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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12915-025-02288-7.

Background

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is central to oxidative phosphorylation in organisms [1]. Complex I converts energy stored in chemical bonds into a proton gradient across the membrane that subsequently drives the synthesis of adenosine triphosphate (ATP). As an essential respiratory enzyme and an important contributor to cellular oxidative stress, complex I dysfunctions arising from mutations in its subunits and assembly factors cause a diverse set of inherited neuromuscular and metabolic diseases [2].

The quinone binding tunnel in complex I is long and amphipathic; the top and bottom sections are hydrophobic, while the central section is surrounded by many charged residues [3]. The charged region may be important in linking redox catalysis to proton translocation because it sits at the start of a chain of charged residues that leads to the membrane plane [4]. This network of charged residues may be involved in propagating energy liberated by quinone-mediated redox catalysis to proton pumping [5]. The former includes the 49-kDa β1-β2 loop and the PSST loop, which form the deepest part of the quinone binding pocket [6]. Many inhibitors of complex I can bind in the distinctively long and narrow channel that leads “upward” from the membrane plane, in which its highly hydrophobic substrate ubiquinone-10 is proposed to bind [3]. Thus far, the ubiquinone-analog complex I inhibitors, piericidin A and pyridaben, have been observed to bind to the top of the channel through conserved interaction with PSST or 49 kDa [7, 8]. These inhibitors hinder electron transfer and lead to the functional impairment of complex I, leading to the death of pests or mites.

Due to the long-term and extensive use of pyridaben, resistance in tetranychids has been reported around the world [9–11]. However, limited reports have uncovered the target-site mutations contributing to pyridaben resistance. Arthropods have evolved pesticide resistance through two main mechanisms: (1) quantitative or qualitative changes in major detoxification enzymes and transporters (pharmacokinetic mechanisms) and (2) decreased sensitivity of pesticide targets caused by amino acid substitutions [12]. The discovery of the mutation H92R (Yarrowia liplytica numbering) in the PSST subunit of complex I implicated this mutation in resistance to mitochondrial electron transport complex I inhibitors (METI-Is) [11]. The H92R mutation (numbered H107R in P. citri) together with a new mutation (A94V) has been identified and their involvement in fenpyroximate resistance and the cross-resistance to pyridaben and tebufenpyrad was confirmed in P. citri [13]. In Tetranychus urticae, bulked segregant analysis (BSA) mapping has revealed genes underlying resistance to fenpyroximate [14]. The known H92R mutation of the PSST subunit was shown to be associated with resistance to this acaricide and, additionally, a nonsynonymous variant of cytochrome P450 reductase (CPR) was associated with resistance to both pyridaben and tebufenpyrad [14].

Panonychus citri is a major cosmopolitan citrus pest that feeds on the leaves, branches, and fruit of citrus crops [9]. Leaf and fruit drop, followed by loss of an entire plant may occur when the mites are not controlled in a timely manner [10]. Thus, the occurrence of P. citri threatens the healthy development of the citrus industry. In this study, we exploited the inherited form of pyridaben resistance based on the P. citri near-isogenic lines using the recombinant self-cross method [9]. BSA mapping was adapted to comprehensively investigate the quantitative (polygenic) genetic architecture of pyridaben resistance. The relationship between potential mutations and pyridaben resistance was then assessed by genotyping different populations and sensitivity detection in P. citri within the homozygous genotype. In addition, the changes in sensitivity in transgenic Drosophila melanogaster flies were assessed after the introduction of single or combined mutations of the PSST subunit. We carried out molecular modelling to determine the location of V103 and H107 relative to the quinone-binding tunnel. Finally, we assessed the complex I activity, ATP level, and fitness in P. citri and transgenic flies.

Results

Mode of inheritance of pyridaben resistance

To determine the inheritance of pyridaben resistance in P. citri, near-isogenic lines were constructed using the recombination self-cross method described in a previous study [9]. Two susceptible strains (Lab_S and Pyr_Control) and two resistant strains (Pyr_R, and Pyr_Rs) were used for hybridization and backcross to obtain the hybrid F1 generation. Subsequently, the backcross F2 generation was obtained by sibling mating the hybrid F1 generation. The D-values of the hybrid F1 generation were all between 0 and 1, indicating incomplete dominance inheritance for pyridaben resistance in P. citri (Table 1). The log concentration-probit response lines showed that the hybrid F1 generation was more convergent with resistant strains (Fig. 1A and D), further indicating incomplete dominant inheritance for pyridaben resistance in P. citri. In addition, there was a coincidence interval in the 95% CI of D-values between orthogonal and counter-crossed F1 progeny, meaning that the related resistance genes were located at the autosome in P. citri. χ² analyses of the backcrosses revealed a significant difference between the observed and expected mortalities, indicating that pyridaben resistance in P. citri was controlled by multiple genes (Additional file 1: Table S1 and Fig. 1B, C, E, and F). In addition, the observed log concentration-probit line of BC-O/BC′-O in the concentration–response data for backcross progeny was separated from the expected lines of BC-E/BC′-E (Fig. 1B, C, E, and F), further revealing that pyridaben resistance was conferred by multiple genes. In conclusion, pyridaben resistance in P. citri resulted from incomplete dominant inheritance controlled by multiple genes located on autosomes.

Table 1.

