3, and Hsp70-5 (Fig. 1). The C-terminus of Hsp70-4 has a conserved endoplasmic reticulum motif “HDEL” (Fig. 2), while Hsp70-2, Hsp70-3, and Hsp70-5 have a conserved cytoplasmic motif “EEVD” (Fig. 1).

Table 1.

Full-length Hsp70 genes identified from Sogatella furcifera

Gene name Accession number Product size (bp) Number of coded amino acids (aa) Molecular weight Theoretical isoelectric point
Hsp70-2 MK168341 1941 646 70,940.21 5.48
Hsp70-3 MN164442 1971 656 71,591.87 5.46
Hsp70-4 MN164443 1968 655 72,874.66 5.27
Hsp70-5 MN164444 1884 627 68,725.93 5.84
Hsp70-6 MN164445 2073 690 74,968.14 5.76
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Fig. 1.

Fig. 1

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Alignment of Hsp70-2, Hsp70-3, and Hsp70-5 sequences of Sogatella furcifera. Hsp70 family signature motifs are denoted by a red box. The green box represents the C-terminal motif EEVD

Fig. 2.

Fig. 2

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Nucleotide sequence of Hsp70-4 and the deduced sequence of Hsp70-4 of Sogatella furcifera. Hsp70 family signature motifs are highlighted in red. The red dot represents the C-terminal motif KDEL

Fig. 3.

Fig. 3

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Nucleotide sequence of Hsp70-6 and the deduced sequence of Hsp70-6 from Sogatella furcifera. Hsp70 family signature motifs are highlighted in red

Phylogenetic analysis of Hsp70 genes

Five Hsp70 sequences of S. furcifera were subjected to a homology search with the selected species indicated in Fig. 4, and a phylogenetic tree was constructed using MEGA 6.0 software with the NJ method to analyze evolutionary relationships among the five Hsp70 proteins of S. furcifera and those of other species. As shown in Fig. 4, the five Hsp70 sequences were divided into three clusters: Hsp70 in the cytosol, endoplasmic reticulum (ER), and mitochondria. The cytosol cluster was divided into two branches: inducible Hsp70 (comprising Hsp70-2 and Hsp70-5) and Hsc70 (comprising Hsp70-3) (Fig. 4). Hsp70-4 and Hsp70-6 were clustered in endoplasmic reticulum and mitochondrial groups, respectively.

Fig. 4.

Fig. 4

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Phylogenetic tree constructed on the basis of the alignment of Hsp70s from insects. Sequences were downloaded from the GenBank protein database. Sequence labels are indicated by the species name and GenBank accession number. The asterisk denotes the Hsp70 sequence of Sogatella furcifera

Expression of Hsp70 genes at different developmental stages

The mRNA expression levels of five Hsp70 genes in S. furcifera at different developmental stages are shown in Fig. 5 (Table S3). Hsp70 was expressed at all developmental stages. Hsp70-2 mRNA levels peaked during the 1st-instar nymphal stage (P < 0.05) and gradually decreased, being the lowest at the 5th-instar third-day stage. Hsp70-2 mRNA expression levels were significantly higher at post-emergence than in the 5th-instar nymph (P < 0.05), peaking on day 1. Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 mRNA expression levels peaked at the 5th-instar second-day stage. Hsp70-3, Hsp70-4, and Hsp70-6 mRNA expression levels peaked on third-day post-emergence, suggesting that they potentially play similar roles in the adults and nymphal stages in S. furcifera. Among all developmental stages, Hsp70-2 mRNA expression levels peaked in the 1st-instar nymph; Hsp70-3 expression levels peaked on the third day of female emergence; Hsp70-4, Hsp70-5, and Hsp70-6 expression levels peaked in the 5th-instar second-day nymphs; these results suggest that these Hsp70 genes play different roles in different developmental stages of S. furcifera.

Fig. 5.

