Toxicity and synergism bioassays
The synergistic effects of PBO, DEM and TPP on sulfoxaflor and acetamiprid were evaluated in Yarkant and Jinghe field strains, and sulfoxaflor- and acetamiprid-resistant strains. Only PBO significantly increased the toxicity of sulfoxaflor in Yarkant-SulR, with a synergism ratio (SR) of 3.16 and a relative synergism ratio (RSR) of 2.79 (Table 1). Furthermore, PBO increased acetamiprid toxicity in Yarkant-AceR, with an SR of 2.04-fold and an RSR of 2.12-fold (Table 1). DEM and TPP had no synergistic effects. The Jinghe strains had similar findings. PBO considerably increased sulfoxaflor toxicity in Jinghe-SulR, with an SR of 2.31 and an RSR of 2.65 (Table 2). Moreover, PBO increased acetamiprid toxicity in Jinghe-AceR, with an SR of 2.91-fold and an RSR of 3.20-fold (Table 2). DEM and TPP had no synergistic effects.
Table 1
Synergistic effects of PBO, TPP and DEM on the toxicity of sulfoxaflor/acetamiprid in the Yarkant-FS, Yarkant-SulR and Yarkant-AceR
Strains | Treatments | LC50 mg L− 1 (95% CI) | Slope ± SE | χ2 (df) | SR† | RSR†† |
Yarkant-FS | Sulfoxaflor | 3.70 (2.67 ~ 4.98) | 1.11 ± 0.09 | 15.03 (13) | - | |
| Sulfoxaflor + PBO | 3.30 (2.32 ~ 4.46) | 1.11 ± 0.10 | 10.28 (13) | 1.13 | |
| Sulfoxaflor + TPP | 3.84 (2.80 ~ 5.14) | 1.15 ± 0.10 | 10.43 (13) | 0.96 | |
| Sulfoxaflor + DEM | 3.49 (2.66 ~ 4.50) | 1.46 ± 0.12 | 7.79 (13) | 1.06 | |
Yarkant-SulR | Sulfoxaflor | 147.48 (119.06 ~ 181.50) | 1.53 ± 0.13 | 11.85 (13) | - | |
| Sulfoxaflor + PBO | 46.88 (36.50 ~ 58.14) | 1.48 ± 0.12 | 18.28 (16) | 3.16 | 2.79 |
| Sulfoxaflor + TPP | 133.98 (114.26 ~ 154.86) | 2.44 ± 0.22 | 7.56 (16) | 1.10 | 1.14 |
| Sulfoxaflor + DEM | 137.58 (119.49 ~ 156.43) | 3.00 ± 0.28 | 9.22 (16) | 1.07 | 1.01 |
Yarkant-FS | Acetamiprid | 7.81 (6.09 ~ 9.90) | 1.51 ± 0.12 | 12.04 (13) | - | |
| Acetamiprid + PBO | 8.09 (6.06 ~ 10.48) | 1.54 ± 0.14 | 5.91 (13) | 0.97 | |
| Acetamiprid + TPP | 7.42 (5.54 ~ 9.52) | 1.78 ± 0.18 | 11.18 (13) | 1.05 | |
| Acetamiprid + DEM | 7.76 (6.03 ~ 9.82) | 1.62 ± 0.14 | 7.94 (13) | 1.00 | |
Yarkant-AceR | Acetamiprid | 225.09 (191.81 ~ 262.58) | 2.29 ± 0.20 | 10.57 (13) | - | |
| Acetamiprid + PBO | 110.05 (87.81 ~ 132.65) | 1.83 ± 0.20 | 9.12 (16) | 2.04 | 2.12 |
| Acetamiprid + TPP | 178.65 (154.97 ~ 204.98) | 2.37 ± 0.20 | 8.51 (16) | 1.26 | 1.20 |
| Acetamiprid + DEM | 201.16 (175.72 ~ 229.21) | 2.69 ± 0.23 | 14.03 (16) | 1.12 | 1.11 |
† Synergism Ratio (SR) = [LC50 of sulfoxaflor or acetamiprid + acetone] / [LC50 of sulfoxaflor or acetamiprid + acetone + synergist]. |
†† Relative synergism Ratio (RSR) = synergistic ratio of Yarkant-SulR or Yarkant-AceR / synergistic ratio of Yarkant-FS. |