Pyridaben bioassay of Lab_S/Pyr_Control, Pyr_R/Pyr_Rs, and their reciprocal crosses, and backcross populations of Panonychus citri

Strains	N	Slope (± SE)	LC50 (mg/L) 95% CI	χ2	RR	D (95% CI)
Pyr_Control	945	1.709 ± 0.128	3.100 (2.667–3.548)	7.902	1
Lab_S	1175	2.574 ± 0.163	0.325 (0.291–0.366)	4.316	1
Pyr_Rs	946	1.348 ± 0.098	1732.525 (1406.561–2140.917)	9.419	558.879
Pyr_R	516	1.586 ± 0.126	844.707 (626.041–1154.176)	7.3638	2599.098
F1RS (Rs♀ × Control♂)	969	2.608 ± 0.164	258.737 (233.037–366.250)	10.742	83.463	0.399 (0.354–0.443)
F1RS (R♀ × Lab_S♂)	1151	1.527 ± 0.089	96.685 (78.634–118.203)	13.601	297.492	0.449 (0.390–0.579)
F1SR (Control♀ × Rs♂)	862	2.347 ± 0.169	212.689 (189.756–239.131)	10.098	68.609	0.337 (0.295–0.378)
F1SR (Lab_S ♀ × R♂)	806	1.216 ± 0.102	46.364 (32.814–69.104)	13.169	142.658	0.262 (0.131–0.392)
BC (F1RS♀ × Rs♂)	944	1.986 ± 0.148	221.577 (197.161–248.343)	5.427	74.476
BC (F1RS♀ × R♂)	882	0.743 ± 0.075	91.580 (58.289–167.497)	10.185	281.785
BC′ (F1SR♀ × Control♂)	1169	1.575 ± 0.108	39.671 (34.369–45.561)	10.893	12.797
BC′ (F1SR♀ × SS♂)	1317	0.941 ± 0.046	6.291 (4.931–7.947)	11.418	19.357

N, the number of test mites; 95% CI, 95% confident limits of LC₅₀; χ², goodness-of-fit test; D, degree of dominance (− 1 completely recessive to + 1 completely dominant)

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

Genetic mapping uncovers some SNPs associated with pyridaben resistance

A BSA approach was employed to locate the genomic regions related to pyridaben resistance in the pyridaben-selected strain (Pyr_Rs). For parent and progeny strains, 20 and 32 Gb of raw data, respectively, were obtained (Additional file 1: Table S2). The rate at which clean reads mapped to the reference genome was greater than 60% (Additional file 1: Table S3). Based on genome-wide allele frequencies between pyridaben-selected and susceptible strains (Pyr_Control), 625 candidate SNPs were detected in the pyridaben-selected strain with a SNP/Indel index of > 0.8/< 0.2 and a probability value P of enriched association sites (FDR < 0.01) (Additional file 1: Table S4 and Fig. S1). Among these SNPs, 159 and 63 were annotated as synonymous and non-synonymous mutations, respectively (Additional file 1: Table S5). In addition, 9 and 4 SNPs were located at the UTR3 and UTR5 regions, respectively (Additional file 1: Table S5). Based on the KOG function annotation analysis, these candidate genes mostly contribute to signal transduction and metabolism (Additional file 1: Fig. S2). Potential genes related to resistance evolution where the SNPs were located were further identified at different contigs (Table 2 and Fig. 2, more details shown in Supplementary data). Among these, a PSST subunit gene encoding NADH dehydrogenase (ubiquinone) iron-sulfur protein 7 was selected to determine its relationship with pyridaben resistance [11].

Table 2.

Candidate gene information

Contig	Genea	Function annotation
Contig00017	EVM0001168	Cuticle protein 14 isoform (Limulus polyphemus)
	EVM0004270	Cuticle protein 14 isoform (Limulus polyphemus)
Contig00216	EVM0000426	Cytochrome P450 307a1 (Drosophila melanogaster)
Contig00221	EVM0011251	ABC transporter G family member 27 (Arabidopsis thaliana)
Contig00228	EVM0005899	Transcription factor SOX-2, partial [Stegodyphus mimosarum]
	EVM0000696	UDP-glycosyltransferase 205A3 (Tetranychus urticae)
Contig00230	EVM0005917	heat shock protein HSP70-12A, putative [Ixodes scapularis]
	EVM0010264	hypothetical protein IscW_ISCW023086 [Ixodes scapularis]
Contig00244	EVM0004275	NADH dehydrogenase [ubiquinone] iron-sulfur protein 7, mitochondrial-like [Megachile rotundata]
	EVM0009739	ABC transporter G family member 23 (Bactrocera dorsalis)
	EVM0002277	Cuticle protein 10.9 (Ixodes ricinus)

^aThese genes were considered as the related resistant factors screened from all-candidate genes (more details shown in supplementary data and the genome location are shown in Fig. 2), which contained the delta-SNPs from the bulked segregant analysis

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

V103I and H107R in the PSST subunit directly contribute to pyridaben resistance

To uncover the contribution of a PSST mutation in pyridaben resistance, we verified mutations in the PSST subunit of P. citri using full-length amplification and identified two mutations in resistant strains at 103_V and 107_H (Fig. 3). The PSST sequence comparison showed that these two mutations were located in a very conserved region among insects (Fig. 3). The genotype of surviving mites treated with the LC₉₀ concentration of pyridaben was determined to demonstrate the relationship between PSST mutations and pyridaben resistance using individual mite DNA extraction. The percentage of the homozygous genotype (103_I/I and 107_R/R) was detected in the four lab strains (Lab_S, Pyr_R, Pyr_Control, and Pyr_Rs) after exposure to LC₉₀ pyridaben (Fig. 4A and B). The percentage of both 103_I/I and 107_R/R increased in alive mites after LC₉₀ pyridaben exposure. Moreover, the mutation frequency at the two sites was detected in different resistant P. citri populations (Fig. 4C and D). The percentage of 107_R/R was higher than 90% in four populations (CQ_TN, CQ_WZ, GX_NN, and GX_GL) and was 100% in the GX_GL and CQ_WZ populations. For the 103_I/I mutation, a frequency of 20% was detected in the GX_NN population. In conclusion, a high frequency of two mutations corresponded to high pyridaben resistance in the field, showing a LC₅₀ value > 100 mg/L for these populations (Additional file 1: Table S6).

