DOI: 10.5061/dryad.bcc2fqzk3
title: Transcriptome analysis of Drosophila suzukii reveals molecular mechanisms conferring pyrethroid and spinosad resistance

description: Drosophila suzukii possess a serrated ovipositor that enables them to lay
 eggs in soft-skinned, ripening fruits, making this insect a serious threat
 to berry production. Since its 2008 introduction into North America,
 growers have used insecticides as the primary approach for D. suzukii
 management, resulting in detections of insecticide resistance in this
 pest. This study sought to identify the molecular mechanisms conferring
 insecticide resistance in these resistant populations. We sequenced the
 transcriptomes of two pyrethroid- and two spinosad-resistant isofemale
 lines. In both pyrethroid-resistant lines and one spinosad-resistant line,
 we identified overexpression of metabolic genes that are implicated in
 resistance in other insect pests. In the other spinosad-resistant line, we
 observed an overexpression of cuticular genes that have been linked to
 resistance. Our findings enabled the development of molecular diagnostics
 that we used to confirm persistence of insecticide resistance in
 California. To validate these findings, we leveraged D. melanogaster
 mutants deficient in either metabolic or cuticular genes that were
 upregulated in resistant D. suzukii to demonstrate that these genes are
 involved in promoting resistance. This study is the first to characterize
 the molecular mechanisms of insecticide resistance in D. suzukii and
 provides insights into how current management practices can be optimized.
description: <strong>Data generated from Illumina short-read sequencing
 (PE150) </strong> RNA was extracted from
 isofemale lines developed from unsprayed Drosophila suzukii collected from
 populations resistant to either spinosad insecticide or resistant
 insecticide on either strawberries (S) or caneberries (C).  Female D.
 suzukii flies were entrained at 25C in 12-hour light:12-hour dark cycles
 for two full days. On the third day, flies were collected on dry ice
 sixteen hours after lights-on. This time point was selected because D.
 suzukii was previously observed to exhibit a low level of cytochrome P450
 expression at this time (Hamby et al., 2013). This means any
 overexpression may be more easily observed. Fly bodies were separated from
 heads using frozen metal sieves (Newark Wire Cloth Company, Clifton, New
 Jersey). Eight to ten female bodies were used per sample
 Pyrethroid-susceptible lines = S7, S8
 Pyrethroid-resistant lines = S3, S4
 Spinosad-susceptible lines = C2, C5
 Spinosad-resistant lines = C3, C4 Differential
 gene expression analysis was performed using sequencing reads derived from
 llumina short-read sequencing. First, rRNA reads were removed using
 SortMeRNA v2.1 (Kopylova et al., 2012). Adapters (ILLUMINACLIP parameters
 2:30:10) and low-quality ends (LEADING: 10, TRAILING:10, MINLEN:36) were
 trimmed using Trimmomatic v0.35 (Bolger et al., 2014). Cleaned reads were
 aligned to the NCBI Drosophila suzukii Annotation Release 102 based on the
 LBDM_Dsuz_2.1.pri assembly (accession no. GCF_013340165.1) (Paris et al.,
 2020) using STAR v2.7.9a (Dobin et al., 2013). Count data from STAR
 (--quantMode GeneCounts) served as input in the DESeq2 package (Love et
 al., 2014) in R to perform differential expression analysis on each
 resistant line vs all susceptible samples. Each resistant line was
 compared to susceptible samples separately as each line might exhibit
 resistance due to different mechanisms. Genes with fold change differences
 between resistant vs susceptible populations with a Benjamini-Hochberg
 adjusted p-value &lt; 0.05 were considered differentially expressed.
 Expression levels of genes were also measured as fragments per kilobase of
 exon per million mapped (FPKM) values calculated with Stringtie v2.0.4
 (Pertea et al., 2015). The consistency between biological replicates was
 calculated with Pearson’s correlation coefficient, which was determined
 with the ‘stats’ package in R version 4.2.1. Expression differences of key
 genes between the resistant and susceptible populations were calculated
 with two-way ANOVA followed by two-stage linear set-up procedure of
 Benjamini, Krieger, and Yekutieli on GraphPad Prism. 