Fig. 5

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Expression of Hsp70 genes at different developmental stages of Sogatella furcifera. Heatmaps were generated using the pheatmap package. 1st: 1st instar, 2nd: 2nd instar, 3rd: 3rd instar, 4th-1d: 4th instar first day, 4th-2d: 4th instar second day, 5th-1d: 5th instar first day, 5th-2d: 5th instar second day, 5th-3d: 5th instar third day, 5th-4d: 5th instar fourth day, Fa-12h: female emergence at 12 h, Fa-1d: female emergence at 1 day, Fa-2d: female emergence at 2 days, Fa-3d: female emergence at 3 days, Fa-4d: female emergence at 4 days, Fa-5d: female emergence at 5 days

Expression of Hsp70 genes under temperature stress

Expression levels of the five Hsp70 genes under temperature stress are shown in Fig. 6 (Table S4). Under high-temperature stress, the five Hsp70 genes were significantly upregulated. Hsp70-2 expression levels were significantly higher at 40 °C than at other temperatures (P < 0.05) and increased significantly with prolonged stress (P < 0.05). However, Hsp70-2 expression levels at 30 °C were not significantly different when compared with the control (at 25 °C). Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 expression levels peaked at 30 min, 30 min, 60 min, and 10 min of stress at 30 °C, respectively. However, Hsp70-4 was significantly downregulated (P < 0.05) at 40 °C in comparison with the control (at 25 °C). Under low-temperature stress, only Hsp70-3 and Hsp70-6 were significantly upregulated (P < 0.05) at 20 °C for 10 min and 30 min, respectively. In contrast, Hsp70-5 was significantly downregulated under low-temperature stress in comparison with the control (at 25 °C).

Fig. 6.

Fig. 6

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Effect of temperature stress on Hsp70 gene expression levels in Sogatella furcifera. Heatmaps were generated using the pheatmap package

Expression of Hsp70 genes under insecticide stress

The 3rd-instar nymphs of S. furcifera were exposed to insecticide stress with thiamethoxam, buprofezin, and abamectin at LC10 and LC25 for 48 h, and the expression levels of the five Hsp70 genes are shown in Fig. 7. Hsp70-2, Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 were significantly downregulated (P < 0.05) under pesticide stress, suggesting that Hsp70-2, Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 could not respond to insecticide stress at LC10 and LC25.

Fig. 7.

Fig. 7

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Effect of insecticide stress on the expression levels of Hsp70 genes in Sogatella furcifera. Mean ± SE values determined using the 2−ΔΔCt method are used to denote relative expression levels under different insecticide concentrations, with non-insecticide treatment (CK) as a reference. Different letters indicate significant differences among treatments for the same duration. a Thiamethoxam. b Buprofezin. c Abamectin

Expression of Hsp70 genes under UV-A stress

The expression levels of the five Hsp70 genes of S. furcifera were quantified via qPCR after UV-A irradiation of 3rd-instar nymphs. The results are shown in Fig. 8 (Table S5). Compared with the control (0 min), Hsp70-3, Hsp70-4, and Hsp70-5 expression levels peaked at 90 min of UV-A irradiation, while Hsp70-6 expression levels peaked at 60 min of UV-A irradiation. On the contrary, UV-A irradiation significantly downregulated (P < 0.05) Hsp70-2 expression levels. After UV-A irradiation, Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 displayed a trend of gradual upregulation, followed by downregulation.

Fig. 8.

Fig. 8

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Effect of UV-A stress on the expression levels of Hsp70 genes in Sogatella furcifera. Heatmaps were generated using the pheatmap package

Discussion

Insects have evolved strategies to adapt to adverse environmental factors, including extreme temperatures and insecticides. Hsps are highly conserved stress response proteins in insects (Feder and Hofmann 1999). In the present study, five Hsp70 genes were identified, and a comparison and analysis of their protein sequences revealed three signature sequences of the Hsp70 family. Our results indicate that members of the Hsp70 family are highly conserved and that their C-terminal regions are generally more distinct than their N-terminal regions. Variations in the C-terminal sequence can determine the functional specificity of individual Hsps (Luo et al. 2015). The conserved C-terminal “EEVD” motif indicates that Hsp70-2, Hsp70-3, and Hsp70-5 are cytoplasmic homologs, and the C-terminus of Hsp70-4 contains a conserved endoplasmic reticulum motif “HDEL” (Maheswar Rao et al. 2010). Phylogenetic analysis revealed that Hsp70-6 is a mitochondrial protein. However, the mitochondrial “PEAEYEEAKK” motif was not detected in Hsp70-6 (Maheswar Rao et al. 2010). In our study, only Hsp70-3 contained a GGMP tetrapeptide (Fu et al. 2010). The function of the GGMP tetrapeptide repeat at the C-terminus of Hsp70 is currently unclear. Several researchers have suggested that whether the C-terminal of the Hsp70 family gene contains a GGMP tetrapeptide repeat is a marker for distinguishing between stress-inducible (Hsp70) and constitutively expressed proteins (Hsc70) (Wu et al. 2001; Piano et al. 2005). The results of this study are consistent with this hypothesis and further demonstrate this view. The similarity between Hsp70 and its counterparts in other insect species suggests that Hsp70 responds to environmental stress in the Hsp chaperone system.

