The population of H. glycines (race 3, HG type 0) was obtained from a naturally infested commercial soybean field in Daqing, Heilongjiang Province. The population was maintained on a susceptible soybean cultivar, ‘Hefeng25’, under greenhouse conditions at the Institute of Pratacultural Science, Heilongjiang Academy of Agricultural Science. Eggs and fresh pre-parasitic second-stage juveniles (J2s) were collected as described previously (Hu et al., 2019). Briefly, cysts were extracted from the nematode-infected soil and soybean roots using floating sieves (850-mm-pore and 250-mm-pore). Cysts were manually crushed on the surface of a 75-mm-pore sieve with a rubber stopper to release intact eggs and J2s that had hatched within cysts. Subsequently, the eggs were collected on a 25-µm pore sieve, rinsed with distilled water, and transferred to a hatching chamber at 26°C for six days for collecting hatched J2s. The seeds of soybean cv. ‘Hefeng25’ were sown in plastic pots containing a sterilized mixture of soil and sand (2:1, v/v) and grown in a greenhouse at a temperature of 21–26°C with a relative humidity at 50–65% under a 16-h light/8-h dark photoperiod.

To identify the CYP genes of H. glycines, the amino acid sequences for all C. elegans CYPs listed in Wormbase (https://wormbase.org/) were utilized as queries to perform BLAST searches in the H. glycines isolate TN10 genome available in the WormBase ParaSite database (https://parasite.wormbase.org/Heterodera_glycines_prjna381081/Tools/Blast) with a significance cut-off of E-value < 10⁻⁵. The putative HgCYP genes were further validated through reciprocal searches of the C. elegans genome in the WormBase database and the non-redundant nucleotide database at the National Centre for Biotechnology Information (NCBI) BLAST server. The closest matched protein for each BLAST hit was documented as the identification of the original H. glycines hit. Software from Interpro (http://www.ebi.ac.uk/interpro/) was employed for functional analysis of predicted proteins. ClustalW was utilized to align the CYP proteins from H. glycines and C. elegans. Phylogenetic analyses were then performed on the comparison results, adopting the neighbor-joining method of MEGA6.0 with a Jones-Taylor/Thornton model (bootstrap test = 500 replicates).

The full-length coding sequence of HgCYP33E1 was amplified from the cDNA of H. glycines J2s using specific primers ( Table S1 ). PCR reactions were performed by adopting Taq PCR MasterMixII (TIANGEN, Beijing, China) with the following cycling conditions: an initial step at 94°C for 3 min, and 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 96 s, followed by a final elongation time of 5 min. The PCR amplicons were purified with a PCR purification kit (TIANGEN, Beijing, China) and ligated into the pLB fast cloning vector (TIANGEN, Beijing, China) according to the manufacturer’s protocol. The resulting recombinant plasmids were transformed into Escherichia coli DH5α chemically competent cells (Biomed, Beijing, China) using a heat shock method. Positive clones were selected through the usage of colony PCR. Subsequently, plasmids were extracted from at least five colonies and verified via sequencing performed by COMATE biological Technology (Jilin, China). Sequence homology comparisons were conducted in Wormbase parasite database (https://parasite.wormbase.org/) and the non-redundant protein databases at the NCBI BLAST server using the BLASTX and BLASTN. Jalview Version 2.1 and ClustalW tools were employed to perform multiple sequence alignment of predicted proteins with conserved motifs across nematode species. Phylogenetic analysis of the obtained data was performed using the neighbor-joining method in MEGA6 software.

The nematicide, abamectin (Sigma, USA), was dissolved in acetone to prepare a 10 mg/mL stock solution. To obtain the desired working concentrations (1 µg/mL, 5 µg/mL, and 10 µg/mL) for abamectin exposure of H. glycines, the stock solution was further diluted in distilled water. Xenobiotic treatment was carried out in a 24-cell culture plate (Corning, USA). Briefly, 500 µL of each concentration of abamectin solution was placed into a 15.6 mm diameter cell, then 20 µL nematode suspension containing approximately 1000 H. glycines J2s was added and gently mixed. Nematodes were incubated in the abamectin solution for 1, 2, 4, 8, 16, and 24 h in an incubator at 25°C under dark condition. The J2s exposed to a diluted acetone solution served as the control group. Then, J2s were collected and rinsed with sterile distilled water for three times, after which nematode samples were frozen in liquid nitrogen and stored at −80°C for RNA extraction.