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

To further explore the contributions of two mutations to pyridaben resistance in P. citri, two homozygous strains within single (107_R/R) or combined (107_R/R and 103_I/I) mutations were obtained by individual mite DNA extraction and genotyping. Compared to WT (PSST_WT), 107_R/R contributed a 19- to 232-fold resistance to 5 METI-Is (Table 3). More importantly, a very high level of resistance to pyridaben (4161.989-fold) occurred when there were two mutations (103_I/I and 107_R/R). Further comparing the double mutant with the single 107_R/R mutant showed that the presence of 103_I/I greatly boosted resistance to tolfenpyrad (from 69- to 672-fold) and fenpyroximate (from 67- to 3790-fold) but 103_I/I provided minimal further contribution towards fenazaquin (from 19- to 24-fold) and tebufenpyrad resistance (from 232- to 368-fold) (Table 3). Taken together, these results indicate that 103_I/I and 107_R/R directly contributed to METI-Is resistance in P. citri.

Table 3.

Toxicity of METIs to Panonychus citri with wild-type PSST or mutations in PSST

Acaricides	Strains	N	Slope (± SE)	LC50 (mg/L) 95% CI	χ2	RRs
Pyridaben	Pc_ WT	602	1.426 ± 0.131	0.361 (0.255–0.506)	5.605	1
	Pc_ H107R	595	2.299 ± 0.202	37.666 (32.772–44.129)	3.458	59.693
	Pc_V103I + H107R	503	2.129 ± 0.297	2626.215 (2247.046–3226.986)	1.589	4161.989
Tolfenpyrad	Pc_ WT	885	1.352 ± 0.097	5.389 (3.772–7.747)	14.196	1
	Pc_ H107R	525	1.358 ± 0.145	369.807 (293.860–476.476)	2.368	68.623
	Pc_V103I + H107R	573	2.232 ± 0.265	3620.795 (3166.871–4267.248)	0.468	671.886
Fenazaquin	Pc_ WT	1031	2.094 ± 0.118	1.005 (0.782–1.271)	13.828	1
	Pc_ H107R	531	1.603 ± 0.154	19.371 (8.104–33.916)	15.507	19.274
	Pc_V103I + H107R	586	2.232 ± 0.180	24.332 (20.840–28.219)	3.909	24.211
Fenpyroximate	Pc_ WT	298	2.260 ± 0.368	0.428 (0.342–0.524)	3.172	1
	Pc_ H107R	706	1.725 ± 0.123	28.828 (24.445–34.184)	4.090	67.355
	Pc_V103I + H107R	623	3.231 ± 0.296	1622.074 (1480.628–1785.820)	1.863	3789.893
Tebufenpyrad	Pc_ WT	779	2.214 ± 0.196	1.181 (0.837–1.602)	4.715	1
	Pc_ H107R	600	1.890 ± 0.171	274.166 (211.697–360.280)	4.744	232.147
	Pc_V103I + H107R	617	2.209 ± 0.230	434.633 (305.226–674.945)	12.141	368.021

N, number of test mites; 95% CI, 95% confident limits of LC₅₀; χ², goodness-of-fit test; RR, LC₅₀ of the mutant strain/LC₅₀ of the wildtype strain

In view of the single V103I mutant, which has not been discovered in P. citri populations by using single mite genotyping methods, and to further investigate PSST mutations, the single and combined mutations were introduced into Drosophila melanogaster. Four transgenic lines (Gal4/UAS_WT, Gal4/UAS_V103I, Gal4/UAS_H107R, Gal4/UAS_V103I + H107R) were obtained, and susceptibility to METI-Is was determined. The knockdown rates of mutant lines (Gal4/UAS_V103I, Gal4/UAS_H107R, Gal4/UAS_V103I + H107R) were significantly lower than those of the control (Gal4/UAS_WT) after exposure to pyridaben, tolfenpyrad, and fenpyroximate for 6 h (Fig. 5). The resistance levels to these three METI-Is follow this order: V103I + H107R > H107R > V103I. 103_I/I did not contribute to fenazaquin or tebufenpyrad resistance (Fig. 5). These results further demonstrate the direct contributions of PSST mutations to METI-Is resistance.

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

The region of the P. citri complex I that encompasses the quinine-binding pocket was modelled with either the wildtype PSST subunit or with the V103I + H107R mutant subunit (Fig. 6). Docking of pyridaben with both models determined that the ligand can be accommodated at the top of the quinone-binding pocket that is lined by the α2 helix of the PSST subunit. Pyridaben adopted a highly similar pose in each model, bound with equivalent estimated free energy of binding (ΔG_b of − 8.4 kcal/mol and − 8.1 kcal/mol for wildtype and mutant PSST, respectively) and formed van der Waals’ contacts with the same set of amino acids (Fig. 6). However, the side chains of residues occupying the 103 and 107 positions are located on the α2 helix face that is orientated away from pyridaben. Therefore, the modelling indicates that resistance conferred by mutation of V103 and H107 is not due to the disruption of ligand-binding interactions with these two side chains.

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

A PSST subunit mutation led to a fitness cost

The PSST subunit carrier of iron-sulfur cluster N2 has been proposed to be a direct electron donor for ubiquinone reduction, and it was hypothesized that the occurrence of an amino acid substitution might lead to its functional impairment. Therefore, mitochondrial complex I activity and the ATP level were determined in three homozygous mite strains (PSST_WT, PSST_H107R, and PSST_V103I + H107R) and four transgenic fly lines (Fig. 7). Compared to WT lines, significant decreases in mitochondrial complex I activity and ATP levels were detected in mutant P. citri strains and transgenic flies (Fig. 7).

