DOI: 10.25338/b8ph17
title: Thesis: Transcriptome analysis of insecticide resistant Drosophila suzukii

description: Drosophila suzukii, also known as spotted wing Drosophila (SWD), is an
 invasive agricultural pest that is a threat to berry production due to a
 serrated ovipositor on female flies that enables them to lay eggs in
 soft-skinned, ripening fruits. Currently, growers rely heavily on the use
 of insecticides to manage D. suzukii. Over the past six years, insecticide
 resistance has been detected in D. suzukii in California, but the
 molecular mechanism underlying this adaptation is unknown. Therefore, we
 sought to identify the molecular mechanism conferring insecticide
 resistance in these pests. We generated isogenic lines from
 field-collected resistant populations and sequenced the transcriptomes of
 two pyrethroid- and two spinosad-resistant lines. In both
 pyrethroid-resistant isogenic lines and one spinosad-resistant line, we
 identified an overexpression of metabolic genes that have been previously
 implicated in insecticide resistance in other insect pests. In the other
 spinosad-resistant line, we observed an overexpression of cuticular genes
 that have been linked to insecticide resistance. Additionally, we observed
 decreased expression of the pyrethroid target gene, paralytic, in both
 pyrethroid-resistant lines. Our findings enabled the development of
 molecular diagnostics that can be used to monitor resistance development
 in the field, specifically by monitoring overexpression of specific target
 genes. Finally, long-read sequencing reveals transcriptome-wide changes in
 the expression of different splice variant isoforms, suggesting that
 alternative splicing can be an additional mechanism enabling insecticide
 resistance. This study is the first to characterize the molecular
 mechanisms of insecticide resistance in field-collected D. suzukii and
 provides insights into how current management practices can be improved.
description: # Transcriptome analysis of Drosophila suzukii reveals molecular
 mechanisms conferring pyrethroid and spinosad resistance CA Tabuloc, CR
 Carlson, F Ganjisaffar, H Zhang, CC Truong, C Chen, KM Lewald, S Hidalgo,
 FG Zalom, JC Chiu --- # Data generated from Illumina short-read sequencing
 (PE150) and PacBio long-read sequencing (Two 8M SMRT cells). RNA was
 extracted from isogenic 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. Both 8M SMRT Cells of Iso-Seq data were pooled together and
 run through the isoseq3 pipeline
 (https://github.com/PacificBiosciences/IsoSeq, v. 3.8.0) to generate
 full-length non-chimeric (FLNC) isoform sequences. PolyA and concatemer
 sequences were removed from PacBio sequencing reads using ‘isoseq refine.’
 To maximize detection of rare isoforms, both high-quality and low-quality
 FLNC isoform sequences were concatenated and mapped to the D. suzukii
 reference genome (accession no. GCF\_013340165.1) (Paris et al., 2020)
 using minimap2 (H. Li, 2018) (v. 2.24-r1122; -ax splice -uf --secondary=no
 -C5). Mapped FLNC isoforms were then filtered and collapsed using
 cDNA\_Cupcake (https://github.com/Magdoll/cDNA\_Cupcake,
 collapse\_isoforms\_by\_sam.py), retaining nonredundant isoforms.
 Nonredundant isoforms were classified against the reference genome and
 transcriptome using SQANTI3 QC v. 5.1 (Tardaguila et al., 2018). All
 short-read RNA-Seq data were used as evidence in SQANTI3 QC to validate
 isoforms. Isoforms flagged as artifacts by SQANTI3 QC were filtered using
 the SQANTI3 “rules” filter, retaining all full-splice matching isoforms
 not flagged as an intrapriming artifact and all non-full-splice matching
 isoforms not flagged as an intrapriming artifact, having a
 reverse-transcriptase-switching junction, and/or non-canonical splice
 junctions without short-read support. After filtering, SQANTI3 rescue was
 run to recover discarded reference transcripts that have long-read
 support, resulting in a final long-read supported transcriptome. This
 long-read transcriptome was then merged with the reference transcriptome
 to create a final, nonredundant expanded transcriptome. Splice junction
 and exon counts were then generated using Quality of RNA-Seq Tool-Set
 (QoRTs) (Hartley & Mullikin, 2015). Differential usage of exons
 and splice junctions was determined with JunctionSeq (Hartley &
 Mullikin, 2016). Features with FDR &lt; 0.05 were considered
 differentially expressed/utilized. # Information for data tables -Suppl.