 <strong>Weighted Gene Co-expression Network Analysis
 </strong> Gene expression (in FPKM) served as
 input for Weighted Gene Co-expression Network Analysis (WGCNA). Genes with
 an expression value of zero for more than six samples were excluded from
 analysis. To explore the modules most correlated with insecticide
 resistance, a correlation analysis using resistance status was performed
 with the WGCNA package (Version 1.72.1) (Langfelder & Horvath,
 2008) on R. Modules with a p-value &lt; 0.05 were considered
 significant. Functional enrichment analysis (described below) was
 performed on the module with the highest correlation with
 resistance.  <strong>Functional enrichment
 analysis </strong> Genes were functionally
 annotated using BLAST against the NCBI Drosophila melanogaster Annotation
 r6.32 based on the Release 6 plus ISO1 mitochondrial genome assembly
 (accession no. GCA_000001215.4) (dos Santos et al., 2015). Gene Ontology
 (GO) enrichment of genes were performed using ShinyGO 0.76.3 (Ge et al.,
 2020). GO terms and pathways were considered enriched if the false
 discovery rate (FDR) &lt; 0.05.  Raw Data can be
 found under BioProject PRJNA983428
description: # Transcriptome analysis of Drosophila suzukii reveals molecular
 mechanisms conferring pyrethroid and spinosad resistance CA Tabuloc, CR
 Carlson, F Ganjisaffar, CC Truong, C Chen, KM Lewald, S Hidalgo, N Nicola,
 C Jones, AA Sial, FG Zalom, JC Chiu ## Data description Data generated
 from Illumina short-read sequencing (PE150). RNA was extracted from
 isofemale lines developed from unsprayed Drosophila suzukii collected from
 populations resistant to either pyrethroid insecticide (specifically
 zeta-cypermethrin) or spinosad insecticide on either strawberries (S) or
 caneberries (C). Female D. suzukii flies were entrained at 25C in 12-hour
 light:12-hour dark cycles for two full days. On the third day, flies were
 collected on dry ice sixteen hours after lights-on (ZT16). This time point
 was selected because D. suzukii was previously observed to exhibit a low
 level of cytochrome P450 expression at this time (Hamby et al., 2013).
 This means any overexpression may be more easily observed. Fly bodies were
 separated from heads using frozen metal sieves (Newark Wire Cloth Company,
 Clifton, New Jersey). Eight to ten female bodies were used per sample.
 Zeta-cypermethrin-susceptible lines = S7, S8 Zeta-cypermethrin-resistant
 lines = S3, S4 Spinosad-susceptible lines = C2, C5 Spinosad-resistant
 lines = C3, C4 #### Differential gene expression (DEG analysis)
 Differential gene expression analysis was performed using sequencing reads
 derived from Illumina short-read sequencing. First, rRNA reads were
 removed using SortMeRNA v2.1 (Kopylova et al., 2012). Adapters
 (ILLUMINACLIP parameters 2:30:10) and low-quality ends (LEADING: 10,
 TRAILING:10, MINLEN:36) were trimmed using Trimmomatic v0.35 (Bolger et
 al., 2014). Cleaned reads were aligned to the NCBI Drosophila suzukii
 Annotation Release 102 based on the LBDM_Dsuz_2.1.pri assembly (accession
 no. GCF_013340165.1) (Paris et al., 2020) using STAR v2.7.9a (Dobin et
 al., 2013). Count data from STAR (--quantMode GeneCounts) served as input
 in the DESeq2 package (Love et al., 2014) in R to perform differential
 expression analysis on each resistant line vs all susceptible samples.
