Susceptibility of the two-spotted spider mite Tetranychus urticae to cyetpyrafen.

(a-d) Collection maps of populations used for bioassay in different years. Red and green points represent resistant and susceptible populations, respectively. (e) LC50 values of each population collected in 2018 (7 populations) (Chen et al., 2019), 2020 (14), 2021 (10), 2024 (13), and a laboratory-selected resistant population (LabR) and a parallel reared susceptible population (LabS) to cyetpyrafen. The green bars show the susceptible populations, while the red bars show the resistant populations.

Signals of selective sweep across genomes of the two-spotted spider mite Tetranychus urticae.

(a) PBE values across the genome for eight historic populations collected in 2017 before the commercial release of cyetpyrafen. Chromosomes are colored alternately dark grey and light grey. (b) PBE values for one lab-selected resistant population, 10 resistant field populations and one susceptible population collected in 2021 and 2024 after the release of cyetpyrafen. Chromosomes are colored alternately dark red and light red for resistant populations, dark green and light green for susceptible populations. Allele frequency (pool-seq data) and haplotypes of mutations are indicated near the corresponding sdhB and sdhD genes of each population in (a) and (b). (c) PBE values for genomic regions around the sdhB gene, comparing values between the historic (left) and recently collected (right) populations. (d) As above but for the sdhD gene. Each point represents a genomic window of 5-kp wide when compared to reference genomes involving the susceptible laboratory line (LabS) and a susceptible field population (NMHH2). The vertical lines indicate the position of sdh genes, while the horizontal lines represent the 1% threshold of PBE values.

Amino acid mutations on sdhB and sdhD genes in populations of the two-spotted spider mite Tetranychus urticae.

(a, b) Sequence logos at mutations related to cyetpyrafen resistance in sdhB (a, 757 sequences) and sdhD (b, 158 sequences). The numbers under logos indicate the position of amino acid residues on protein chains. Excluding T. urticae, conserved amino acids without any alternative are labeled with asterisks. Amino acids of T. urticae are shown below. Amino acids in red indicate mutations. (c) Amino acid mutation sites on a 3D structure of the wild-type succinate dehydrogenase of T. urticae. The sdhA, sdhB, sdhC and sdhD chains are shown in yellow, blue, green, and red, respectively. Solid 3D shapes indicate mutations on five residues identified in this study (His258, Ile260, Asp116, Arg119, and Pro120) and one residue (Ser56) reported by previous studies (Njiru et al., 2022, Sugimoto et al., 2020) that confer resistance to SDH inhibitors. (d) Temporal variation in allele frequencies of resistance mutations in populations of T. urticae determined through Sanger sequencing of target genes. Each dot represents one population. Allele frequencies of populations collected from 2013-2018, 2020, 2021, and 2024 are shown as different colors. A258S was omitted as it was only present in one population (SDRZ) sampled in 2017.

Genetic structure and haplotype networks of sdhB and sdhD genes of the two-spotted spider mite Tetranychus urticae.

(a) Treemix result showing genetic structure and gene flow among populations. The mutations detected by pool-seq data were labeled near the corresponding population. (b) Haplotype network of sdhB gene inferred from homozygous individuals. In figures (a) and (d), the red population codes show resistant populations, the green ones show the susceptible populations, while the black ones show populations of unknown resistance status. (c) Haplotype network of sdhD gene inferred from homozygous individuals. The colored haplotypes are those carrying the resistant mutation. (d) Presence of each haplotype carrying the target mutations in two periods. In the early resistance development stage of 2020-2021, each haplotype was mainly distributed across a limited range, while in 2024, the same haplotype was distributed across a wide geographical range. The filled rectangle indicates that the entire haplotype is present in the population, while a colored border suggests that only the mutations were detected, but the haplotype was not assigned. The number of populations carrying the corresponding mutation are listed in the right column for the two time periods.

Function of target-site mutations in the sdhB and sdhD genes and survival of two-spotted spider mite Tetranychus urticae in cyetpyrafen resistance.

(a-c) Association between frequency of target-site mutations in the sdhB and sdhD genes and survival of two-spotted spider mite T. urticae exposed to 1000 mg/L of cyetpyrafen. (a) Allele frequencies are measured as the predominant mutation in the population at either the sdhB or sdhD genes. (b) Allele frequency is the proportion of individuals carrying a resistant allele at either the sdhB or sdhD genes. (c) Allele frequency is the proportion of individuals carrying a homozygous genotype for either the sdhB or sdhD genes. Red and dark green points represent resistant and susceptible populations, respectively. Blue dashed lines indicate the expected association between two variables, i.e. y = x. (d) Mortality of mite lines carrying one of the homozygous mutations or a combination of two mutations exposed to 1000 mg/L of cyetpyrafen. V, I260V; G, D116G; E, R119E; L, R119L; V and C, I260V and R119C; S, susceptible populations LabS. Populations with the same homozygous mutations are shown in the same color. Error bars show standard error of the mean.