Fig 1.
Circos plot of beet armyworm genome.
Tracks from innermost to outermost are: I. Long terminal repeat elements (LTRs), II. Long interspersed nuclear elements (LINEs), III. Short interspersed nuclear elements (SINEs), IV. DNA transposons, V. Gene density, VI. GC content, and VII. Chromosome ideogram with numbers indicating the chromosome nomenclature order and size (in Mb). A 10 kb sliding window is shown for the GC content track and 100 kb for all other tracks.
Table 1.
Genome assembly metrics for four Noctuidae species.
Table 2.
Genome annotation statistics of the beet armyworm.
Fig 2.
Experimental design for crosses, bioassays and bulked segregant analysis (BSA) to map emamectin benzoate resistance.
(A) Single-pair crosses between JZ-S and WH-EB virgin adults produced families of hybrid F1 offspring. A male F1 was backcrossed to a female JZ-S to produce the backcross (BC) family. Progeny from the BC family were exposed to a diagnostic concentration of EB of which 120 survivors and their F1 parents were analyzed by next-generation sequencing (NGS). (B) BSA mapping revealed a 1 Mb genomic region in chromosome 17 highly correlated with EB resistance in WH-EB. The mean frequency deviation values obtained from a sliding window analysis (1 Mb window with 100 Kb step) were plotted across all 31 chromosomes of the genome. The red dashed line corresponds to the top 1% threshold of the SNP-index likely containing changes linked with EB resistance. (C) Expanded map of chromosome 17 (from 15 Mb to 16 Mb) that includes the CYP9A gene cluster (15.71 Mb to 15.81Mb).
Fig 3.
Genome mapping of the CYP9A gene cluster by CRISPR/Cas9 gene editing.
Deletions in the CYP9A gene cluster are indicated with dotted boxes and include ΔCYP9A40-CYP9A98, ΔCYP9A40-CYP9A107, ΔCYP9A107-CYP9A98, and ΔCYP9A186. Resistance ratios to EB for each of the homozygous strains harboring both the natural CYP9A186-F116V mutation in resistant WH-EB strain and the CRISPR/Cas9-edited strains are shown.
Table 3.
Response to abamectin and emamectin benzoate of beet armyworm larvae from a susceptible strain (WH-S), a resistant strain (WH-EB), and four CRISPR/Cas9 gene-edited CYP9A knockout strains (dA40-A98, dA40-A107, dA107-A98, and dA186).
Fig 4.
Identification of the CYP9A186 T346G mutation resulting in the F116V substitution and quantitative real-time PCR transcriptional analysis of CYP9A186.
(A) Alignment of the deduced CYP9A186 amino acid sequences from WH-EB and WH-S with a translated human cytochrome P450 (e.g., CYP3A4). The F116V substitution is shown with a red arrow. The predicted locations of alpha helical secondary structures are marked based on the template of the CYP3A4 protein structure (1TQN) using ESPript 3.0. (B) Representative chromatograms from direct sequencing of CYP9A186 PCR products from WH-EB and WH-S showing the T346G mutation. (C) qRT-PCR analysis of CYP9A107, CYP9A27, CYP9A11, CYP9A186 and CYP9A98 in midgut and fat body from WH-S and WH-EB. Data shown represent means ± SE derived from four biological replicates. Asterisks indicate significant differences between the strains (Student’s t-test, p<0.05).
Fig 5.
Metabolism of emamectin benzoate and abamectin by microsomes containing recombinant CYP9A186 and CYP9A186-F116V.
(A) Specific activity (pmol/min/pmol P450) of EB and abamectin hydrolysis. Data are mean values ± SEM (n = 6). Dotted lines show mean limits of detection (LOD). There is no significant difference between WT and LOD (One sample t-test, p = 0.69 and 0.83 for emamectin benzoate and abamectin, respectively). (B) MRM signals of emamectin benzoate and its metabolites. (C) MRM signals of abamectin and its metabolites.