 Each resistant line was compared to susceptible samples separately as each
 line might exhibit resistance due to different mechanisms. Genes with fold
 change differences between resistant vs susceptible populations with a
 Benjamini-Hochberg adjusted p-value &lt; 0.05 were considered
 differentially expressed. Expression levels of genes were also measured as
 fragments per kilobase of exon per million mapped (FPKM) values calculated
 with Stringtie v2.0.4 (Pertea et al., 2015). The consistency between
 biological replicates was calculated with Pearson’s correlation
 coefficient, which was determined with the ‘stats’ package in R version
 4.2.1. Expression differences of key genes between the resistant and
 susceptible populations were calculated with two-way ANOVA followed by
 two-stage linear set-up procedure of Benjamini, Krieger, and Yekutieli on
 GraphPad Prism. #### Weighted Gene Co-expression Network Analysis Gene
 expression (in FPKM) served as input for Weighted Gene Co-expression
 Network Analysis (WGCNA). Genes with an expression value of zero for more
 than six samples were excluded from analysis. To explore the modules most
 correlated with insecticide resistance, a correlation analysis using
 resistance status was performed with the WGCNA package (Version 1.72.1)
 (Langfelder & Horvath, 2008) on R. Modules with a p-value &lt; 0.05
 were considered significant. Functional enrichment analysis (described
 below) was performed on the module with the highest correlation with
 resistance. #### Functional enrichment analysis Genes were functionally
 annotated using BLAST against the NCBI Drosophila melanogaster Annotation
 r6.32 based on the Release 6 plus ISO1 mitochondrial genome assembly
 (accession no. GCA_000001215.4) (dos Santos et al., 2015). Gene Ontology
 (GO) enrichment of genes were performed using ShinyGO 0.76.3 (Ge et al.,
 2020). GO terms and pathways were considered enriched if the false
 discovery rate (FDR) &lt; 0.05. ## Information for data tables -Suppl.
 Table 1: Statistics for zeta-cypermethrin bioassays Bioassays were
 performed to identify isofemale lines resistant to zeta-cypermethrin
 (Mustang® Maxx). Eight isofemale lines were tested (indicated as S#). Two
 isofemale lines from an untreated orchard (Wolfskill, W#) served as the
 susceptible control. Each biological replicate consisted of 5 males and 5
 females (n=8). Significance was determined by one-way ANOVA followed by
 Tukey’s multiple comparison test. -Suppl. Table 2: Statistics for spinosad
 bioassays Bioassays were performed to identify isofemale lines resistant
 to spinosad (Entrust®). Eight isofemale lines were tested (indicated as
 C#). Two isofemale lines from an untreated orchard (Wolfskill, W#) served
 as the susceptible control. Each biological replicate consisted of 5 males
 and 5 females (n=8). Significance was determined by one-way ANOVA followed
 by Tukey’s multiple comparison test. -Suppl. Table 4: DEGs in
 zeta-cypermethrin-resistant Drosophila suzukii isofemale line (line S3)
 relative to susceptible lines (lines S7 and S8) developed from the same
 field-collected population List of differentially expressed genes (DEGs)
 in zeta-cypermethrin resistant line S3 as compared to susceptible lines S7
 and S8. "SWD gene ID" and "SWD Gene Description" are
 based off of NCBI Drosophila suzukii Annotation Release 102 based on the
 LBDM_Dsuz_2.1.pri assembly (accession no. GCF_013340165.1) (Paris et al.,
 2020). "Fbgn" is the FlyBase Gene ID that is most similar (by
 sequence similarity) to the SWD gene ID. "Dmel gene ID" is the
 Drosophila melanogaster gene symbol associated with each Dmel gene ID.
