Mutations of the complex I PSST target gene confers acaricide resistance and a fitness cost in Panonychus citri

Abstract

Background: Pesticide resistance is a serious problem that threatens crop industries. Major resistance towards pyridaben, an acaricidal inhibitor of mitochondrial electron transport complex I (METI-Is), has been reported in tetranychids following its extensive use worldwide. Understanding mechanisms of pyridaben resistance is crucial for sustainable resistance management.

Results: The inheritance of pyridaben resistance was incompletely recessive and controlled by multiple genes in P. citri, which was determined by reciprocal crosses and backcross experiments. Bulked segregant analysis was performed to identify gene loci underlying pyridaben resistance. Subsequently, the two PSST-subunit mutations H107R and the previously undiscovered V103I mutation were positively correlated with pyridaben resistance in different populations or strains by single mite genotyping. The bioassay further showed that H107R contributed to moderate resistance, while V103I in combination with H107R was responsible for a very high level of resistance in homozygous P. citri strains. These contributions to pyridaben resistance were also verified in transgenic Drosophila through the introduction of the wildtype, single- or double-mutated P. citri PSST subunit. In addition, life-table analysis and behavioral measures were conducted to assess the fitness cost associated with resistance development. Accompanied by reduced ATP levels and complex I activity, a fitness cost was observed as reduced fecundity and lower mobility due to PSST mutations.

Conclusions: Our findings provide direct evidence that PSST mutations conferred the evolution of pyridaben resistance but simultaneously led to a fitness cost due to functional defects in complex I. These data provide theoretical insights into sustainable resistance management in agricultural production.

Keywords: Bulked segregation analysis; Citrus red mite; Fitness cost; Inheritance; Target-site mutation.

Fig. 1

Log concentration–probit lines of resistant/susceptible parents, their reciprocal offspring (F₁RS/F₁SR), and backcross progeny (BC/BC'). Lab_S/Pyr_Control: susceptible parents, Pyr_Rs/Pyr_R: resistant parents, RS and SR: the F1 progeny of reciprocal crosses, BC/BC′-O/E: the observed and excepted mortality, respectively, of backcross progeny

Fig. 2

The genome locations and delta-SNPs of candidate genes. The blue spots were SNPs; the dotted line was the 95% threshold; the red triangle was the gene location on contig

Fig. 3

Sequence alignment of insect PSST subunits. Lab_S/Pyr_Control: susceptible Panonychus citri strains, Pyr_Rs/Pyr_R: pyridaben-resistant Panonychus citri strains. Tetranychus urticae, tetur07g05240 (https://bioinformatics.psb.ugent.be/orcae/overview/Tetur); Drosophila melanogaster, NP_001285313.1; Ixodes scapularis: XP_029835921.4; Halotydeus destructor: KAI1301479.1; Dermatophagoides pteronyssinus: XP_027199782.1; Haemaphysalis longicornis: KAH9369938.1; Tyrophagus putrescentiae: KAH9392596.1; Galendromus occidentalis: XP_003746867.1; Aedes albopictus: XP_019534269.1; Bactrocera dorsalis: XP_011206258.1; Plutella xylostella: XP_011549720.1; Bombyx mori: XP_012550867.2; Bemisia tabaci: CAH0394144.1; Aphis gossypii: XP_027842069.1; Tribolium castaneum: XP_008201665.2

Fig. 4

Mutation frequency of V103I and H107R in Panonychus citri. A and B Mutation frequency analysis of V103I and H107R in four lab strains. Lab_S/Pyr_Control: susceptible Panonychus citri strains, Pyr_Rs/Pyr_R: pyridaben resistant Panonychus citri strains. LC₉₀ denotes the mites treated with LC₉₀ of pyridaben and CK are the control mites treated with ddH₂0. C and D Mutation frequency of V103I and H107R in different Panonychus citri populations collected from seven citrus gardens. The coordinates of the populations are listed in Table S6

Fig. 5

Susceptibility to METIs in four transgenic Drosophila. The acaricides contact bioassay in transgenic flies was performed as previously described [15]. Acaricide filter papers with a concentration of 1000, 2000, 1200, 1000, and 1600 mg/L of pyridaben, fenpyroximate, tolfenpyrad, fenazaquin, and tebufenpyrad, respectively, were evaluated. Data are presented as the mean ± SEM. Asterisks indicate a significant difference; ***, P < 0.001; ns means no significant difference. Significant differences between wild-type and mutant lines were determined using a Student’s t-test

Fig. 6

Docking predictions of pyridaben in models of Panonychus citri mitochondrial complex I. Pyridaben (black sticks) docked in models of complex I with A wildtype PSST subunit or B the V103I + H107R double mutant. Amino acids within < 4.5 Å of pyridaben are shown as white sticks. The amino acids at positions 103 and 107 of the PSST subunit are shown as red sticks

Fig. 7

ATP level and mitochondrial complex I activity detection in Panonychus citri and transgenic Drosophila. A, B, and C Transgenic flies with single or combined P. citri PSST mutations; D and E Panonychus citri strains with wild-type and mutant homozygous PSST. Data indicate the mean ± SEM; different letters indicate significant differences based on one-way ANOVA

Fig. 8

Survival rate and fecundity assessment in Panonychus citri. A Age-specific survival rate (l_x); B age-stage fecundity of females (f_x5) (eggs/female); C age-specific fecundity (m_x);D age-specific net reproductive rate of the population (l_xm_x) in PSST_WT, PSST_H107R, and PSST_V103I + H107R

Fig. 9

Behavioral assays. A Climbing rate of Panonychus citri. “*” indicates a significant difference, P < 0.05, Student’s t-test. B High temperature (38 °C) tolerance of transgenic flies. “*” indicates a significant difference, P < 0.05, Student’s t-test. C and D Locomotion of transgenic (C) male and (D) female flies during 24 h cycles (gray bars, light; black bars, darkness)