The Bacillus strain Bv-DS1 isolated previously in our laboratory (Hu et al., 2022) was used in this study. The fresh culture filtrate was prepared by growing the bacterial strain in 50 mL of LB liquid medium for 48 h at 28°C on a shaking incubator (200 rpm/min). The fermented bacteria were centrifuged at 2,500 g for 10 min, and the supernatant of the Bv-DS1 strain was collected and filtered using a 0.22-µM Millipore filter. The prepared culture filtrate was used for nematode treatment in a 24-cell culture plate. 100 µL of undiluted culture filtrate or was added to each well, followed by the addition of a nematode suspension (approximately 1000 H. glycines J2s in 900 µL of water per well). The well containing 100 µL of sterilized culture broth was used as the control group. After incubation for 2, 4, 6, 8, 12, 16, and 24 h at 25°C (dark treatment), respectively; J2s were collected and washed with sterile distilled water three times. Next, nematodes were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction and gene expression analysis.

Root exudates of two soybean cultivars. ‘KangXian12’ and ‘Hefeng25’ were prepared according to the method described by Gao et al. (2018). Five seedlings for each soybean cultivar grown for four weeks were collected from the 2-L pot and gently washed with deionized water to remove vermiculites. Then, soybean seedlings were transferred to a 1-L plastic cup containing 0.5 μM CaCl₂. In order to collect root exudates, all root systems were absolutely immerged in the solution under the dark condition for 12 h. Then, all exudate collection solutions were filtered through a 0.22-μm filter and lyophilized for subsequent treatment. A stock solution of root exudates was prepared by dissolving 10 mg of lyophilized powder in 1 mL of distilled water. Subsequently, 10 µL crude root exudate was added to each well of a 24-cell culture plate containing approximately 1000 H. glycines J2s in 900 µL of water per well. The plate was incubated at 25°C in the dark for 2, 4, 6, 8, 16, and 24 h, respectively. J2s exposed to sterile water were set as the control group. Nematode samples were collected and immediately frozen in liquid nitrogen for RNA extraction.

Above experiments were repeated at least three times and each treatment had at least three biological replicates. Nematode samples for RNA extraction were collected from at least three wells at each time point as a biological replicate.

Total RNA was extracted from approximately 2000 H. glycines nematodes in each sample using an RNAPrep Pure Micro Kit (TianGen Biotech, Beijing, China) following the manufacturer’s instructions. The quality and quantity of the extracted RNA were evaluated through a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). An equal amount (0.5 µg) of purified RNA was reverse-transcribed into cDNA using a FastKing gDNA Dispelling RT SuperMix FastKing Kit (TianGen Biotech, Beijing, China). Gene expression levels were analysed in the LightCycler® 480 real-time PCR System with AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The qRT-PCR procedure involved a preheat cycle of 95°C for 5 min, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. The primers were designed based on sequences of HgCYP genes retrieved from H. glycines database (https://scnbase.org/) using Primer 5.0 software. The sequences for the forward and reverse primers used for qRT-PCR are listed in Supplemental Table S1 . After amplification, a melting curve analysis was performed to confirm the specificity of target genes. For each nematode sample, three biological and three technical replicates were conducted. Gene expression was normalized to the levels of a reference gene, glyceraldehyde-3-phosphate dehydrogenase (HgGAPDH) of H. glycines (Wang et al., 2014). The fold change in expression was calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001).