 "Log2FoldChange" is the difference in expression between the
 resistant line (S3) to both susceptible lines (S7 and S8--pooled
 together). Positive values indicate that the gene is upregulated in the
 resistant line while negative values indicate that the gene is
 downregulated in the resistant line. "padj" is the
 Benjamini-Hochberg adjusted p-value, and -log10(padj) is the log
 transformation of the adjusted p-values, where higher values indicate more
 significant fold change differences. Data is sorted into 3 tabs: genes
 that are upregulated, downregulated, and not significant. Genes with fold
 change differences between resistant vs susceptible populations with a
 Benjamini-Hochberg adjusted p-value &lt; 0.05 were considered
 differentially expressed. -Suppl. Table 5: DEGs in
 zeta-cypermethrin-resistant Drosophila suzukii isofemale line (line S4)
 relative to susceptible lines (lines S7 and S8) developed from the same
 field-collected population List of differentially expressed genes (DEGs)
 in zeta-cypermethrin resistant line S4 as compared to susceptible lines S7
 and S8. "SWD gene ID" and "SWD Gene Description" are
 based off of NCBI Drosophila suzukii Annotation Release 102 based on the
 LBDM_Dsuz_2.1.pri assembly (accession no. GCF_013340165.1) (Paris et al.,
 2020). "Fbgn" is the FlyBase Gene ID that is most similar (by
 sequence similarity) to the SWD gene ID. "Dmel gene ID" is the
 Drosophila melanogaster gene symbol associated with each Dmel gene ID.
 "Log2FoldChange" is the difference in expression between the
 resistant line (S3) to both susceptible lines (S7 and S8--pooled
 together). Positive values indicate that the gene is upregulated in the
 resistant line while negative values indicate that the gene is
 downregulated in the resistant line. "padj" is the
 Benjamini-Hochberg adjusted p-value, and -log10(padj) is the log
 transformation of the adjusted p-values, where higher values indicate more
 significant fold change differences. Data is sorted into 3 tabs: genes
 that are upregulated, downregulated, and not significant. Genes with fold
 change differences between resistant vs susceptible populations with a
 Benjamini-Hochberg adjusted p-value &lt; 0.05 were considered
 differentially expressed. -Suppl. Table 6: Statistics comparing the
 expression of metabolic genes in zeta-cypermethrin-resistant vs.
 susceptible Drosophila suzukii Relative expression (FPKM) of cytochrome
 P450 genes (Cyp), heat shock proteins (Hsp), the carboxylesterase Cricklet
 (Clt), and glutathione-s-transferase E3 (GstE3) in the susceptible (S7 and
 S8) vs resistant (S3 and S4) groups. There are a total of 3 biological
 replicates of 8-10 females per line. Significance was determined by 2-way
 ANOVA. -Suppl. Table 7: Enrichment of DEGs in zeta-cypermethrin-resistant
 Drosophila suzukii (line S3) Enrichment pathways within the Kyoto
 Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) Biological
 Processes (bio proc) categories for genes up- or down-regulated in line S3
 (Suppl. Table 4). The table was produced by ShinyGo (Ge et al., 2020).
 "Enrichment FDR" (where FDR is the false discovery rate) is
 adjusted from the hypergeometric test. "nGenes" is number of
 differentially expressed genes that belong to the indicated pathway while
 "Pathway Genes" indicate the total number of genes that make up
 that pathway. FDR tells us how likely the enrichment is by chance.
 "Fold Enrichment" indicates how drastically genes of a certain
 pathway is overrepresented. "Pathway" is the name of the
 enriched pathway, and "URL" provides information on that
 pathway. Finally, "Genes" provides a list of differentially
 expressed genes that are in each pathway. -Suppl. Table 8: Enrichment of
 DEGs in zeta-cypermethrin-resistant Drosophila suzukii (line S4)
 Enrichment pathways within the Kyoto Encyclopedia of Genes and Genomes
 (KEGG) and Gene Ontology (GO) Biological Processes (bio proc) categories
 for genes up- or down-regulated in line S4 (Suppl. Table 5). The table was
 produced by ShinyGo (Ge et al., 2020). "Enrichment FDR" (where
 FDR is the false discovery rate) is adjusted from the hypergeometric test.