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

Based on life table analysis, the deutonymph stage of mutant P. citri strains were significantly prolonged compared to the PSST_WT line (Table 4). In addition, the egg, larva, and protonymph stages of the PSST_V103I + H107R strain were obviously shortened compared to WT. However, both mutant strains showed a significantly prolonged mean generation time (T) (Table 4). Furthermore, the fecundity and population parameters were compared between WT and mutant strains. A significant decrease in fecundity occurred in the two mutant strains; however, there were no significant differences in the number of eggs in transgenic flies for three days (Additional file 1: Fig. S3). In addition to those population parameters, the net reproductive rate (R₀), intrinsic rate of increase (r), finite rate of increase (λ) and gross reproductive rate (GRR) were obviously decreased in the two mutant strains (Table 4). The life table results showed that the age-specific survival rate (lx) and the survival rate of females (s_xj) almost overlapped in the whole life history of the three test strains, indicating that PSST mutations did not affect the survival rate (Fig. 8A and Additional file 1: Fig. S4). The peak values of female fecundity (f_x5), age-specific fecundity (m_x), and age specific net fecundity (l_xm_x) for PSST_WT were significantly higher than those of the two mutant strains with values of 8.545, 5.755, and 5.755, respectively (Fig. 8B, C, and D). The decrease in ATP in P. citri with PSST mutations may have contributed to the reduced fecundity.

Table 4.

Developmental duration, fecundity, and population parameters of wild-type and mutant PSST strains of Panonychus citri

Stage and parameters	PSST_WT	PSST_H107R	PSST_V103I + H107R
Egg	3.91 ± 0.05a	3.85 ± 0.07a	3.80 ± 0.07b
Larva	1.43 ± 0.09a	1.21 ± 0.07a	1.17 ± 0.07b
Protonymph	1.82 ± 0.07a	1.69 ± 0.08a	1.66 ± 0.09b
Deutonymph	1.57 ± 0.09a	1.64 ± 0.09b	1.66 ± 0.08b
Adult	18.87 ± 0.63a	18.92 ± 0.71b	19.63 ± 0.61b
Fecundity (eggs/female)	67.75 ± 3.08a	57.25 ± 2.38b	61.33 ± 3.09ab
Net reproductive rate (R0) (d−1)	45.63 ± 4.98a	41.10 ± 4.46b	43.81 ± 4.78ab
Intrinsic rate of increase (r) (d−1)	0.26 ± 0.01a	0.25 ± 0.01b	0.25 ± 0.01ab
Mean generation time (T) (d)	14.27 ± 0.14a	14.56 ± 0.16b	14.77 ± 0.14b
Finite rate of increase (λ) (d−1)	1.31 ± 0.01a	1.29 ± 0.01b	1.29 ± 0.01ab
Gross reproductive rate (GRR)	52.24 ± 5.12a	47.31 ± 4.17b	49.09 ± 4.26ab

The initial numbers of PSST_WT, PSST_H107R, and PSST_V103I + H107R strains used for constructing the life table were 49, 39, and 42, respectively. The number of each life stage is given in parentheses. Different letters indicate significant differences using the paired bootstrap test

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

The behavioral assays were performed to assess the movement ability and high temperature tolerance. In the climbing assay, the percentage of P. citri mutant strain individuals arriving at the marker was significantly less than that of PSST_WT after 30 min (Fig. 9A). In addition, compared to PSST_WT, the transgenic flies with single and combined mutations showed lower tolerance at 38 °C (Fig. 9B); however, no significant differences in high temperature tolerance were found between Gal4/UAS_V103I and Gal4/UAS_WT. The locomotion assay showed that the male flies with PSST mutations had a lower crossing frequency in 30 min (Fig. 9C). Nevertheless, a distinct difference was presented in female flies in that the crossing lines of four transgenic fly strains overlapped within three days (Fig. 9D). These results indicate that PSST mutations had adverse impacts on the moving capability and high temperature tolerance in P. citri or transgenic flies.

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)

Discussion

The global overuse of mitochondrial electron transport inhibitors (METIs) has triggered widespread resistance evolution in major agricultural pests, including P. citri [9]. Previous work demonstrated that the overexpression of CYP4CL2 mediated the metabolic pyridaben resistance in P. citri via transcriptomic profiling, RNAi silencing, and Drosophila transgenic models [16]. In this study, reciprocal crosses between resistant and susceptible P. citri strains revealed the autosomal polygenic inheritance of pyridaben resistance. Bioassays and behavioral analyses identified two PSST subunit mutations (H107R and the novel V103I) in mitochondrial complex I conferring target-site resistance, though with a concomitant fitness cost.

The PSST subunit H107R mutation is a recognized pyridaben resistance determinant across multiple mite species [11, 13, 17]. In T. urticae, this mutation (numbered H92R) correlates with METI-I resistance [11, 13, 17] and high pyridaben resistance levels are related to the combination of this PSST mutation and overexpressed CYPs [17]. In P. citri, the association of H107R with METI-I resistance was first described by Alavijeh et al. [13], and A109V substitution (numbered A94V in [13]) was also identified but failed to confer resistance when singly introduced into transgenic Drosophila [13]. Here, we discovered a novel V103I mutation co-occurring with H107R in resistant P. citri, rather than A94V. Genotypic screening revealed strong selection for 103_I/I and 107_R/R homozygotes under pyridaben pressure. Both homozygotes were prevalent in different field populations with high resistance levels. The role of these two PSST mutations in pyridaben resistance was investigated by generation of homozygous P. citri strains and also transgenic Drosophila. We found that PSST H107R directly provided moderate or high resistance to METI-Is and its combination with V103I further increased resistance towards three of five tested METI-Is. These findings were reproduced with transgenic flies and, as Drosophila can be genome-edited, the single V103I mutation could be studied in isolation and was found to provide low resistance to three of five METI-Is tested, confirming its functional role.