The specific primers for dsRNA synthesis were designed based on the CDS sequences of HgCYP33E1 and an unrelated GFP gene, which contains a T7 promoter sequence appended to the 5’ end ( Table S1 ). The 787-bp fragment of HgCYP33E1 was amplified by PCR from the recombinant pLB plasmids that contained HgCYP33E1 inserts. Subsequently, the amplified products were used as a template for synthesizing dsRNA with a MEGAscript^® RNAi Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. The synthesized dsRNAs were examined using 1% agarose gel electrophoresis and were stored at -80°C until further use. The method of dsRNA soaking of H. glycines J2s was performed according to the protocol established by Urwin et al. (2002). Briefly, approximately 4,000 freshly hatched J2s were mixed with a soaking buffer containing 2 mg/mL dsRNA, 3 mM spermidine, and 50 mM octopamine, and then incubated in the dark at room temperature on a slowly shaking rotator. J2s socked in dsRNA of GFP gene and those soaked in buffer without dsRNA were used as control groups. After the 24-h incubation, nematodes were washed four times with the nucleoid acid-free water and collected by brief centrifugation for behavior observation, RNA extraction and qRT-PCR analysis, nematicide treatment, or infection assay. The uptake of dsRNA by H. glycines J2s was visualized under a fluorescence microscope (Olympus, Japan) using fluorescein isothiocyanate (FITC, 0.1 mg/ml, Sigma, USA) as a fluorescent tracer.

To examine the impact of silencing of HgCYP33E1 on H. glycines infectivity and development, approximately 200 J2s were socked in dsRNA_HgCYP33E1 solution, dsRNA_GFP solution, or buffer and subsequently inoculated on 12-day-old soybean seedlings (‘Hefeng25’) grown in pot (12-cm-diameter × 8-cm-deep) with a sterilized sand-to-soil ratio of 2:1 in a growth chamber (16 h of light and 8 h of darkness, 24°C). At two-day post-inoculation (dpi), the root systems were collected and stained with acid fuchsin solution (0.013% acid fuchsin in 0.8% acetic acid) to observe nematodes inside the roots under a stereomicroscope. After 18 dpi, the nematodes in different stages per root system were counted after acid fuchsin staining, using a microscope. The experiment was conducted three times with eight replicates for each treatment in a completely randomized experimental design.

Approximately 100 H. glycines J2s were soaked in dsRNA_HgCYP33E1 solution, dsRNA_GFP solution, or buffer (as control) for 24 h before being transferred into each well in a 12-well culture plate containing 1 mL of 5 µg/mL abamectin aqueous solution or 1 mL of 0.05% acetone solution (as control treatment). All plates were placed in an incubator at room temperature in the dark for 24 h. Subsequently, to determine the mortality rate of J2s, microscopic observations were conducted as previous method (Hu et al., 2019). Briefly, one drop of 1N sodium bicarbonate was added to each well. J2s that exhibited no movement during the 30-second observation period after being stimulated with sodium bicarbonate were considered dead, while any nematode that altered its body shape in response to sodium bicarbonate was classified as alive. This experiment was performed twice with six replicates for each treatment in a completely randomized experimental design.

To evaluated H. glycines infectivity, freshly hatched J2s were treated with dsRNA_HgCYP33E1, dsRNA_GFP, or socking buffer (as control) for 24 h before being incubated in abamectin (5 µg/mL) aqueous solutions or in acetone solution (0.05%) for 24 h at room temperature in the dark. After incubation, the nematodes were thoroughly washed with distilled water to remove abamectin. Approximately 200 J2s were inoculated on 12-day-old ‘Hefeng25’ seedlings grown in pots under the same condition as described above. At 2 dpi, each root system was collected and stained with acid fuchsin solution (0.35 acid fuchsin, 25% acetic acid) to count the number of J2s that penetrated into roots using a stereomicroscope. This experiment was conducted twice with eight replicates for each treatment in a completely randomized experimental design.