 "nGenes" is number of differentially expressed genes that belong
 to the indicated pathway while "Pathway Genes" indicate the
 total number of genes that make up that pathway. FDR tells us how likely
 the enrichment is by chance. "Fold Enrichment" indicates how
 drastically genes of a certain pathway is overrepresented.
 "Pathway" is the name -Suppl. Table 9: Genes in all WGCNA
 modules for zeta-cypermethrin-resistant Drosophila suzukii (line S3) To
 identify which genes in line S3 are most correlated with zeta-cypermethrin
 resistance, WGCNA was performed. Modules with a p-value &lt; 0.05 were
 considered significant. The data table was produced by the WGCNA package
 (Version 1.72.1) (Langfelder & Horvath, 2008) on R.
 "Transcript" is the NCBI Reference Sequence ID, and
 "LOC" is the SWD gene ID based off of NCBI Drosophila suzukii
 Annotation Release 102 based on the LBDM_Dsuz_2.1.pri assembly (accession
 no. GCF_013340165.1) (Paris et al., 2020). "Fbgn" is the FlyBase
 Gene ID that is most similar (by sequence similarity) to the SWD gene ID.
 "GeneSymbol" is the Drosophila melanogaster gene symbol
 associated with each Fbgn. "Module Color" is the clusters of
 highly interconnected genes. Modules correspond to clusters of genes with
 high absolute correlations. Gene significance (GS) is the correlation
 between gene expression and insecticide susceptibility.
 "p.GS.Susceptibility" is the p-value associate with the GS.
 Module membership (MM) is the association between gene expression and each
 module eigengene. The eigengene can be thought of as a weighted average
 expression profile. Finally, "p.MM" is the p-value associated
 with the Module Membership. -Suppl. Table 10: Enrichment of the turquoise
 module in zeta-cypermethrin-resistant Drosophila suzukii (line S3)
 Enrichment pathways within the Kyoto Encyclopedia of Genes and Genomes
 (KEGG) and Gene Ontology (GO) Biological Processes (bio proc) categories
 for genes within the turquoise cluster. Turquoise was the cluster most
 correlated with zeta-cypermethrin resistance in line S3. The table was
 produced by ShinyGo (Ge et al., 2020). "Enrichment FDR" (where
 FDR is the false discovery rate) is adjusted from the hypergeometric test.
 "nGenes" is number of differentially expressed genes that belong
 to the indicated pathway while "Pathway Genes" indicate the
 total number of genes that make up that pathway. FDR tells us how likely
 the enrichment is by chance. "Fold Enrichment" indicates how
 drastically genes of a certain pathway is overrepresented.
 "Pathway" is the name of the enriched pathway, and
 "URL" provides information on that pathway. Finally,
 "Genes" provides a list of differentially expressed genes that
 are in each pathway. -Suppl. Table 11: Genes in all WGCNA modules for
 zeta-cypermethrin-resistant Drosophila suzukii (line S4) To identify which
 genes in line S4 are most correlated with zeta-cypermethrin resistance,
 WGCNA was performed. Modules with a p-value &lt; 0.05 were considered
 significant. Data table was produced by the WGCNA package (Version 1.72.1)
 (Langfelder & Horvath, 2008) on R. "Transcript" is the NCBI
 Reference Sequence ID, and "LOC" is the SWD gene ID based off of
 NCBI Drosophila suzukii Annotation Release 102 based on the
 LBDM_Dsuz_2.1.pri assembly (accession no. GCF_013340165.1) (Paris et al.,
 2020). "Fbgn" is the FlyBase Gene ID that is most similar (by
 sequence similarity) to the SWD gene ID. "GeneSymbol" is the
 Drosophila melanogaster gene symbol associated with each Fbgn.
 "Module Color" is the clusters of highly interconnected genes.
 Modules correspond to clusters of genes with high absolute correlations.
