The full-length mMnSOD cDNA of B. tabaci (GenBank accession number JQ867105) consists of 1210 bp, with a 675 bp open reading frame (ORF), which encodes 224 amino acids. The full-length nucleotide sequences and the deduced amino acid sequences are shown in Figure 1. The Bt-mMnSOD cDNA sequence contains a 5′ untranslated region (UTR) of 185 nucleotides, a long 3′ UTR of 350 nucleotides consisting of a stop codon (TAG), a putative polyadenylation consensus signal (AATAAA) and a poly (A) tail. The predicted molecular mass is 24.5 kDa and the estimated isoelectric point of this protein is 8.97. SignalP program analysis showed no putative signal peptide in Bt-mMnSOD. However, TargetP and MITOPROT revealed that it contains a putative mitochondrial targeting sequence of 25 amino acids and is located inside the mitochondria.

Multiple sequences alignment of Bt-mMnSOD with other MnSODs of vertebrate and invertebrate animals indicated high conservation of four putative manganese binding sites (H54, H102, D186 and H190) and a MnSOD signature from 186 to 193 (DVWEHAYY) (Figure 2). The major differences between the mitochondrial and cytosolic MnSODs (cMnSODs) are located at the N-terminal region (Figure 2). cMnSODs have a conserved N-terminal extension, which is not present in mMnSODs, but lack a mitochondrial targeting sequence. MnSOD sequences of whitefly and other species were obtained from GenBank (Table 1).

A phylogenetic tree of the amino acid sequences of selected MnSODs was constructed by the neighbor-joining distance method using MEGA version 4 (Figure 3). The phylogenetic analysis indicated that MnSODs can be classified into two groups. All the mMnSODs were clustered together as one subgroup and all cMnSODs as another group (Figure 3). Bt-mMnSOD was in the same group of other mMnSODs, suggesting that it is an mMnSOD.

The expression of Bt-mMnSOD protein was considerably increased after induction with 0.2 mM IPTG at 18 °C for 18 h (Figure 4, lanes 1, 2). Moreover, the proteins were expressed both in the inclusion bodies and as soluble protein (Figure 4, lanes 3, 4). The soluble Bt-mMnSOD protein was then purified with GST purification system and resolved by SDS-PAGE, with the result of a 50.5 kDa single band (Figure 4, lane 5). The molecular mass of the purified product was consistent with the predicted molecular weight of the recombinant protein, which consisted of Bt-mMnSOD (24.5 kDa) and GST tag (26 kDa).

Thermostability of purified Bt-mMnSOD was studied by incubating the enzyme at 37, 45, 55, 65, 75 and 85 °C for various intervals. The residual activities of Bt-mMnSOD were measured (Figure 5). The enzymatic activity of Bt-mMnSOD was stable after incubation at 37 °C and 45 °C for 30 min. At the temperature of 55 °C, the Bt-mMnSOD activity decreased significantly after 20 min (p < 0.01), but still retained up to 70% after 60 min incubation. The activity reduced by 50% when incubated for 10 min at 65 °C and decreased rapidly in the initial 10 min (p < 0.01) at 75 and 85 °C. The enzyme was completely inactivated when incubated for 50 min at 85 °C and 60 min at 75 °C, respectively.

To better understand the physiological roles of Bt-mMnSOD, its expression was detected by qPCR in different developmental stages. The mRNA levels of Bt-mMnSOD were significantly higher in the 4th instar than in egg and nymph or adult (Figure 6). The result of qPCR suggests that Bt-mMnSOD might play important roles in the development of B. tabaci, especially in the 4th instar.

To evaluate the in vivo activity of the MnSOD in B. tabaci stimulated by external temperature stresses, B. tabaci adults were exposed to low (4 °C), medium (26 °C) and high (40 °C) temperatures for 0 (control), 30, 60 and 120 min, respectively (Figure 7). The activity of MnSOD significantly increased after incubation at low (4 °C) and high (40 °C) temperatures for 60 and 120 min, compared with control (p < 0.01). However, no significant differences were detected when the B. tabaci adults were exposed at 26 °C from 0 to 120 min or incubated at 4 °C and 40 °C in the initial 30 min.