Hsps are developmentally regulated in some insects (Sharma et al. 2007). In this study, Hsp70-2 expression levels peaked in the first-instar nymph and gradually decreased until the 5th-instar stage, concurrent with N. lugens homologs (Lu et al. 2017). Furthermore, in Tribolium castaneum, Hsp70III was significantly upregulated in young larvae compared to that in old larvae (Mahroof et al. 2005). In contrast, Hsp70-4, Hsp70-5, and Hsp70-6 expression levels peaked at the nymphal stage on the 5th-instar second day, concurrent with CpHSP70-2 expression levels in Cydia pomonella, in 5th-instar larvae (Yang et al. 2016). Our results show that Hsp70-3 was gradually upregulated during the first-instar nymphal stage to adulthood and peaked on the third day of female emergence, concurrent with NlHsc70 in N. lugens, which was most upregulated in female insects (Lu et al. 2016a). The difference in Hsp70 expression patterns at different developmental stages suggests that they potentially play different roles in protection against environmental stress at different developmental stages.

Climatic factors, particularly temperature, potentially influence the distribution and abundance of insects (Zhang and Denlinger 2010). Hsp overexpression in response to heat stress is directly associated with the acquisition of heat tolerance in insects, although these reactions are different in different insects. In this study, five Hsp70 genes of S. furcifera were significantly upregulated under high- and low-temperature stress compared with the control, although the induction time and temperature were different. In particular, Hsp70-2 expression levels peaked at a 40 °C temperature stress for 60 min, its expression levels being approximately 320-fold that of the control group, indicating that Hsp70-2 plays an important role in adapting to high-temperature stress in S. furcifera. Similarly, RpHsp70-1 and RpHsp70-2 expression levels of apterous adult aphids of R. padi were significantly higher than those of the control (Li et al. 2017) at 36 °C. A study on Corythucha ciliata revealed that CcHSP70 expression levels increased gradually with an increase in temperature and peaked at 41 °C (Ju et al. 2018). Similar results were obtained with Leptinotarsa decemlineata upon heat treatment (Dumas et al. 2019). In addition, the transcriptome analyses of three rice planthoppers found that a high temperature can induce the expression of the heat shock protein 70 gene (Huang et al. 2017). These results further confirmed that heat stress can induce Hsp70 gene expression and helps insects adapt to temperature stress. Furthermore, Hsp70-3 and Hsp70-6 were significantly upregulated at 20 °C for 10 min and 30 min, respectively, in comparison with the control (25 °C). However, under temperature stress at 10 °C, all genes were inhibited. Similar results were obtained with L. decemlineata. Similar results were found in the L. decemlineata study. Compared to the control (maintained at 15 °C), when L. decemlineata were exposed to 5 °C, the expression level of the Hsp70 gene was significantly induced; however, when L. decemlineata were exposed to − 5 °C, the expression level of the Hsp70 gene was significantly inhibited (Dumas et al. 2019). A study of Apolygus lucorum found that the expression of the Alhsc70 gene was significantly reduced by the 4 °C treatment when compared with the control (Sun et al. 2016). However, Hsp70 induction has not been reported in Drosophila melanogaster (Nielsen et al. 2005). Nonetheless, exposure to a low temperature significantly induces Hsp70 gene expression in other insects (Paim et al. 2016; Hu et al. 2018). These studies indicate that Hsp70 plays different roles in the adaptation to low- and high-temperature environments.