 Gene significance (GS) is the correlation between gene expression and
 insecticide susceptibility. "p.GS.Susceptibility" is the p-value
 associate with the GS. Module membership (MM) is the association between
 gene expression and each module eigengene. The eigengene can be thought of
 as a weighted average expression profile. Finally, "p.MM" is the
 p-value associated with the Module Membership. -Suppl. Table 12:
 Enrichment of the turquoise module in zeta-cypermethrin-resistant
 Drosophila suzukii (line S4) Enrichment pathways within the Kyoto
 Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) Biological
 Processes (bio proc) categories for genes within the turquoise cluster.
 Turquoise was the cluster most correlated with zeta-cypermethrin
 resistance in line S4. The table was produced by ShinyGo (Ge et al.,
 2020). "Enrichment FDR" (where FDR is the false discovery rate)
 is adjusted from the hypergeometric test. "nGenes" is number of
 differentially expressed genes that belong to the indicated pathway while
 "Pathway Genes" indicate the total number of genes that make up
 that pathway. FDR tells us how likely the enrichment is by chance.
 "Fold Enrichment" indicates how drastically genes of a certain
 pathway is overrepresented. "Pathway" is the name of the
 enriched pathway, and "URL" provides information on that
 pathway. Finally, "Genes" provides a list of differentially
 expressed genes that are in each pathway. -Suppl. Table 13: DEGs in
 spinosad-resistant Drosophila suzukii isofemale line (line C3) relative to
 susceptible lines (lines C2 and C5) developed from the same
 field-collected population List of differentially expressed genes (DEGs)
 in zeta-cypermethrin resistant line C3 as compared to susceptible lines C2
 and C5. "SWD gene ID" and "SWD Gene Description" are
 based off of NCBI Drosophila suzukii Annotation Release 102 based on the
 LBDM_Dsuz_2.1.pri assembly (accession no. GCF_013340165.1) (Paris et al.,
 2020). "Fbgn" is the FlyBase Gene ID that is most similar (by
 sequence similarity) to the SWD gene ID. "Dmel gene ID" is the
 Drosophila melanogaster gene symbol associated with each Dmel gene ID.
 "Log2FoldChange" is the difference in expression between the
 resistant line (C3) to both susceptible lines (C2 and C5--pooled
 together). Positive values indicate that the gene is upregulated in the
 resistant line while negative values indicate that the gene is
 downregulated in the resistant line. "padj" is the
 Benjamini-Hochberg adjusted p-value, and -log10(padj) is the log
 transformation of the adjusted p-values, where higher values indicate more
 significant fold change differences. Data is sorted into 3 tabs: genes
 that are upregulated, downregulated, and not significant. Genes with fold
 change differences between resistant vs susceptible populations with a
 Benjamini-Hochberg adjusted p-value &lt; 0.05 were considered
 differentially expressed. -Suppl. Table 14: DEGs in spinosad-resistant
 Drosophila suzukii isofemale line (line C4) relative to susceptible lines
 (lines C2 and C5) developed from the same field-collected population List
 of differentially expressed genes (DEGs) in zeta-cypermethrin resistant
 line C4 as compared to susceptible lines C2 and C5. "SWD gene
 ID" and "SWD Gene Description" are based off of NCBI
 Drosophila suzukii Annotation Release 102 based on the LBDM_Dsuz_2.1.pri
 assembly (accession no. GCF_013340165.1) (Paris et al., 2020).
 "Fbgn" is the FlyBase Gene ID that is most similar (by sequence
 similarity) to the SWD gene ID. "Dmel gene ID" is the Drosophila
 melanogaster gene symbol associated with each Dmel gene ID.