To characterize the effect of host shift on the in vivo activity of the MnSOD in B. tabaci, whitefly adults were transferred from cotton (a favorable host plant) to tobacco (an unfavorable host plant) for 0, 1, 3 and 5 days. When whiteflies were transferred from cotton to tobacco for 5 days, the activity of MnSOD significantly increased compared with 0 days (p < 0.01), whereas in the control (transfer from cotton to cotton), the level of activity remained unchanged (Figure 8).

Furthermore, the activities of MnSOD were measured after the whitefly feeding on the imidacloprid-treated cotton plants for various durations (Figure 9). No significant differences were observed in the initial 12 h. However, after 24 h feeding on imidacloprid-treated cotton plants, a significant increase of MnSOD activity was detected compared with 0 h (p < 0.01).

Stock cultures of the invasive whitefly MEAM1, collected from Zhejiang, China, were established in the laboratory. Details of the methods for maintaining whitefly cultures were described by Jiu et al.[43]. The cultures were maintained on cotton plants (Gossypium hirsutum L. cv. Zhemian 1793) and regularly monitored for purity using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with the primer H16 (5′-TCTCAGCTGG-3′) [44].

Cotton (cv. Zhemian 1793) and tobacco (Nicotiana tabacum cv. NC89), a suitable and an unsuitable host plant for the whitefly, respectively [45,46], were used in this study. All plants were cultivated with a potting mix (a mixture of peat moss, vermiculite organic fertilizer and perlite in a 10:10:10:1 ratio by volume) in plastic pots in whitefly-proof glasshouses at 27 ± 1 °C, 14 h light: 10 h darkness and 40%–60% relative humidity (RH). Plants were used for experiments at the 5–6 true leaf stage.

Total RNA was isolated using the SV total RNA isolation system (Promega, Madison, WI, USA), following the manufacturer’s protocol. cDNA was synthesized using total RNA as a template with the SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). Initially, partial Bt-mMnSOD cDNA sequence was obtained from the whitefly transcriptome deposited in NCBI (GenBank accession no. HP662057) [47]. The 5′ and 3′ ends of Bt-mMnSOD were amplified using the SMART RACE cDNA Amplification Kit with the 5′ primer (5′-CTAAACAGCTGCTGAATATC-3′) and 3′ primer (5′-ATGTTTGCGTGCTCCAAAAA-3′), following the manufacturer’s instructions. The amplified products were analyzed on 1.5% agarose gel, and the target band was purified by a PCR purification kit (Promega, Madison, WI, USA). Then the products were cloned into the pMD18-T vector (Takara, Dalian, China) for sequencing.

The nucleotide and deduced amino acid sequences of the Bt-mMnSOD were analyzed by DNAMAN version 6 [48]. The sequence similarity of MnSODs from different species was analyzed and compared using the BLAST search program [49]. Theoretical isoelectric points and predicted molecular masses were calculated using Prot-Param tools [50]. The signal peptide was predicted by Signal P4.0 program [51]. The MITOPROT [52] and TargetP 1.1 [53] were used to predict whether the sequence contains any targeting sequence and where it locates. To compare MnSOD of whitefly and other species, related sequences were obtained from GenBank (Table 1) and aligned using Clustal W [54]. The phylogenetic relationship among the sequences was determined by reconstructing a protein phylogeny using MEGA4 [55]. The tree topology was evaluated by the bootstrapping method (1000 replications).

A pair of primers were designed to clone the open reading frame of the Bt-mMnSOD and then ligated to the pGEX-4T-3 expression vector. The sense primer was designed as 5′-GGATCCATGTTTGCGTGCTCCAAAAA-3′ with a BamH1 site and the antisense primer 5′-GTCGACCTAAACAGCTGCTGAATATC-3′ containing Sal1 site. The ligated product was then transformed into E. coli BL21 (DE3) pLysS cells for recombinant protein expression (Novagen, Madison, WI, USA).