As molecular chaperones, Hsps are potentially induced upon insecticide treatment and contribute to insecticide resistance (Yoshimi et al. 2002), although these reactions differ among insect species. For example, the treatment of R. padi with beta-cypermethrin at LC30 significantly upregulated RpHsp70-1 and RpHsp70-2 after 24 h and 36 h (Li et al. 2017). Treatment of Apolygus lucorum with cyhalothrin at LD50 significantly upregulated AlHSC70; however, chlorpyrifos significantly downregulated AlHSC70 (Sun et al. 2016). Furthermore, in N. lugens, exposure to imidacloprid (0.02 ng and 0.04 ng per female) significantly upregulated N1Hsp70; however, this gene was significantly downregulated with an increase in the treatment concentration (Lu et al. 2017). Moreover, a study on L. decemlineata reported no significant effect on Hsp70 expression levels upon imidacloprid and chlorantraniliprole treatment (Dumas et al. 2019). Our results show that thiamethoxam, buprofezin, and abamectin treatment at LC10 and LC25 significantly downregulated the five Hsp70 genes after 48 h in S. furcifera nymphs. These results confirm that Hsp70 genes have different functions in response to insecticides in different insects, and their response levels are different for different insecticides. In a study of R. padi, it was found that after treatment of apterous aphids with beta-cypermethrin LC10 and LC30, the expression of RpHsp70-1 and RpHsp70-2 first showed inhibition, then was significantly upregulated and, finally, gradually returned to a normal level (Li et al. 2017). In a study of N. lugens, it was also found that the expression level of N1Hsp70 was first induced then returned to a normal level after each newly emerged female adult was treated with 0.04 ng per female imidacloprid (Lu et al. 2017). There was a significant time effect. In this study, we found that the expression levels of five SfHsp70 genes were significantly inhibited after 48 h of insecticide stress. The reason for this result may be due to the time effect, and its specific reasons require further study.

For herbivorous insects directly interacting with plants, sunlight is an important source of abiotic stress (Zhao and Jones 2012). UV-A radiation constitutes the long-wavelength band of ultraviolet radiation, which causes harmful effects through the generation of reactive oxygen species (Schauen et al. 2007). This study shows that UV-A radiation can upregulate Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6, concurrent with previous reports on Drosophila melanogaster, showing that UV-A radiation upregulates DmHsp70 (Wang et al. 2014). Hsp70-3, Hsp70-4, Hsp70-5, and Hsp70-6 expression levels displayed a decreasing trend with a prolonged duration of UV-A irradiation. A similar phenomenon has been reported in T. castaneum and D. melanogaster (Sang et al. 2012; Wang et al. 2014). The reduction in Hsp70 transcription levels may have occurred because insects are exposed to UV radiation for a long period, and UV-induced cell damage exceeds the maximum tolerance restored by the upregulation of Hsp70 genes (Kim et al. 2011). Hsp70 is detrimental to normal cell growth in Drosophila (Feder et al. 1992), requiring a specific negative feedback regulatory mechanism for Hsp70 to balance normal cellular metabolism (Li and Duncan 1995). Hsp70 expression levels first increase and then decrease after prolonged exposure to UV-A radiation, indicating that it may be regulated by a negative feedback regulation mechanism to prevent the accumulation of harmful substances affecting normal cell growth. Specific reasons warrant further investigation.

In this study, we identified five Hsp70 genes in S. furcifera and quantified their expression levels at different developmental stages, different temperature stress, insecticide stress, and UV-A stress. Our results show that the five Hsp70 genes were expressed at different levels at different developmental stages, different temperatures, and UV-A radiation, indicating that different Hsp70 genes may function differently in responding to abiotic stress in S. furcifera. However, our results also show that under stress conditions with three insecticides, the five Hsp70 genes were significantly downregulated, probably because the Hsp70 genes assessed herein are only some of the Hsp70 genes in S. furcifera. Therefore, future studies are required to comprehensively assess Hsp70 family genes through insect genomic data to further the current understanding of the role of Hsp70 in insect adaptation to environmental factors.

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Funding information

This study was supported by the National Natural Science Foundation of China (Grant No. 31560522 and 31960537), Provincial Key Project for Agricultural Science and Technology of Guizhou (NY20133006 and NY20103064), International Cooperation Base for Insect Evolutionary Biology and Pest Control ([2016]5802), and Graduate Education Innovation Project of Guizhou Province (Qian Jiao He YJSCXJH, No. [2018]043).

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