We hypothesized that V103I could form different ligand-binding contacts that could explain its cross-resistance to some but not all METI-Is. However, molecular modelling indicates that neither V103 nor H107 side chains are positioned to interact with ligands. Instead, these two amino acids are located on the same side of the PSST α2 helix – separated by one turn of the helix – but are on the opposite side of α2 that lines the top of the quinone-binding tunnel. Both the V103I and H107R mutations involve replacing the original side chain with a larger substitution and one possibility is that these mutations perturb conformational dynamics in that region, as structural studies with the Thermus thermophilus complex I with METI-Is bound including pyridaben show that this region is dynamic and that ligand binding induces conformational changes [7]. Cryo-electron microscopy studies link conformational changes around the quinone-binding cavity with global conformational changes that drive activity of the complex [6] while mutations along the PSST loop that is contiguous with the α2 helix were found to reduce activity of Yarrowia lipolytica complex I [5]. These studies reveal changes in this region can impact the enzyme’s activity, which is consistent with the reduced P. citri complex I activity and ATP generation found with the individual V103I and H107R mutations and the additive effect of the combined mutations. A reduction in available ATP may be the molecular basis for the observed fitness costs effects, as the presence of mutations V103I and H107R led to reduced fecundity and weaker locomotion in P. citri and lower high temperature tolerance and weaker motivity in transgenic Drosophila.

Pyridaben resistance in P. citri was found to be determined by multiple genes at an autosome based on the log-probit lines and chi-square values. These results differ from those in Tetranychus kanzawai [18] and T. urticae [19], in which resistance inheritance is controlled by a monogenic factor, indicating differences in the inheritance of pyridaben resistance among different mite species. Furthermore, the involvement of multiple genes in P. citri raises the possibility that resistance mechanisms other than those of target-site and detoxication may be involved. For example, in this study SNPs were identified in genes encoding cuticle proteins (Table 2). Thickening and reduced permeability of the cuticle has been implicated in the resistance of mosquitoes and aphids to different classes of insecticide [20–22] and so changes to the P. citri cuticle that reduce permeability towards the contact-acaricide pyridaben may play a role in its resistance. Unraveling the complexity of pyridaben resistance in P. citri caused by the complicated acaricides’ application background and different habitats and determining the relative contribution of different mechanisms to METI-I resistance will ultimately provide valuable information for management strategies of this crop pest.

Conclusions

In summary, utilizing the genetic strategy, genome mapping, functional analysis, and fitness evaluation, we verified that PSST subunit mutations V103I and H107R conferred high pyridaben resistance and a fitness cost that putatively resulted from decreased ATP production. These findings demonstrate that resistance evolution mediated by target-site mutations involves a significant fitness cost. This groundwork supports an applied resistance management strategy using rotations of acaricides with different modes of action to inhibit field populations.

Methods

Mite strains and chemicals

Two recombinant inbred strains, a pyridaben-susceptible strain (Pyr_Control) and a pyridaben-resistant strain (Pyr_Rs) were obtained in a previous study through the recombinant inbred method between a relatively susceptible strain (Lab_S) and pyridaben-resistant population (Pyr_R) collected from a citrus orchard in Nanning, Guangxi Province [9]. Simply, the offsprings were obtained through the cross between the female of Lab_S and the male of Pyr_R. Offsprings were divided into two groups; one group was not treated with pyridaben (Pyr_Control) and another group (Pyr_Rs) was exposed to LC₅₀ concentration of pyridaben for screening in each generation. When one concentration of pyridaben led to more than 90% mortality in Pyr_Control but less than 10% in Pyr_Rs, it meant a significant resistance trait occurred in Pyr_Rs as compared to Pyr_Control.

Four strains (Lab_S, Pyr_R, Pyr_Control and Pyr_Rs) were reared in the laboratory on bean leaves at 27 ± 1 °C with 60 ± 5% relative humidity (RH) under a 16:8 h light:dark photoperiod. The Pyr_Rs strain was treated with 1000 mg/L pyridaben monthly to maintain its resistance level.

Pyridaben (98%), tolfenpyrad (97%), and tebufenpyrad (98%) were obtained from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fenpyroximate (96%) and fenazaquin (96%) were obtained from Meryer Chemical Technology Co., Ltd. (Shanghai, China) and ACMEC Biochemical Co., Ltd. (Shanghai, China), respectively. Each chemical was first dissolved in acetone and subsequently diluted by distilled water containing 0.1% Triton X-100.

Bioassays

Acaricide bioassays were conducted via leaf-dipping as described previously by Yamamoto et al. [23]. Unsprayed sweet orange leaves from sweet orange (Citrus sinensis (L.) Osbeck) were cut into 25-mm discs and subsequently were placed on wet sponges. Twenty five to thirty female adults were transferred to the leaf discs using a soft brush. After 2–3 h, each disc with mites was dipped into acaricide solutions or distilled water (control, containing 1% acetone and 0.1% Triton X-100) for 5 s. More than three replicates (one disc = one replicate) were performed for each concentration, and seven concentrations were prepared for each acaricide. The excess solution around the mites was absorbed with absorbent paper. All tested mites were placed in an incubator at 27 ± 1 °C with 60 ± 5% RH under a 16:8 h light: dark photoperiod. The mortality was evaluated after 24 h, and the mites were recorded as dead if they did not move normally when touched with a soft brush. Tests were valid if the mortality of control was < 10%.