Bacteria were grown in 800 mL LB medium containing 0.1 g/mL ampicillin at 37 °C until an optical density of 0.6 at 600 nm was reached. IPTG was added to a final concentration of 0.2 mM, and the culture was grown at 18 °C for 18 h for the induction of protein expression. Protein samples were analyzed by 12% SDS-PAGE, followed by staining with Coomassie brilliant blue. The Bt-mMnSOD protein with the GST tag was purified using the GST gene fusion system, as described [56]. The eluted protein content was measured by UV absorbance at 280 nm, and the purity of the protein was evaluated by 12% SDS-PAGE.

The activity of the purified recombinant Bt-mMnSOD was determined using the CuZn/Mn SOD Assay Kit (Beyotime, Shanghai, China). The kit applied the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) method. The WST-1 could react with superoxide anion to generate water-soluble formazan dye. However, this reaction can be inhibited by SOD. Through colorimetric analysis of the WST-1 product, we can calculate the SOD enzyme activity. The absorbance was measured at 450 nm using spectrophotometer at room temperature. One unit of enzyme activity was defined as the amount of enzyme that results in 50% inhibition of the xanthine oxidase coupled reaction under the assay condition. To determine the thermal stability of Bt-mMnSOD, reactions were carried out at 37, 45, 55, 65, 75 and 85 °C for 0 (control), 10, 20, 30, 40, 50 and 60 min and the relative activity of Bt-mMnSOD was measured, respectively. Each treatment was replicated for three times. A multiple comparisons (LSD) test was conducted to detect significant differences (p = 0.01) between the treatments using the DPS software [57].

Quantitative real-time PCR (qPCR) was performed on the ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems, USA). Total RNA was isolated from different developmental stages of B. tabaci, including egg & nymph, 4^th instar and adult. RNA concentration was measured by NanoDrop 2000c (Thermo, Minneapolis, MN, USA). cDNA was synthesized using PrimeScript^® RT reagent Kit (Takara, Dalian, China). The following primers, qPCR-F (5′-ACCACCGCTAATCAAGATCC-3′) and qPCR-R (5′-TGCAGGTAGTAGGCGTGTTC-3′), designed by the GenScript Real-time PCR Primer Design Software [58], were used for qPCR. As an endogenous control, the expression of β-actin was measured in parallel.

Whiteflies were independently exposed to three different temperatures (4, 26, 40 °C) for 0, 30, 60 and 120 min in climatic chambers. The control and treated whiteflies were kept in liquid nitrogen immediately after treatment and were stored at −80 °C until use. Fifty whiteflies were transferred into a tissue grinding tube with 200 μL cell lysis buffer (20 Mm Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100). After grinding, the lysate was centrifuged at 4 °C and the supernatant was used for enzymatic assays. The activity of the MnSOD was determined using the CuZn/Mn-SOD Assay Kit (Beyotime, Shanghai, China). During the assay, CuZnSOD inhibitor A and B were added in the sample according to the manufacturer’s protocol, so the result of the assay was only about MnSOD. The detailed procedures can be referred to in the protocol (Beyotime, Shanghai, China). Each treatment was replicated for three times. The statistical analysis is described in “Thermostability of purified Bt-mMnSOD”.

Whiteflies collected from cotton plants were transferred to tobacco plants and new cotton plants (control). After 0 (control), 1, 3 and 5 days, 50 whiteflies were collected in a 5 mL tube from tobacco and new cotton plants, respectively. The whiteflies were then treated with liquid nitrogen and stored at −80 °C. Each treatment was replicated for three times. The method used for preparing samples and the detection of enzyme activity is described in “Effect of thermal stresses on the MnSOD activity of the whitefly”. The statistical analysis is described in “Thermostability of purified Bt-mMnSOD”.

Three cotton plants were sprayed with 15 mL of imidacloprid (Dashifeng, Beijing, China). The concentration of active ingredients was 20 mg/L. After 6 h, two thousand adult whiteflies were transferred to these cotton plants. Thereafter, 50 whiteflies were collected in a 5 mL tube after 0 (control), 6, 12 and 24 h, respectively. The whiteflies were then treated with liquid nitrogen and stored at −80 °C. Each treatment was replicated three times. The method used for preparing samples and the detection of enzyme activity is described in “Effect of thermal stresses on the MnSOD activity of the whitefly”. The statistical analysis is described in “Thermostability of purified Bt-mMnSOD”.