*2.3. Detoxification Enzyme Activity*

To further determine the potential role of detoxification enzymes in the development of resistance of *S. furcifera* to sulfoxaflor, both the Sus-Lab strain, either untreated or treated with a synergistic agent (DEM, TPP or PBO), and the SF-Sel strain, either untreated or treated with a synergistic agent (DEM, TPP or PBO), were analyzed to determine the activities of CarEs, GSTs and P450s. As shown in Figure 1, the activity of CarEs in the SF-Sel strain treated with TPP was the highest (0.9616 mmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1), followed by those in the SF-Sel strain (0.8257 mmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1), the Sus-Lab strain treated with TPP (0.8187 mmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1) and the Sus-Lab strain (0.7397 mmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1). However, there was no significant difference in CarE activity under any of the treatments (*p* > 0.05). The GSTs activity of the SF-Sel strain was the greatest, at 1.0103 mmol <sup>×</sup> min−1<sup>×</sup> mg pro−1, which was significantly different from the other three treatments (*p* < 0.05), where the activities ranged from 0.6824 to 0.8113 mmol <sup>×</sup> min−1<sup>×</sup> mg pro−<sup>1</sup> and did not significantly differ from each other (*p* > 0.05). P450s activity was also the greatest in the SF-Sel strain (13.5345 nmol <sup>×</sup> min−1<sup>×</sup> mg pro−1, which was significantly different from those under the other treatments (*p* < 0.05)), followed by the SF-Sel strain treated with PBO (10.7384 nmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1). The P450s activities of the Sus-Lab strain and Sus-Lab strain treated with PBO were the weakest (5.6181 and 4.8470 nmol <sup>×</sup> min−1<sup>×</sup> mg pro<sup>−</sup>1), and both of these values were significantly different from those under the other treatments (*p* < 0.05) (Figure 1).

**Figure 1.** Synergistic effects of TPP, DEM and PBO on activity of detoxification enzymes: CarE (**A**), GST (**B**) and P450 (**C**) in 3rd-instar nymph of *S furcifera.* The activities of CarE, GST and P450 in 3rd-instar nymph of *S furcifera* are presented as the mean of three replications ± *SE*. Means followed by the same letters did not differ significantly (*p* > 0.05) according to the ANOVA test. The F3, 8 values of different treatments on CarE, GST and P450 in 3rd-instar nymph of *S furcifera* were 1.818, 5.410, 610.745, and the *p* values on CarE, GST and P450 in 3rd-instar nymph of *S furcifera* were = 0.222 > 0.05, = 0.025 < 0.05, = < 0.0001, respectively.

#### *2.4. Illumina Sequencing and Read Assembly*

The Sus-Lab and SF-Sel strains were each assessed in triplicate. The total numbers of reads (150 bp) obtained were 335,119,142 in the six samples, with over 50,653,904 reads for each sample. The proportion of reads containing duplicate sequences was 0.37% ~ 0.42%, and the proportion of low-quality reads was 1.23%~1.35%, including reads with > 10% Ns and abase number of Q ≤ 10 in > 50% of the total reads. After filtering out the linker sequences or low-quality reads, 329,449, and 482 clean reads were obtained, and the Q20 and Q30 base percentages of clean reads of were over 98.69% and 96.04%, respectively (Supplementary Table S1).

#### *2.5. Transcriptome Data Splicing*

The clean reads from the transcriptomes of the Sus-Lab and SF-Sel strains were composed of the mixed pools and were assembled into approximately 74,119 unigenes, with a longest unigene of 34,340 nt and a shortest unigene of 201 nt (Supplementary Figure S1A). The N50 value, at which the cumulative fragment length reaches 50% of the total fragment length, was 2043 nt, and there were 5552 unigenes of over 3000 nt. When the reads were compared with the unigenes, 3971 of the unigenes were found to exhibit more than 10,000 reads, while 34, 847 of unigenes presented only 11~100 reads (Supplementary Figure S1B).

#### *2.6. Transcriptome Annotation*

From to the 74,119 assembled unigenes, 24,719 of which were successfully annotated in the Nr database, and the species with the greatest number of homologous sequences included *Zootermopsis nevadensis* (2001 genes), *Bemisia tabaci* (1620 genes), *Cimex lectularius* (1500 genes), and *Halyomorpha halys* (1424 genes) (Supplementary Figure S2A). Additionally, 19,050 unigenes were annotated with the Swissprot database, 17,119 unigenes with the KOG database, and 11,788 unigenes with the KEGG database, and 10,516 unigenes were annotated in all four databases (Supplementary Figure S2B), among the 17,119 unigenes annotated with the KOG database, 6671 unigenes were classified as general function prediction only, accounting for the largest proportion, and 4867 unigenes were classified as being associated with signal transduction mechanisms (Supplementary Figure S2C).

#### *2.7. Analysis of Gene Expression*

The results indicated that the correlations of gene expression levels in the three samples (M1, M2 and M3) of the Sus-Lab strain (with correlation index of 0.99 to 1.00) were significantly higher than those in the SF-Sel strain (S1, S2 and S3 samples, with a correlation index of 0.85 to 0.94) (Figure 2A). Principal component analysis (PCA) of the six samples also showed that there was a significant clustering relationship on PC1 between the gene expression levels in the samples of the susceptible strains and the SF-Sel strain. Additionally, the degree of the contribution of PC1 (88.7%) was significantly higher than that of PC2 (7.9%) (Figure 2B).

**Figure 2.** (**A**) Heat map of gene expression levels in the six samples. The darker the color is, the greater the correlation is. Three samples (M1, M2 and M3) are in the Sus-Lab strain, and other three samples (S1, S2 and S3) are in the SF-Sel strain. The same means as followed. (**B**) Principal component analysis (PCA) of six samples.