Genetic cross and backcross experiments

To determine the inheritance of pyridaben resistance, reciprocal crosses were performed between the pyridaben-resistant strains (Pyr_R and Pyr_Rs) and the susceptible strains (Lab_S and Pyr_Control), generating F1 reciprocal cross lines (F₁RS, offsprings from the mating between the female of resistant strains and the male of susceptible strains; F₁SR, offsprings from the mating between the male of resistant strains and the female of susceptible strains). Virgin female mites were paired with a sex ratio of 1:1 for the crosses, and these mites were placed on fresh leaves to lay eggs. The 3–5-day-old females of the F1 reciprocal cross lines were used to determine the median lethal concentration (LC₅₀) values. The resistance degree of dominance (D) was calculated based on the LC₅₀ values following Stone’s description [24] and the formula is as follows (X₁, X₂, and X₃ means the logarithm of the LC₅₀ values of Pyr_R/Pyr_Rs, F₁RS/F₁SR and Lab_S/Pyr_Control, respectively):

$$D=\frac{2X_2-X_1-X_3}{X_1-X_3}$$

Based on the arrhenotokous characteristics of tetranychid mites, unmated female mites produced male mites, meaning that haploid males inherited only maternal genetics [25]. Therefore, the F2 females generated from F1 sibling mating offspring were equivalent to the backcross females. F1 heterozygous lines were transferred to fresh leaves and backcrossed to obtain F2 females (BC, offsprings from sibling mating in F₁RS; BC′, offsprings from sibling mating in F₁SR). Three- to 5-day-old females were sampled for bioassay. Chi-square (χ²) tests and concentration–response curve assessment between the observed and expected mortality were estimated to identify monogenic or multiple gene resistance. The expected mortality was calculated following Georghiou et al. [26] description as follows (W1 and W2 means the actual mortality of heterozygous lines (F₁RS/F₁SR) and resistant/susceptible parents responding to one concentration of pyridaben):

$$\text{E}\left(,\text{BC or BC'}\right)=0.5\times \text{W}1\left({\text{F}}_1,\text{RS or},,{\text{F}}_1,\text{SR}\right)+0.5\times \text{W}2(\text{resistant or susceptible})$$

Moreover, the χ² analysis for estimating the hypothesis of monogenic inheritance was performed following Sokal and Rohlf’s model [27] description as follows (F, the actual mortality; n, the number of treated mites; p, the expected mortality; q = 1-p; r, the number of the pyridaben concentration):

$${X_i}^2=\frac{{(F-pn)}^2}{\mathit{pqn}}$$

$$X^2=\sum _{i=r}^r{X_i}^2$$

Genomic DNA preparation, DNA quantification, and qualification

The DNA samples extracted from Lab_S and Pyr_R strains were used as parental pools. Meanwhile, DNA samples of Pyr_Control and Pyr_Rs were prepared for offspring pools. Approximately 2000 adult females were collected for each of the 4 strains for DNA extraction using the Mollusk DNA Kit following the manufacturer’s protocol (Omega Bio-Tek, GA, USA). DNA degradation and contamination were monitored on 1% agarose gel. The DNA purity and concentration were measured using a Nano Photometer® spectrophotometer (IMPLEN, CA, USA) and Qubit® DNA Assay Kit with a Qubit® 2.0 Flurometer (Life Technologies, CA, USA), respectively.

Bulked segregant analysis (BSA)

Library preparation

DNA libraries were constructed from 1.5 μg input DNA using a Truseq Nano DNA HT Sample Preparation Kit (Illumina, CA, USA) following the manufacturer’s recommendations, and index codes were used to distinguish each sample. Briefly, the DNA sample was fragmented to a size of 350 bp, and then the DNA fragments were end polished, A-tailed, and ligated with the full-length adapter through further PCR amplification. Purified PCR products (AMPure XP system, Beckman, CA, USA) were quality controlled via Agilent 2100 Bioanalyzer (Agilent, CA, USA) and real-time PCR. Libraries were sequenced on the Illumina HiSeq 4000 platform (Illumina, CA, USA), yielding 150 bp paired-end reads with 350 bp inserts.

Quality control and genome mapping

Raw FASTQ reads underwent stringent quality control: filtering sequences with ≥ 10% unidentified nucleotides (N), > 50% low-quality nucleotides (Phred score < 5), > 10 bp adapter overlap (≤ 10% mismatch), and PCR duplicates (identical paired-end reads).

The clean data for each sample were mapped to the P. citri genome (JAAABK000000000, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_014898815.1/) using the Burrows-Wheeler Aligner (BWA) [15]. Alignment files were converted to BAM files using SAMtools software [28]. PCR duplicates were removed, and potential PCR duplications were simultaneously removed, retaining the highest mapping quality alignments for reads with colliding genomic coordinates.

SNP and InDel detection, annotation, and indexing

Variant calling (SNPs/InDels) was conducted using the unified genotype function in GATK software [29]. ANNOVAR [30] was used to annotate SNPs or InDels based on the GFF3 files for the reference genome. Homozygous variants between parents (Lab_S and Pyr_R) were identified, and offspring pool read depth data were used to calculate SNP/InDel indices (ratio of differing reads to total reads) [31]. The genotype of one parent (Lab_S) was used as the reference to calculate the read number statistics for this parent’s genotype and the others in the offspring pool. Sites with SNP/InDel indices < 0.2 or > 0.8 in both pools were excluded. Genome-wide indices were determined using 1 Mb sliding windows (10 Kb step), averaging SNP/InDel indices within each window. The delta SNP/InDel index was derived from differences between the two pools and these were considered as the candidate SNPs within more than a 95% threshold.

Detection of candidate target site mutations and sequence alignment

To identify the candidate SNPs in four lab strains (Lab_S, Pyr_R, Pyr_Control, and Pyr_Rs), 200 adult females were sampled for RNA extraction using MicroElute® Total RNA (Omega Bio-Tek, GA, USA). RNA from each sample (1 μg) was treated with RQ1 RNase-free DNase (Promega, WI, USA) and used to generate cDNA using the PrimeScript TM 1 st Strand cDNA Synthesis Kit (Takara, Dalian, China) in accordance with the manufacturer's instructions. cDNA products were stored at − 20 °C.