All data were subjected to one-way ANOVA with Turkey post hoc test (for multi-group comparisons) using GraphPad Prism version 8.0.2 software. Error bars in all figures were expressed as the mean ± the standard deviation (SD). The significance level was set at P < 0.05. All graphs were generated using GraphPad Prism 8

The genome information of H. glycines (isolate TN10) (Masonbrink et al., 2019), available in the WormBase ParaSite database, was used to identify the CYP genes in H. glycines. Amino acid sequences of CeCYP genes from C. elegans in the WormBase were used as queries to screen for putative CYP proteins in H. glycines. Total of 36 CYP gene loci were identified in the 123-Mb pseudomolecule assembly of the H. glycines genome, responsible for encoding at least 31 full-length and 5 partial CYP proteins ( Table S2 ). Among HgCYP gene loci, Hetgly04863 has two transcripts. The gene IDs, gene names, and protein sequences of all identified HgCYP members are listed in Table S2 . Our analysis revealed that HgCYP genes were relative evenly distributed across the eight chromosomes, with the exception of chromosome 07. The majority of HgCYPs (13 genes, accounting for 36.11 of the total) were clustered on chromosome 05. All identified HgCYPs possess the conserved domains of the cytochrome P450 family, including the typical heme binding loop (FxxGxxxCxG), helix-K (ExxR), and helix-C (WxxxR). After conducting phylogenetic analysis of 34 HgCYPs and 81 CYPs from C. elegans, a neighbor-joining phylogenetic tree was generated to visualize the results. The CYP proteins from H. glycines were clustered in four major sub-groups, namely I, III, IV, and V ( Figure 1 ). Sub-group IV was the largest group and clustered 14 H. glycines CYPs together with 18 CeCYPs. Sub-group I was the second largest group, containing 7 HgCYP and 21 CeCYPs. Sub-group III consisted of 3 HgCYPs and 10 CYPs from C. elegans. Sub-group IV clustered 10 HgCYPs together with 10 CeCYPs. Notably, no H. glycines CYPs were found in sub-group II, which only contained 22 CYPs from C. elegans. These findings imply that specific CYP members may have evolved independently after the divergence of H. glycines and C. elegans.

To assess whether the HgCYP superfamily is involved in the xenobiotic detoxification of nematicides, the expression levels of individual HgCYP genes were examined in H. glycines J2s exposed to abamectin, a well-known nematicide. Note that only the expression data for the 26 HgCYPs are shown in Figure S2 by evaluating the primer specificity and amplification efficiency. Due to the high degree of sequence similarity, it is difficult to design specific primers to amplify Hetgly07572 and Hetgly07628 separately. Therefore, the qRT-PCR results for Hetgly07572/Hetgly07628 represent the combined expression values of these two genes and do not exhibit statistical significance relative to the control. As a result, exposure of H. glycines to 1 µg/mL abamectin for 2 and 8 hours generated 10 significantly (*P < 0.05, **P < 0.01) differentially expressed HgCYPs, with 5 up-regulated and 5 down-regulated genes ( Figure S2 ). In sub-group I of HgCYPs, abamectin treatment resulted in an average increase in expression of Hetgly10349 and Hetgly13553 between 1.70- and 2.74-fold, with the exception of expression of Hetgly08781 which was downregulated 1.95-fold at 8 h. Interestingly, Hetgly08425 of sub-group III showed a fluctuating change in expression pattern, which was downregulated 3.98-fold after exposure 2 h and then upregulated 3.81-fold at 8 h. For all tested members of sub-group IV, abamectin treatment only caused the significant (P < 0.01) change in expression of Hetgly02475, with upregulation 3.93-fold at 8 h ( Figure S2 ). Additionally, of the four differentially expressed HgCYPs in sub-group V, all but Hetgly13473 was downregulated by abamectin ( Figure S2 ).