Table 2
Synergistic effects of PBO, TPP and DEM on the toxicity of sulfoxaflor/acetamiprid in the Jinghe-FS, Jinghe-SulR and Yarkant-AceR
Strains | Treatments | LC50 mg L− 1 (95% CI) | Slope ± SE | χ2 (df) | SR† | RSR†† |
Jinghe-FS | Sulfoxaflor | 9.46 (6.91 ~ 12.89) | 0.99 ± 0.08 | 16.83 (13) | - | |
| Sulfoxaflor + PBO | 10.93 (7.34 ~ 16.02) | 0.87 ± 0.10 | 9.76 (13) | 0.87 | |
| Sulfoxaflor + TPP | 12.73 (8.75 ~ 18.58) | 0.85 ± 0.09 | 9.48 (13) | 0.74 | |
| Sulfoxaflor + DEM | 11.18 (8.31 ~ 15.09) | 1.05 ± 0.09 | 13.05 (13) | 0.85 | |
Jinghe-SulR | Sulfoxaflor | 139.44 (110.50 ~ 174.54) | 1.37 ± 0.12 | 7.74 (13) | - | |
| Sulfoxaflor + PBO | 60.43 (49.42 ~ 71.96) | 1.99 ± 0.17 | 18.83 (16) | 2.31 | 2.65 |
| Sulfoxaflor + TPP | 128.24 (112.16 ~ 145.42) | 2.69 ± 0.22 | 13.03 (16) | 1.09 | 1.30 |
| Sulfoxaflor + DEM | 134.86 (118.25 ~ 152.69) | 2.79 ± 0.23 | 12.02 (16) | 1.03 | 1.22 |
Jinghe-FS | Acetamiprid | 30.75 (24.07 ~ 39.34) | 1.35 ± 0.11 | 16.40 (13) | - | |
| Acetamiprid + PBO | 33.62 (25.55 ~ 42.27) | 1.52 ± 0.13 | 20.41 (16) | 0.91 | |
| Acetamiprid + TPP | 33.54 (25.80 ~ 41.79) | 1.62 ± 0.14 | 20.62 (16) | 0.92 | |
| Acetamiprid + DEM | 32.42 (25.03 ~ 40.27) | 1.67 ± 0.14 | 11.91 (16) | 0.95 | |
Jinghe-AceR | Acetamiprid | 193.32 (153.57 ~ 242.36) | 1.39 ± 0.12 | 8.42 (13) | | |
| Acetamiprid + PBO | 66.23 (50.11 ~ 83.52) | 1.41 ± 0.14 | 14.35 (16) | 2.91 | 3.20 |
| Acetamiprid + TPP | 157.74 (136.10 ~ 181.28) | 2.38 ± 0.20 | 6.42 (16) | 1.23 | 1.33 |
| Acetamiprid + DEM | 161.62 (140.54 ~ 184.47) | 2.57 ± 0.22 | 7.54 (16) | 1.20 | 1.26 |
† Synergism Ratio (SR) = [LC50 of sulfoxaflor or acetamiprid + acetone] / [LC50 of sulfoxaflor or acetamiprid + acetone + synergist]. |
†† Relative synergism Ratio (RSR) = synergistic ratio of Jinghe-SulR or Jinghe-AceR / synergistic ratio of Jinghe-FS. |
Metabolic enzyme activity
The relative activity of carboxylesterases (CESs), glutathione transferases (GSTs), and cytochrome P450 (P450s) in the Yarkant and Jinghe strains were presented in Fig. 1. It was observed that the relative activity of P450s in Yarkant-SulR and Yarkant-AceR was 1.59- and 1.62-fold higher (P < 0.0001), respectively, compared to Yarkant-FS. However, there were no significant differences in GSTs and CESs activity between Yarkant-FS, Yarkant-SulR, and Yarkant-AceR (P = 0.211). In the Jinghe strains, the activity of P450s was found to be 1.89- and 2.01-fold greater in Jinghe-SulR and Jinghe-AceR (P < 0.0001), respectively, as compared to Jinghe-FS. But there were no significant differences in GSTs and CESs activity were observed among Jinghe-FS, Jinghe-SulR, and Jinghe-AceR (P = 0.146).