The primers for full-length cloning were obtained from Primer Premier 5 software (Additional file 1: Table S7), and PCR was performed using PrimeSTAR® Max DNA Polymerase (Takara, Dalian, China) following the manufacturer’s instructions. The PCR products were purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian, China) and sequenced by BGI (BGI Genomics Co., Ltd, Shenzhen, China). The sequences were aligned to the reference gene to determine the candidate site mutation using BioEdit version 7.0. Using MAFFT version 7 (online service), the protein sequences of the PSST subunit were aligned to those from different insect species, which were downloaded from NCBI [32]. The alignment results were viewed with Jalview [33].

Individual mite DNA extraction and genotyping

To genotype individual mites from the four lab strains (Lab_S, Pyr_R, Pyr_Control and Pyr_Rs) and seven filed populations (YN_BS, CQ_TN, GX_GL, GX_NN, SC_LZ, CQ_WZ, and CQ_LP) for V103I and H107R mutations, individual adult females were homogenized in 20 μL STE buffer (10 mM Tris–HCL, 100 mM NaCl, 1 mM EDTA, pH 8) and 2 μL of (10 mg mL⁻¹) proteinase K (Sigma-Aldrich, MO, USA). Homogenates were incubated at 60 °C for 30 min, followed by proteinase K inactivation at 95 °C for 5 min. The supernatant was obtained after centrifuging and used as a template for PCR. The primers are listed in Additional file 1: Table S7. An approximately 300-bp DNA fragment was produced using PrimeSTAR® Max DNA Polymerase. For amplification confirmation, 5 µL PCR products were run on a 1% agarose gel at 110 V for 25 min. DNA fragments were sequenced by BGI and visualized using BioEdit 7.0 for genotyping.

Lethal concentration exposure

To identify the PSST mutations after exposure to lethal pyriadben concentrations, adult female mites from four lab strains (Lab_S, Pyr_R, Pyr_Control, and Pyr_Rs) were treated using the leaf-dipping method described above. Briefly, female adult mites were transferred onto a fresh sweet orange leaf disc (2-cm diameter). All leaf discs with mites were subsequently dipped into pyridaben solution after 2–3 h. The excess solution around the mites was removed using filter paper. All tests were placed in an artificial chamber. After 24 h, live mites were collected for individual mite DNA extraction and genotyping.

Introgression of two PSST mutations into the susceptible background

Based on the previous resistance gene introgression into a susceptible background, the recombinant inbred strains were obtained in previous study [9]. Female adult mites were reared on a single leaf disc for 3 days to lay eggs. Individual mite DNA extraction and genotyping were performed to screen out mites of the homozygous genotype, wild type (103_V/V and 107_H/H), and mutant genotypes (107_R/R and 103_I/I + 107_R/R). Mites with the single mutation of V103I were not detected in any of the tested strains. Thus, three homozygous lines were obtained: PSST_WT, PSST_H107R, and PSST_V103I + H107R. These lines were reared separately for more than 6 generations for bioassay and fitness assessments.

Transgenic fly construction and bioassay

To further assess the contributions of the single or combined mutations to pyridaben resistance, PSST subunit mutations were introduced into Drosophila melanogaster (W1118) to construct transgenic flies. A previous study indicated that the H92R mutation (a homologous site of H107R) is lethal in Drosophila flies [11]. Thus, the PSST genes of the wild type (WT) and mutant (V103I and H107R) were expressed in D. melanogaster via the Gal4/UAS system from Fungene Biotech (http://www.fungene.tech/) (Beijing, China) following Pan et al. [34]. Four Gal4/UAS_PSST lines were finally obtained, and PCR was used to validate the PSST mutation sites (Additional file 1: Fig. S5 and Table S7). All fly lines were reared on an artificial diet at 25 °C, 65% RH, and a 12 h:12 h light: dark photoperiod.

Bioassays were conducted to assess the susceptibility of transgenic flies to METI-Is (pyridaben, fenpyroximate, tolfenpyrad, fenazaquin, and tebufenpyrad) [34–36]. Acaricide filter papers with 1000, 2000, 1200, 1000, and 1600 mg/L of pyridaben, fenpyroximate, tolfenpyrad, fenazaquin, and tebufenpyrad, respectively (Additional file 1: Fig. S6), were prepared in distilled water with 1% acetone and 0.1% Triton-X100. Filter papers of corresponding size were placed into a glass vial, and the filter paper was placed against the wall of the bottle. Then, the filter papers were soaked evenly by sucking 50 μL of the drug solution through a pipettor. Filter papers soaked in double distilled water with 1% acetone and 0.1% Triton-X100 were used as the control group. Filter paper was placed in a glass vial with 25–30 transgenic flies, and the vials were plugged with cotton soaked in 20% (w/v) sucrose. The flies were recorded at 1, 3, 6, 12, and 24 h and considered knocked down if their legs could not move or convulse. Five replicates were used for each treatment. All test flies were reared at 25 °C, 65% RH, and with a 12 h:12 h light: dark photoperiod. A Student’s t-test was used to compare differences in the knockdown rate between mutant and WT lines.

Modelling and ligand docking

The region of the P. citri mitochondrial complex I that encompasses the quinine-binding pocket was modelled using Chai-1 software [37], which was accessed via the Neurosnap webserver (https://neurosnap.ai/). The following four subunits were modelled and assembled: PSST (Accession number XP_053210093.1), 49 kDa (XP_053214783.1) and subunits 1 (ADJ68020.1) and 3 (YP_003795545.1). Chai-1 was run with MSA mode set to mmseqs2, 10 recycling steps, 200 diffusion steps, and with the inter-chain restraint that each subunit must be < 5.5 Å with at least one other subunit. Separate models were generated with the wildtype PSST subunit or the V103I + H07R double mutant, and their quinone-binding pocket was predicted using Caver 3 [38] with a probe radius of 1.5 Å.