Of the five abamectin-induced HgCYP genes, Hetgly10349, Hetgly02475, and Hetgly13553 were inferred to encode full-length CYP proteins. Hence, Hetgly10349, Hetgly02475 and Hetgly13553 were selected as representatives to further explore the potential function of the CYP superfamily in H. glycines parasitism and response to xenobiotics. Finally, only a full-length 1602-bp open reading frame (ORF) of Hetgly13553 was successfully amplified from J2s cDNA of H. glycines using the gene-specific primers based on its transcript in the H. glycines genome (Masonbrink et al., 2019) ( Figure 2A ). The resulting ORF fragment (GenBank accession OQ260000.1) encoded a protein of 534 amino acids. High nucleotide sequence similarity was observed between the cloned ORF fragment and Hetgly13553 (85% query cover and 99% identity) in H. glycines TN10 genome, as well as a transcript evm.model.chr1.1377 (100% query cover and 99% identity) in H. glycines X12 genome (https://scnbase.org/). We used the deduced amino acid sequence of Hetgly13553 as a query to perform BLASTp analysis in the nonredundant protein database of NCBI and WormBase Parasite databases. It was found that Hetgly13553 exhibited a 31.78% homology to CeCYP33E1 (NP_501480.2) of C. elegans, and a 35.32% identity to CYP33E1 (KAH7715121.1) of Aphelenchus avenae. Therefore, the CYP protein encoded by Hetgly13553 was designated as HgCYP33E1. Furthermore, multiple homologous sequences of HgCYP33E1 were identified within phylum Nematoda, including free-living, animal-parasitic, and plant-parasitic species. Specifically, it shared 95.69%, 64.39%, and 62.71% high sequence similarity with three CYP33E1 proteins of CN species including H. schachtii (Hsc_gene_9892), Globodera rostochiensis (Gr22_v10_g11627), and Globodera pallida (KAI3419411.1), respectively. Additionally, HgCYP33E1 displayed a relative high sequence identity with two homologues from root-knot nematode, Meloidogyne graminicola (KAF7627337.1, 48.08%) and Meloidogyne javanica (Mjav1s04115g032950, 52.34%). A comparison of protein homology of CYP33E1 from H. glycines and other nematode species further revealed that HgCYP33E1 had several typical conserved domains of the cytochrome P450 family, such as heme binding loop (FxxGxxxCxG), helix-K (ExxR), and helix-C (WxxxR). Its N-terminal also contained a conserved region of proline ( Figure 2B ). A phylogenetic tree was constructed to analyze the evolutionary relationship of CYP33E1 among H. glycines and other species. It was observed that HgCYP33E1 was first clustered into one group with CYP33E1 proteins of PPNs. In contrast, HgCYP33E1 shared higher percentage of sequence with CYP33E1 proteins from Globodera and Heterodera nematodes, and then formed a subgroup ( Figure 2C ).

The expression levels of HgCYP33E1 were assessed via qRT-PCR analysis of RNA samples extracted from eggs, pre-parasitic J2s, parasitic J2s, J3s, females, and males of H. glycines. HgCYP33E1 was found to be expressed in all developmental stages of H. glycines. Notably, the pre-parasitic J2s and parasitic J2s exhibited significantly (P < 0.05) higher expression levels of HgCYP33E1 compared to other parasitic stages, with transcript levels being approximately 2.58- and 3.45-fold higher than those of eggs. HgCYP33E1 expression level was found to be similar among eggs, J3s, and females, while a lower expression was observed in the males ( Figure 3 ).

Due to significantly elevated transcript levels of HgCYP33E1 observed in abamectin-treated J2s at two time points, 2-h and 8-h ( Figure S2 ), this suggests that abamectin may continuously activate HgCYP33E1 expression in H. glycines. To further explore the sensitivity of HgCYP33E1 transcript to abamectin treatment, we determined the expression levels of HgCYP33E1 in H. glycines J2s exposed to different concentrations of abamectin (1 µg/mL, 5 µg/mL, and 10 µg/mL) for various time periods (1, 4, 8, 16, and 24 hours). After 1 hour exposure, both 1 µg/mL and 5 µg/mL abamectin did not cause significant changes in HgCYP33E1 transcript levels. However, high concentrations of abamectin (10 µg/mL) significantly (P < 0.05) induced the expression of HgCYP33E1 at 1 h. Significant up-regulation of HgCYP33E1 was found at low dose of abamectin (1µg/mL) after 8 and 16 hours treatment. The transcript levels of HgCYP33E1 increased significantly by 6.88-fold (P < 0.01) and 9.11-fold (P < 0.001), respectively, compared to nematodes treated with acetone solution (control). On the other hand, the expression levels of HgCYP33E1 consistently increased and remained significantly up-regulation following 4, 8, 16, and 24 hours of exposure to 5 µg/mL and 10 µg/mL abamectin ( Figure 4A ). These findings indicate that the changes in expression of HgCYP33E1 induced by abamectin are dose-dependent.