 Table 2: Statistics for zeta-cypermethrin bioassay Bioassays were
 performed to identify isogenic lines resistant to zeta-cypermethrin
 (Mustang® Maxx). Eight isogenic lines were tested (indicated as S#). Two
 isogenic 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 3: Statistics for spinosad
 bioassay Bioassays were performed to identify isogenic lines resistant to
 spinosad (Entrust®). Eight isogenic lines were tested (indicated as C#).
 Two isogenic 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 5: DEGs in
 zeta-cypermethrin-resistant Drosophila suzukii (line S3) 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 6:
 DEGs in zeta-cypermethrin-resistant Drosophila suzukii (line S4) 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 7:
 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 8: 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 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 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 9: 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 6). 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 10: 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 11:
 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 12: 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 13: 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 14: DEGs in spinosad-resistant
 Drosophila suzukii (line C3) 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 15: DEGs in spinosad-resistant
 Drosophila suzukii (line C4) 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 16: 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 17: Enrichment of DEGs in
 spinosasd-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 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: 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 15). 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 19: 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 20: 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 21:
 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 22: Differential usage of splice
 junctions, exons, and introns in insecticide-resistant Drosophila suzukii
 Differential usage of splice junctions, exons, and introns for each
 resistant D. suzukii line to both susceptible lines was determined using
 JunctionSeq (Hartley & Mullikin, 2016). Alternative splicing (AS)
 events for each line is in a separate tab. AS categories include Exon
 Skipping (ES), Exon Inclusion (EI), Intron Retention (IR), and Alternative
 splice site (Altss). "Gene ID" is the D. suzukii gene ID from
 the updated transcriptome reference. "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). "Novel" indicates that the gene was not previously
 annotated in the LBDM\_Dsuz\_2.1.pri assembly. "Fbgn" is the
 FlyBase Gene ID that is most similar (by sequence similarity) to the SWD
 gene ID. "GeneSym" is the Drosophila melanogaster gene symbol
 associated with each Fbgn. The remainder of the table is an output from
 JunctionSeq. A full description of each column can be found on pages 14-15
 of the JunctionSeq User Manual:
 http://hartleys.github.io/JunctionSeq/doc/JunctionSeq.pdf. -Suppl. Table
 23: Pairwise comparisons of differentially spliced genes Differential
 usage of splice junctions, exons, and introns for pairwise comparisons
 between each resistant D. suzukii line to each susceptible line was
 determined using JunctionSeq (Hartley & Mullikin, 2016).
 Alternative splicing (AS) events for each line is in a separate tab. AS
 categories include Exon Skipping (ES), Exon Inclusion (EI), Intron
 Retention (IR), Alternative splice site (Altss). "Gene ID" is
 the D. suzukii gene ID from the updated genome annotation. "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). "Novel" indicates that
 the gene was not previously annotated in the LBDM\_Dsuz\_2.1.pri assembly.
 "Fbgn" is the FlyBase Gene ID that is most similar (by sequence
 similarity) to the SWD gene ID. "GeneSym" is the Drosophila
 melanogaster gene symbol associated with each Fbgn. The remainder of the
 table is an output from JunctionSeq. A full description of each column can
 be found on pages 14-15 of the JunctionSeq User Manual:
 http://hartleys.github.io/JunctionSeq/doc/JunctionSeq.pdf. -Suppl. Table
 24: Enrichment of differentially spliced genes Enrichment pathways within
 the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)
 Biological Processes (bio proc) categories for differentially spliced
 genes from each pairwise comparison (Suppl. Table 23). The table was
 produced using ShinyGo. "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. "Fold
 Enrichment" is defined as the percentage of genes in your list
 belonging to a pathway, divided by the corresponding percentage in the
 background. 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. Note: Missing values are denoted
 by NA. # Raw Data can be found under BioProject PRJNA983428