A structure file for pyridaben was generated ab initio using MarvinSketch v19.22 of the ChemAxon suite (https://chemaxon.com/). Ligand docking was performed using Autodock Vina following methods described by Yin et al. [39]. Figures were produced using PyMOL (https://pymol.org).

Complex I activity assay and ATP content detection

A total of 1000 female adult mites or 10 female flies were collected and used in the complex I activity assay, which was performed using the CheKine™ Micro Mitochondrial Complex I Activity Assay Kit (Abbkine, Wuhan, China) following the manufacturer’s protocols [40]. In addition, the ATP levels were determined in macrophages using an ATP Assay Kit (Beyotime, Shanghai, China) following the manufacturer’s protocol [41].

Two-sex life table construction

Three P. citri strains (PSST_WT, PSST_H107R, and PSST_V103I + H107R) were used to construct age-stage, two-sex life tables. More than 30 pairs of mites were prepared, and 1 pair of mites was transferred onto a single fresh sweet orange leaf disc and left for 24 h to lay eggs. The male mites were removed the next day. One egg was randomly retained, and the extra eggs were removed. The development stages, survival, and number of eggs laid were recorded daily until all mites died. The leaf discs were replaced every week.

Behavioral assays

Climbing ability

Ten 3-day-old female mites were transferred to a plastic pipe of 15 cm in length, and 10 cm was marked from the bottom. After the mites were shaken to the bottom, the number of mites that reached the mark was recorded after 5, 10, 20, and 30 min. Ten replicates were performed in total.

Locomotion assays

The locomotor behavior was determined following a previously described method [42]. Individual 5- to 7-day-old flies were loaded into tubes with 2% (w/v) agarose and 5% (w/v) sucrose and monitored using the Drosophila Activity Monitoring System (DAMS, Trikinetics, MA, USA) in 24 h light/dark cycles. The flies were allowed to adapt to the new environment for 1 day, and data were collected every 30 min. All flies were monitored for 3 days, and 32 flies (female: male 1:1) were recorded.

Temperature sensitivity assays

Twelve 5–7-day-old female flies were transferred into new empty glass fly vials by aspiration. The flies were exposed to a temperature of 38 °C and recorded with a video camera for 12 min. The knockdown rate of flies in each vial was measured every 2 min, and 6 vials were prepared for each line.

Egg-laying assays

Newly emerged flies with a female: male ratio of 2:1 were reared on conventional cornmeal-agar-molasses medium in single vials for mating. After 7 days, the female flies were transferred to 35-mm glass vials containing fresh food for 3 days. The number of eggs was manually counted using a stereomicroscope (Chongqing Optec Instrument Co., Ltd, Chongqing, China).

Statistical analysis

The bioassay data passing the χ² goodness-of-fit test were analyzed using Polo Plus 1.0 software to determine the LC₅₀ values, slopes (± SE), and 95% confidence limits. For the age-stage, two-sex life table analysis, the developmental duration, fecundity, and population parameters (the net reproductive rate (R₀), intrinsic rate of increase (r), finite rate of increase (λ) and gross reproductive rate (GRR)) were calculated and assessed using TWOSEX-MSChart program [43] within the bootstrap method using 100,000 random re-samplings [44, 45]. The significance of life table parameters for the three P. citri strains was assessed using a paired bootstrap test in TWOSEX-MSChart [45, 46]. One-way ANOVA was used to analyze data from the complex I activity, ATP content detection, locomotion assays and egg-laying assays. The climbing ability and temperature sensitivity assays were analyzed using an independent sample t-test. All analyses were performed and figures generated using GraphPad Prism 8.0 (GraphPad software).

Abbreviations

METI-Is

Mitochondrial complex I electron transport inhibitors

ATP

Adenosine triphosphate

BSA

Bulked segregant analysis

CPR

Cytochrome P450 reductase

Lab_S

A relative susceptible strain reared in laboratory from 2016

Pyr_R

A pyridaben resistant population collected from Nanning, China, 2019

Pyr_Control

Pyridaben-susceptible strain was obtained through the recombinant inbred method

Pyr_Rs

Pyridaben-resistant strain was obtained through the recombinant inbred method

WT

Wildtype

T

Mean generation time

R₀

The net reproductive rate

r

Intrinsic rate of increase

λ

Finite rate of increase

GRR

Gross reproductive rate

lx

The age-specific survival rate

s_xj

The survival rate of females

f_x5

The peak values of female fecundity

m_x

Age-specific fecundity

l_xm_x

Age specific net fecundity

D

Dominance

F₁RS

Offsprings from the mating between the female of resistant strains and the male of susceptible strains

F₁SR

Offsprings from the mating between the male of resistant strains and the female of susceptible strains

LC₅₀

The median lethal concentration

BC

Offsprings from sibling mating in F₁RS

BC′

Offsprings from sibling mating in F₁SR

Authors’ contributions

Conceived and designed the experiments: DP, GY, JW, WD.; Performed the experiment: DP and MX; Contributed the materials/data analysis: DP, MX, CL, XL; Homology modelling and ligand docking: LA and AOO; Wrote and modified the paper: DP, AOO, JW, WD. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Fundamental Research Funds for the Central Universities (SWU-XDPY22001) of China and the China Agriculture Research System of MOF and MARA. AOR was supported by the Medical Research Council (MRC) (grant MR/W002159/1).

Data availability

The raw data for bulked segregant analysis were submitted to the NCBI database with BioProject accession number of PRJNA1177840 (BioSample: SAMN44455280- SAMN44455283). https://figshare.com/articles/dataset/Supplementary_file_All_genes_within_delta-SNP_index_locating_txt/27907821?file

Supplementary data to this article can be found online at https://figshare.com/articles/dataset/Supplementary_file_All_genes_within_delta-SNP_index_locating_txt/27907821?file=50809026.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.