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).