Identification of cytochrome P450 genes involved in sulfoxaflor and acetamiprid resistance
The cDNA sequences encoding putative cytochrome P450 genes of Yarkant and Jinghe strains identified by Aphis gossypii reference genome sequence (NCBI: ASM2018417v2) were aligned with the Non-redundant protein sequences. In the Yarkant strains, a total of 38 cytochrome P450 genes were annotated, of which, 6 were from the CYP2 family, 17 from the CYP3 family, 11 from the CYP4 family and 4 from the MitoCYP family. In the Jinghe strain, a total of 37 cytochrome P450 genes were annotated, of which, 5 were from the CYP2 family, 17 from the CYP3 family, 11 from the CYP4 family and 4 from the MitoCYP family. Among them, CYP6CY19 was only annotated in the Jinghe strains, while CYP307C1 and CYP6UN1 were only annotated in the Yarkant strains.
The heatmap of differential expression of cytochrome P450 genes in Yarkant and Jinghe resistant strains was shown in Fig. 2. In Yarkant resistant strains, nine cytochrome P450 genes were differentially expressed in Yarkant-SulR, six cytochrome P450 genes were significantly upregulated, such as CYP6CY59, CYP6CY20, CYP6DC1, CYP6CY13, CYP380C44 and CYP380C45, and three cytochrome P450 genes significantly downregulated, such as CYP18A1, CYP6CY12 and CYP380C46 (|log2 Fold change| ≥ 1 and P-value < 0.05). Five cytochrome P450 genes were differentially expressed in Yarkant-AceR, three cytochrome P450 genes were significantly upregulated, such as CYP6CY59, CYP6DC1, and CYP380C45, and two cytochrome P450 genes were significantly downregulated, such as CYP6CY12 and CYP380C46 (|log2 Fold change| ≥ 1 and P-value < 0.05). In Jinghe resistant strains, four cytochrome P450 genes were differentially expressed in the Jinghe-SulR, three cytochrome P450 genes were significantly upregulated, including CYP6CY24, CYP380C46, and CYP380C45, and one cytochrome P450 gene was significantly downregulated, e.g., CYP6DC1 (|log2 Fold change| ≥ 1 and P-value < 0.05). The Jinghe-AceR had three cytochrome P450 genes differentially expressed, two cytochrome P450 genes were significantly upregulated, including CYP6CY9 and CYP380C46, and one cytochrome P450 gene was significantly downregulated, such as CYP6DC1 (|log2 Fold change| ≥ 1 and P-value < 0.05).
In Yarkant-SulR and Yarkant-AceR, CYP6CY59, CYP6DC1 and CYP380C45 were both significantly upregulated, whereas CYP6CY12 and CYP380C46 were significantly downregulated. But CYP380C46 was significantly upregulated in both Jinghe-SulR and Jinghe-AceR strains, while both strains exhibited a significant downregulation of CYP6DC1. Moreover, the expression of CYP6DC1 was significantly upregulated in both Yarkant-SulR and Yarkant-AceR, but significantly downregulated in both Jinghe-SulR and Jinghe-AceR. While CYP380C46 was significantly upregulated in both Jinghe-SulR and Jinghe-AceR, but significantly downregulated in both Yarkant-SulR and Yarkant-AceR.
qRT-PCR validation of RNA-seq
The expression levels of up-regulated P450 genes in the transcriptome were evaluated using qRT-PCR in the resistant strains of Yarkant and Jinghe to validate the veracity of the transcriptome sequencing results. CYP380C44 (P = 0.014), CYP6CY13 (P = 0.026), CYP6CY20 (P = 0.006), CYP380C45 (P = 0.008), CYP6DC1 (P = 0.022) and CYP6CY59 (P = 0.020) were considerably expressed in Yarkant-SulR compared to Yarkant-FS (Fig. 2A), while CYP380C45 (P = 0.001), CYP6DC1 (P = 0.005) and CYP6CY59 (P = 0.029) were significantly expressed in Yarkant-AceR compared to Yarkant-FS (Fig. 2B). The relative expression of CYP6CY24 (P = 0.026), CYP380C46 (P = 0.004) and CYP380C45 (P = 0.015) in Jinghe-SulR was substantially greater than that of Jinghe-FS (Fig. 2C), whereas CYP380C46 in Jinghe-AceR was significantly higher than that of Jinghe-FS (P = 0.039) (Fig. 2D). The expression of CYP6CY9 was greater in Jinghe-AceR than in Jinghe-FS, although the difference was not statistically significant (P = 0.109) (Fig. 2D). Except CYP6CY9, the results obtained from qRT-PCR were consistent with the transcriptome sequencing results, indicating the reliability of the transcriptome data.