 "Log2FoldChange" is the difference in expression between the
 resistant line (C4) to both susceptible lines (C2 and C5--pooled
 together). Positive values indicate that the gene is upregulated in the
 resistant line while negative values indicate that the gene is
 downregulated in the resistant line. "padj" is the
 Benjamini-Hochberg adjusted p-value, and -log10(padj) is the log
 transformation of the adjusted p-values, where higher values indicate more
 significant fold change differences. Data is sorted into 3 tabs: genes
 that are upregulated, downregulated, and not significant. Genes with fold
 change differences between resistant vs susceptible populations with a
 Benjamini-Hochberg adjusted p-value &lt; 0.05 were considered
 differentially expressed. -Suppl. Table 15: Statistics comparing the
 expression of metabolic and cuticular genes in spinosad-resistant vs.
 susceptible Drosophila suzukii Relative expression (FPKM) of metabolic and
 cuticular genes (twdl: tweedle; cpr: cuticular protein) in the susceptible
 (C2 and C5) and resistant (C3 and C4) groups.There are a total of 3
 biological replicates of 8-10 females per line. Significance was
 determined by 2-way ANOVA. -Suppl. Table 16: Enrichment of DEGs in
 spinosad-resistant Drosophila suzukii (line C3) Enrichment pathways within
 the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)
 Biological Processes (bio proc) categories for genes up- or down-regulated
 in line C3 (Suppl. Table 13). The table was produced by ShinyGo (Ge et
 al., 2020). "Enrichment FDR" (where FDR is the false discovery
 rate) is adjusted from the hypergeometric test. "nGenes" is
 number of differentially expressed genes that belong to the indicated
 pathway while "Pathway Genes" indicate the total number of genes
 that make up that pathway. FDR tells us how likely the enrichment is by
 chance. "Fold Enrichment" indicates how drastically genes of a
 certain pathway is overrepresented. "Pathway" is the name of the
 enriched pathway, and "URL" provides information on that
 pathway. Finally, "Genes" provides a list of differentially
 expressed genes that are in each pathway. -Suppl. Table 17: Enrichment of
 DEGs in spinosad-resistant Drosophila suzukii (line C4) Enrichment
 pathways within the Kyoto Encyclopedia of Genes and Genomes (KEGG) and
 Gene Ontology (GO) Biological Processes (bio proc) categories for genes
 up- or down-regulated in line C4 (Suppl. Table 14). The table was produced
 by ShinyGo (Ge et al., 2020). "Enrichment FDR" (where FDR is the
 false discovery rate) is adjusted from the hypergeometric test.
 "nGenes" is number of differentially expressed genes that belong
 to the indicated pathway while "Pathway Genes" indicate the
 total number of genes that make up that pathway. FDR tells us how likely
 the enrichment is by chance. "Fold Enrichment" indicates how
 drastically genes of a certain pathway is overrepresented.
 "Pathway" is the name of the enriched pathway, and
 "URL" provides information on that pathway. Finally,
 "Genes" provides a list of differentially expressed genes that
 are in each pathway. -Suppl. Table 18: Genes in all WGCNA modules for
 spinosasd-resistant Drosophila suzukii (line C3) To identify which genes
 in line C3 are most correlated with spinosad resistance, WGCNA was
 performed. Modules with a p-value &lt; 0.05 were considered significant.
 Data table was produced by the WGCNA package (Version 1.72.1) (Langfelder
 & Horvath, 2008) on R. "Transcript" is the NCBI Reference
 Sequence ID, and "LOC" is the SWD gene ID based off of NCBI
 Drosophila suzukii Annotation Release 102 based on the LBDM_Dsuz_2.1.pri
 assembly (accession no. GCF_013340165.1) (Paris et al., 2020).
 "Fbgn" is the FlyBase Gene ID that is most similar (by sequence
 similarity) to the SWD gene ID. "GeneSymbol" is the Drosophila
 melanogaster gene symbol associated with each Fbgn. "Module
 Color" is the clusters of highly interconnected genes. Modules
 correspond to clusters of genes with high absolute correlations. Gene
 significance (GS) is the correlation between gene expression and
 insecticide susceptibility. "p.GS.Susceptibility" is the p-value
 associate with the GS. Module membership (MM) is the association between
 gene expression and each module eigengene. The eigengene can be thought of
 as a weighted average expression profile. Finally, "p.MM" is the
 p-value associated with the Module Membership. -Suppl. Table 19: Genes in
 all WGCNA modules for spinosasd-resistant Drosophila suzukii (line C4) To
 identify which genes in line C4 are most correlated with spinosad
 resistance, WGCNA was performed. Modules with a p-value &lt; 0.05 were
 considered significant. Data table was produced by the WGCNA package
 (Version 1.72.1) (Langfelder & Horvath, 2008) on R.