Considering of the potential influence of chemicals derived from host plant, such as root exudates or defence secondary metabolites, on the behavior of the infective-juveniles of H. glycines in soil and the development of parasitic stages in soybean roots, it is possible that HgCYP33E1 plays a role in the biotransformation of xenobiotics derived from host plant. To investigate this hypothesis, the relative expression levels of HgCYP33E1 were assessed in J2s treated with root exudates from a resistant soybean cv. ‘Kangxian 12’, and a susceptible cv. ‘Hefeng25’, for different duration (2, 4, 6, 8, 16, and 24 hours). The results demonstrated that HgCYP33E1 was significantly up-regulated after exposure to 0.1 mg/L of ‘Kangxian 12’ root exudates for 4 and 8 hours, displaying a 2.86-fold (P < 0.01) and 2.39-fold (P < 0.05) increase, respectively, compared to J2s in the control group ( Figure 4B ). In contrast, J2s exposed to root exudates of ‘Hefeng25’ for 6 hours showed significantly (P < 0.05) higher levels of HgCYP33E1 transcript compared to the control worms. These results suggest that HgCYP33E1 may be involved in responding to and processing xenobiotics present in the root exudates of soybean plants, potentially contributing to the interaction between H. glycines and its host plant.

Previous research suggested that certain metabolites produced by microorganisms possess nematicidal activity against H. glycines J2s (Lai et al., 2014). To investigate whether HgCYP33E1 is involved in the metabolism of these xenobiotic substances from biocontrol agents, we assessed the expression levels of HgCYP33E1 when J2s were exposed to the fermentation broth of Bv-DS1, known for its the biocontrol efficiency against M. incognita (Hu et al., 2022).The results revealed that Bv-DS1 fermentation treatment caused a mortality rate of 76.4% in H. glycines J2s ( Figure S3 ), suggesting that the Bv-DS1 metabolites display toxic effects on H. glycines similar to those observed for M. incognita. J2s exposed to Bv-DS1 fermentation did not show significant dereference in the transcript levels of HgCYP33E1 compared to nematodes treated with the sterilized culture broth (control) at 2, 4, 6, 8, or 16 hours. However, HgCYP33E1 expression significantly (P < 0.05) increased after 12 h of incubation with Bv-DS1 fermentation. Interesting, after 24 hours of treatment, there was a remarkable reduction in HgCYP33E1 expression ( Figure 4C ).

