Differential regulation of antioxidant enzymes in Frankliniella occidentalis (Thysanoptera: Thripidae) exposed to thermal stress

Insects and temperature treatments

Frankliniella occidentalis populations were collected in Hangzhou, China, in 2008 and were reared with kidney bean, Phaseolus vulgaris Linn, at 25 ± 0.5 °C and 70 ± 5% relative humidity with a 16:8 h light:dark photoperiod as outlined by Li et al. (2011b). Newly emerged 2^nd instar larvae were collected, and pools of 100 were exposed to high (31, 33, 35, 37, 39 or 41 °C) or low (0, –2, –4, –6, –8 and –10 °C) temperatures for 1 h in glass tubes as described (Chang et al., 2017). Through the results of the pre-experiment, 35 and –4 °C were decided as the model temperature on F. occidentalis, which was further explored by subjecting groups of individuals to 0, 0.5, 1, and 2 h of thermal stress; controls were maintained at 26 °C (0 h time point). Following thermal stress, larvae were incubated at 26 °C for 30 min and used a brush to touch it gently, thrips would be identified as surviving if it respond to the stimulus. Survivors were frozen in liquid nitrogen and stored at –80 °C for future use. Four replicate pools were used for each temperature and time period.

Determination of enzyme activity

The assay kit used for protein extraction was from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. Treated samples were homogenized in 0.9% saline and then centrifuged at 2,500 × rpm for 10 min (Jia et al., 2011). Supernatants containing the enzyme fractions were collected, and protein content was determined using the Bradford (1976) method.

POD and SOD activities were assessed with commercially available kits (Qin et al., 2017). Absorbance values were obtained using the BioTek PowerWave HT Microplate Spectrophotometer (Bio-Tek Instruments Inc., Winooski, Vt., USA). GST activity was measured as a function of reduced glutathione (GSH) using 10 mg of cytosolic protein and 1-chloro-2,4-dinitrobenzene (CDNB; Shanghai Chem, Shanghai, China) as a substrate (Habig, Pabst & Jakoby, 1974; Attig et al., 2014). GST activity was determined at A₃₄₀ with a microplate spectrophotometer (Shanghai Xinmao Instrument, Shanghai, China), and results are shown as μmol GSH-CDNB/min/mg protein.

RNA isolation, partial cloning of SOD, POD and GST1, and qRT-PCR

The SV Total RNA Isolation System was used to isolate RNA from F. occidentalis as recommended by the manufacturer (Promega, San Luis Obispo, CA, USA). RNA quality and concentration were determined, and cDNA was generated from total RNA with the First Strand cDNA Synthesis Kit (Clontech, Mountain View, CA, USA) as outlined previously (Zhang et al., 2019).

Transcriptome sequencing was performed on F. occidentalis exposed to low temperature (–13 °C), high temperature (40 °C) and normal temperature control (26 °C) at the Shanghai Biotechnology corporation by Illumina sequencing platform. The RNA-seq data were deposited with the Sequence Read Archives PRJNA73493 at NCBI. To remove adapter contamination, low-quality bases and bases artificially introduced during library construction, we trimmed all raw reads using Trimmomatic 0.32 (http://www.usadellab.org/cms/index.php?page=trimmomatic) before transcript assembly, while the unpaired reads were discarded (Bolger, Lohse & Usadel, 2014). The clean reads were mapped to the sequences in the rRNA database of all published insects downloaded from NCBI to discard rRNAs using SOAP. Only the clean reads with the standard of Q30 > 85% and processed with Trimmomatic 0.32 and SOAP were used for further analysis (Gu et al., 2019). Clean data were assembled with Trinity to obtain a high-quality unigene library (Grabherr et al., 2011). The unigene library of three species were first assembled to obtain individual UniGene databases; the general UniGene library was obtained by clustering the three individual databases through CD-Hit to facilitate comparison of expression patterns (Fu et al., 2012). The transcripts selected in the clustering united as unigenes using the De Bruijn graph algorithm; CD-Hit was used to reduce sequence redundancy and improve the performance of other sequence analyses (Fu et al., 2012; Yang & Smith, 2013). For functional annotation, we obtained information on unigenes using BLAST (cut-off e-value of 10–5) with protein databases such as NR (NCBI nonredundant database), Swiss-Prot, GO (Gene Ontology), COG (Clusters of Orthologous Groups), KOG (euKaryotic Orthologous Groups), eggNOG, Pfam (Protein family) and KEGG (Kyoto Encyclopedia of Genes and Genomes). “Superoxide Dismutase”, “Peroxidase” and “glutathione-s-transferase” were used as key words to search related gene fragments in the transcriptome database of F. occidentalis, respectively. The fragment gene cDNA of the SOD, GST1 and POD was submitted to GenBank (accession no. MZ364120, MZ364118 and MZ364119, respectively). According to the obtained gene fragments, the corresponding primers (Table 1) were designed for fragment verification. PCR products were cloned and sequenced as described (Zhang et al., 2019).