 "Transcript" is the NCBI Reference Sequence ID, and
 "LOC" is the SWD gene ID based off of NCBI Drosophila suzukii
 Annotation Release 102 based on the LBDM_Dsuz_2.1.pri assembly (accession
 no. GCF_013340165.1) (Paris et al., 2020). "Fbgn" is the FlyBase
 Gene ID that is most similar (by sequence similarity) to the SWD gene ID.
 "GeneSymbol" is the Drosophila melanogaster gene symbol
 associated with each Fbgn. "Module Color" is the clusters of
 highly interconnected genes. Modules correspond to clusters of genes with
 high absolute correlations. Gene significance (GS) is the correlation
 between gene expression and insecticide susceptibility.
 "p.GS.Susceptibility" is the p-value associate with the GS.
 Module membership (MM) is the association between gene expression and each
 module eigengene. The eigengene can be thought of as a weighted average
 expression profile. Finally, "p.MM" is the p-value associated
 with the Module Membership. -Suppl. Table 20: Enrichment of the green
 module in spinosasd-resistant Drosophila suzukii (line C4) Enrichment
 pathways within the Kyoto Encyclopedia of Genes and Genomes (KEGG) and
 Gene Ontology (GO) Biological Processes (bio proc) categories for genes
 within the green cluster. Green was the cluster most correlated with
 spinosad resistance in line C4. The table was produced by ShinyGo (Ge et
 al., 2020). "Enrichment FDR" (where FDR is the false discovery
 rate) is adjusted from the hypergeometric test. "nGenes" is
 number of differentially expressed genes that belong to the indicated
 pathway while "Pathway Genes" indicate the total number of genes
 that make up that pathway. FDR tells us how likely the enrichment is by
 chance. "Fold Enrichment" indicates how drastically genes of a
 certain pathway is overrepresented. "Pathway" is the name of the
 enriched pathway, and "URL" provides information on that
 pathway. Finally, "Genes" provides a list of differentially
 expressed genes that are in each pathway. -Suppl. Table 21: Statistics for
 quantitative PCR (qPCR)-based assays with 2022 D. suzukii populations
 Results of a one sample T and Wilcoxon Test comparing the expression of
 cytochrome P450 (cyp) genes, tweedle (twdl) genes, and ecdysone receptor
 (ecR) in 2022 field-collected populations compared to the average gene
 expression of the susceptible (S7 and C2) and resistant (S3 and C3)
 isofemale lines (established from 2019 collections) (n=5 biological
 replicates of 8-10 females). One-way ANOVA followed by Holm-Sidak’s
 multiple comparisons test was also performed to assess expression
 differences between the 2022 F1 populations and the 2019 resistant
 controls. -Suppl. Table 22: Statistics for bioassays with D. melanogaster
 mutants To confirm knockdown of genes associated with insecticide
 resistance, the relative expression of cyp4d14, cyp4d8, and cpr6D in
 wild-type (w1118) and mutant fly lines was compared to a hypothetical mean
 of 1 using a one-sample t and Wilcoxon Test (n=4). Next, mortality of
 w1118 and mutant flies when exposed to sublethal concentrations of
 zeta-cypermethrin and spinosad (n=5 biological replicates of 5 males and 5
 females) were compared using two-way ANOVA followed by Holm-Sidak’s
 multiple comparisons test. Note: Missing values are denoted by NA. ##
 Sharing/Access Raw Data can be found under BioProject PRJNA983428