Effects of RNAi of P450 genes on resistance of Aphis gossypii to sulfoxaflor and acetamiprid
Based on the results of transcriptional analysis, five P450s genes were selected for functional validation. CYP6DC1 and CYP6CY13 were selected in the Yarkant-resistant strains, in which CYP6DC1 was significantly upregulated in Yarkant-SulR and Yarkant-AceR, and CYP6CY13 was significantly upregulated in Yarkant-SulR only. Similarly, CYP380C46 and CYP6CY24 were selected among the Jinghe resistant strains, where CYP380C46 was significantly upregulated in Jinghe-SulR and Jinghe-AceR, and CYP6CY24 was significantly up-regulated in Jinghe-AceR only. CYP380C45 was also selected as it was significantly upregulated in Yarkant-SulR, Yarkant-AceR and Jinghe-SulR.
The relative expression of CYP6DC1, CYP380C45 and CYP6CY13 was significantly lower than that of the dsGFP and DEPC treatments after Yarkant-SulR was fed a diet incorporated with dsRNA for 48 h (CYP6DC1: P = 0.009; CYP380C45: P < 0.0001; CYP6CY13: P = 0.024). Compared to dsGFP treatment, the relative expression of CYP6DC1, CYP380C45 and CYP6CY13 was reduced by 0.29-, 0.19- and 0.51-fold, respectively, in Yarkant-SulR (Fig. 4). The toxicity of sulfoxaflor was significantly increased after Yarkant-SulR A. gossypii feeding dsCYP6DC1, dsCYP380C45 and dsCYP6CY13 after 48 h, the mortality rates were 62.34%, 80.52%, and 75.32%, respectively, which were significantly higher than those of the DEPC (32.47%) and dsGFP (33.77%) treatments at the diagnostic dose of sulfoxaflor (P < 0.001) (Fig. 5A). After 48 h of feeding dsCYP6DC1 and dsCYP380C45, Yarkant-AceR A. gossypii was significantly more susceptible to acetamiprid, with mortality rates of 68.12% and 78.26%, respectively. These mortality rates were significantly higher than those of the DEPC (26.09%) and dsGFP treatments (30.43%), respectively, at the diagnostic dose of acetamiprid (P < 0.001) (Fig. 5B). However, after 48 h of feeding dsCYP6CY13, the mortality rate (40.58%) of Yarkant-AceR A. gossypii was not significantly different from the DEPC and dsGFP treatments.
In Jinghe-SulR, after 48 h of RNAi, the relative expression of both CYP6CY24 and CYP380C46 was significantly lower compared to the dsGFP and DEPC treatments (CYP6CY24: P = 0.006; CYP380C46: P = 0.009). Compared to the dsGFP treatment, the expression of CYP6CY24 and CYP380C46 was reduced by 0.48- and 0.23-fold, respectively, in Jinghe-SulR strain (Fig. 4). After feeding dsCYP6CY24, dsCYP380C45 and dsCYP380C46 for 48 h, sulfoxaflor toxicity was significantly increased in Jinghe-SulR, with mortality rates of A. gossypii were 71.60%, 79.01% and 74.07%, respectively, which were significantly higher than those of the DEPC (34.57%) and dsGFP (37.04%) treatments at the diagnostic dose of sulfoxaflor (P < 0.001) (Fig. 5C). After feeding dsCYP380C45 and dsCYP380C46 for 48 h, Jinghe-AceR was significantly more susceptible to acetamiprid, with mortality rates of A. gossypii were 60.98% and 73.17%, which were significantly higher than those of the DEPC (30.49%) and dsGFP (32.93%) treatments at the diagnostic dose of acetamiprid (P < 0.001) (Fig. 5D). However, after 48 h of feeding the dsCYP6CY24 treatment, the mortality rate (43.90%) of Jinghe-AceR A. gossypii was not significantly different from that of the DEPC and dsGFP treatments.