To explore the role of HgCYP33E1 in H. glycines parasitism, a 787-bp double-stranded RNA (dsRNA) was synthesized, containing a conservative region of HgCYP33E1 (at 328–1160 bp). This dsRNA was then used to inhibit HgCYP33E1 expression by soaking H. glycines J2s in a dsRNA solution. To ensure the specificity of silencing, the targeted fragment of HgCYP33E1 was confirmed by BLAST search in NCBI and the H. glycines genome database. No significant nucleotide match for other genes was found in H. glycines. In addition, the sequence corresponding to targeted dsHgCYP33E1 was further aligned with its selective three sequences (Hetgly13554, Hetgly15919, and Hetgly20860) with the highest scores in the results of BLAST search. No stretches of identical sequences of >6 nucleotides between the targeted HgCYP33E1 ^328–1160 and three no-targeted sequences were detected ( Figure S4 ). Microscopic examination revealed efficient adsorption of the dsRNA labelled with fluorescein isothiocyanate, a fluorescent tracer, into J2 bodies after 24 h of soaking in the dsRNA buffer ( Figure 5A ). qRT-PCR analysis was conducted to assess the impact of RNAi on the transcript levels of HgCYP33E1 and other HgCYP genes, which were clustered in sub-group I. The results showed a significant (P < 0.05) reduction in transcript levels of HgCYP33E1 in nematodes that were soaked in dsRNA_HgCYP33E1 (2 mg/mL), which was 2.5-fold and 1.9-fold lower than those in the soaking buffer without dsRNA (the control) and dsRNA_GFP solution (non-native control treatment) ( Figure 5B ). However, the expression levels of Hetgly10349 were slightly up-regulated in dsHgCYP33E1-treated J2s relate to the control nematodes, but no statistical significance was found ( Figure 5B ). Subsequently, the infectivity and development of H. glycines on a susceptible cv. ‘Hefeng25’ was further analysed in the pot assay. The number of nematodes soaking in dsRNA_HgCYP33E1 of buffer per root system at 2 dpi was 49.2 ± 6.8, which was slightly decreased by 10.2% when compared to the dsRNA_GFP-treated nematodes (54.8 ± 4.7), although this difference was not statistically significant ( Figure 5C ). At 18 dpi, the proportion of H. glycines of developmental stages in the root system was further evaluated. The roots that were inoculated with the dsRNA_HgCYP33E1-treated nematodes exhibited a slightly higher population of J2s and J3s, while the number of J4s was significantly (P < 0.05) lower, when compared to the control and dsRNA_GFP-treated group. However, there was no significant difference observed in the total number of nematodes per root system among the control, dsRNA_GFP-, or dsRNA_HgCYP33E1-treated nematodes ( Figure 5D ).

The analysis of gene expression suggested the induction of HgCYP33E1 upon exposure to abamectin. Notably, a similar expression pattern of HgCYP33E1 was observed when H. glycines J2s were treated with 5 µg/mL and 10 µg/mL abamectin. Consequently, we proceeded to investigate whether the knockdown of HgCYP33E1 had any impact on H. glycines susceptibility to 5 µg/mL abamectin. After incubation in 0.05% acetone (v/v) solution, J2s treated with dsRNA (for both HgCYP33E1 and GFP) as well as the soaking buffer control displayed similar mortality rates ( Figure 6A ). However, in contrast to J2s exposed to the buffer or GFP_dsRNA, those treated with HgCYP33E1_dsRNA showed significantly (P < 0.05) higher mortality rates after 24-h incubation in 5 µg/mL abamectin. The mortality rate for the HgCYP33E1_dsRNA-treated nematodes was 24.8%, whereas the GFP_dsRNA-treated and buffer-treated groups were 14.2% and 14.8%, respectively ( Figure 6A ). After 48-h abamectin treatment, the mortality rates of J2s soaked in the buffer and GFP_dsRNA significantly increased to 27.4% and 26.5%, respectively, compared to the control group exposed to acetone solution, but the J2 mortality for HgCYP33E1_dsRNA-treated group escalated from 5.8% to 35.9%. The findings indicate that HgCYP33E1 may be involved in the resistance of H. glycines to abamectin. To further examine this hypothesis, 200 non-dsRNA or dsRNA-treated J2s, which had previously been incubated for 24 hours in 5 µg/mL of abamectin solution, were inoculated on 12-days-old ‘Hefeng25’ roots. The effect of abamectin on root invasion of H. glycines after HgCYP33E1 RNAi was then assessed at 2 dpi. We observed that transient exposure to abamectin decreased H. glycines J2s infectivity compared to the control (0.05% acetone). The number of J2s soaked with dsRNA of HgCYP33E1 solution per root was found to be 11.6, which was lower than the GFP_dsRNA-soaked (n = 19.0) and the buffer group (n = 20.4) following abamectin treatment ( Figure 6B ). However, statistical analysis revealed no significant difference between the control and the dsRNA of HgCYP33E1 group.