Quantitative real-time reverse transcriptase PCR (qRT-PCR) was conducted using the protocols described by Zhang et al. (2019). Specific primers (Table 1) were designed according to the above verified fragments for qRT-PCR. Melting curve analysis was executed to analyze the specificity of PCR products. According to the evaluation results of Zheng et al. (2014) on the reliability of reference genes in F. occidentalis, expression levels were normalized using reference genes GAPDH, RPL32 and EF-1, 18S for high and low temperature stress, respectively.

Statistical analyses

qRT-PCR data analyzing was conducted in Bio-Rad CFX Manager 3.1 software. The average Ct values of biological replicates were used to calculate the relative expression levels. The results of qPCR were analyzed with the 2^–ΔΔCt method (Livak & Schmittgen, 2001). Firstly, for all test samples and calibration samples, the Ct value of the housekeeping gene were used to normalize the Ct value of the target gene. Normalized results were ΔCt(test) and ΔCt (calibrator), respectively. And using ΔCt (calibrator) to normalize ΔCt (test), ΔΔCt was obtained. The ratio of expression level was calculated by 2^–ΔΔCt. The lg(X) method was used to transform the expression level data for normality and homogeneity of variance. Significant differences were detected by one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons test. Data were analyzed with SPSS v. 16.0 and considered significant at P < 0.05.

Effect of high temperature stress on antioxidant activity

SOD activity increased with rising temperature from 31 to 37 °C and was highest at 37 °C. The activity of SOD activity began to decline at 39 °C, and the level at 41 °C was significantly lower than 37 °C (F_6,19 = 4.245, P < 0.05) (Fig. 1A). A similar pattern was observed with POD, where activity rose with increasing temperature, peaked at 35 °C and was significantly lower at 41 °C than 35 °C (F_6,21 = 7.089, P < 0.05) (Fig. 1B). GST activity was highest at 35 °C (Fig. 1C) and began to decline with increasing temperature (F_6,21 = 8.312, P < 0.05).

Temporal changes in antioxidant enzyme activity at 35 °C

Antioxidant enzyme activity was significantly higher than the control (0 h, 26 °C) when insects were exposed to 35 °C for 0.5, 1 and 2 (SOD: F_3,10 = 10.005, P < 0.05; POD: F_3,12 = 8.037, P < 0.05; GSTs: F_3,10 = 5.815, P < 0.05). No significant differences in antioxidant activity were detected between 0.5, 1 and 2 h of exposure (Fig. 2).

Expression of antioxidant genes in response to heat and cold stress

The expression of antioxidant genes was evaluated at 31, 33, 35, 37, 39 and 41 °C; 26 °C served as a control. SOD expression showed significant decreases in expression at 35–37 °C; however, expression peaked at 39 °C and was comparable to the control (26 °C) (Fig. 3A). With the exception of 35–37 °C, SOD expression was not significantly changed by high temperatures (F_6,18 = 29.203, P < 0.05). In contrast, POD expression levels at 33, 37 and 39 °C were significantly higher than the control at 26 °C; however, except that the expression level was significantly decreased at 35 °C, there was no significant difference in the expression level of 31 °C compared with the control (F_6,18 = 51.745, P < 0.05) (Fig. 3B). GST1 expression was suppressed or unaffected relative to the control at all elevated temperatures (F_6,17 = 32.682, P < 0.05) (Fig. 3C). All three antioxidant genes shared a common insensitivity in response to high temperature. Even under some temperatures, the expression level was higher than that of the control, but the relative expression level was not very high.

Expression of the three antioxidant genes was also evaluated in response to low temperature stress at 0, –2, –4, –6, –8 and –10 °C. SOD expression showed a significant decline at all temperatures relative to the control at 26 °C; although the expression of –4 °C was higher than other temperatures, it was also inhibited by low temperature (Fig. 4A) (F_6,20 = 243.607, P < 0.05). POD expression was also strongly inhibited, with the lowest expression appeared at –6 °C. (F_6,18 = 51.909, P < 0.05) (Fig. 4B). Like SOD and POD, the GST1 expression were decreased compared with the control relative with low temperature. (F_6,17 = 32.682, P < 0.05) (Fig. 4C).

Temporal changes in the expression of antioxidant genes

Compared to the control (0 h, 26 °C), SOD expression decreased significantly when 2^nd instar larvae were exposed to 35 °C for 0.5, 1 and 2 h (SOD: F_3,12 = 31.689, P < 0.05) and was lowest at the 1 h exposure period (Fig. 5A). POD expression was significantly upregulated at 0.5 and 2 h and was higher than expression levels at 0 (control) and 1 h, with the peak appeared at 2 h. (F_3,12 = 72.243, P < 0.05) (Fig. 5B). GST1 expression pattern was similar to POD (Fig. 5C). Although there was no significant difference at 0.5 h compared to the control (GST1: F_3,11 = 1709.476, P < 0.05).

After exposure to −4 °C, the expression levels of the three antioxidant genes decreased significantly when compared to the control (SOD: F_3,10 = 201.898, P < 0.05; POD: F_3,11 = 204.420, P < 0.05; GST1: F_3,10 = 72.835, P < 0.05). Interestingly, all three genes showed a peak in expression after a 1 h exposure to −4 °C; however, it should be noted that expression at 1 h was lower than the control (Fig. 6).