Effects of Pesticide Selection and Dual Exposure to Pesticides and Pathogens on Gene Expression
Next, we introduced the pesticide-exposed samples into the analysis, still treating OP and Pyr-associated treatments separately, to investigate interactions between pesticide selection regime, pesticide exposure, and Btt exposure on gene expression. For both pesticide class analyses, pesticide exposure had the greatest effect on gene expression (Figs. 3B-C; Pesticide Tx OP or Pyr) and induced differential expression of many canonical detoxification genes, including several cytochrome P450s, esterases, GSTs, UDP-glucuronosyltransferases (UGTs), and ABC transporters (Suppl. Fig. 5; OP or Pyr PTx). Genes differentially expressed after both OP and Pyr exposure were commonly enriched for GO processes involved in xenobiotic and detoxification responses including oxidoreductase and serine hydrolase activity (Suppl. Fig. 4B).
Genes differentially expressed with respect to pesticide selection regime and to the interaction between selection regime and pesticide treatment can identify genes associated with pesticide resistance. Overall, Pyr-regime had a greater effect on differential gene expression compared to OP-regime (Fig. 3; Regime OP or Pyr). Similarly, a greater number of genes were differentially expressed with the interaction between Pyr regime and Pyr exposure compared to OP regime and OP exposure (Figs. 3B [Regime OP, Pesticide Tx OP], 3C [Regime Pyr, Pesticide Tx Pyr]). Both OP-regime and Pyr-regime induced differential expression of canonical detoxification (e.g., P450s, esterases, GSTs, and ABC transporters) and cuticle-associated genes (Suppl. Figs. 5, 6; OP or Pyr Reg). GO enrichment analyses revealed the importance of structural constituent of cuticle for genes differentially expressed with OP regime and the interaction between OP regime and OP exposure (Suppl. Fig. 4B; OP Reg and OP Reg:PTx). Pyr-regime individuals differentially expressed genes enriched for molecular functions involved in oxidoreductase activity, heme binding, and structural constituent of cuticle, while genes differentially expressed with the interaction between Pyr regime and Pyr exposure were enriched for these functions and also serine hydrolase activity (Suppl. Fig. 4B; Pyr Reg and Pyr Reg:PTx). Thus, GO enrichment analyses revealed the common differential expression of cuticle-related genes in both OP and Pyr resistance, while Pyr resistance was additionally associated with differential expression of oxidoreductase metabolic detoxification processes.
In the models containing pesticide exposure samples, no genes were differentially expressed with the interaction between OP regime andBtt treatment (Fig. 3B; Regime OP, Bt Tx +) providing further evidence that, here, evolved OP resistance had minimal effects on immune responses to Btt . In contrast, 61 genes were differentially expressed with the interaction between Pyr selection regime andBtt treatment (Fig. 3C; Regime Pyr, Bt Tx +), although there was no significant GO term enrichment. Mirroring patterns observed in the no-pesticide model, the model that includes pesticide exposure samples also indicates that Pyr regime larvae had lower induced expression of a defensin-like transcript compared to control-regime larvae when exposed to Btt (Fig. 5B).
When considering the interaction effect between pesticide exposure andBtt infection, 175 genes were differentially expressed with dual OP and Btt exposure (Fig. 3B; Pesticide Tx OP, Bt Tx +), and only one gene (an upregulated pupal cuticle protein) was differentially expressed with dual exposure to Pyr and Btt (Fig. 3C; Pesticide Tx Pyr, Bt Tx +). OP pesticide exposure interacted with Bttexposure to induce differential gene expression of immune, detoxification, cuticle, and development genes compared to expression induced by the main effect of Btt exposure (Fig. 4A; compare BtTx and OP PTx:BtTx). Further analysis revealed that these interaction effects were mainly driven by significantly dampened upregulation upon combined OP and Btt exposure compared to upregulation induced with Btt alone; transcripts upregulated with Btt alone were not differentially expressed or were downregulated with combined OP and Btt exposure (Fig. 4A). For example, while Bttexposure alone induced upregulation of two attacins, three cytochrome P450s, and several cuticular proteins, larvae exposed to both OP andBtt had more modest differential expression of these same transcripts (e.g. attacin-1 in Fig. 5A). Genes differentially expressed with the interaction between OP treatment and Bttexposure were enriched for GO terms involved in the molecular functions of oxidoreductase activity and structural constituent of cuticle (Suppl. Fig. 4B; OP PTx:BtTx).