The effect of chlorantraniliprole on the transcriptomic profile of Spodoptera frugiperda: a typical case analysis for the response of a newly invaded pest to an old insecticide

doi:10.21203/rs.3.rs-2196987/v1

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Research Article

The effect of chlorantraniliprole on the transcriptomic profile of Spodoptera frugiperda: a typical case analysis for the response of a newly invaded pest to an old insecticide

https://doi.org/10.21203/rs.3.rs-2196987/v1

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published 31 Dec, 2022

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Background

Chlorantraniliprole is a diamide insecticide widely used in China over the last 15 years. The fall armyworm (FAW), Spodoptera frugiperda, newly invaded China in 2019. The response of FAW to chlorantraniliprole deserves more attention, in the context of many destructive lepidopteran species are resistant to diamide insecticides and the patent on core chemical of chlorantraniliprole in China expired in August 2022.

Methods and results

This study investigated the response profile in larvae under chlorantraniliprole-induced (LC₅₀) stress using methods of bioassay, RNA-Seq and qPCR. We observed growth inhibition and lethal effects in FAW larvae, but at a relatively high LC₅₀ value compared to other several pests. Additionally, under chlorantraniliprole-induced stress, 3309 unigenes were found to be differentially expressed genes (DGEs). The impacted genes included 137 encoding for detoxification enzymes, 29 encoding for cuticle proteins, and 20 key enzymes involved in the chitin metabolism, which all associated with metabolic resistance. Finally, we obtained the single nucleotide polymorphisms (SNPs) of two RyR genes, which are the target proteins for chlorantraniliprole. We also investigated the causes of the high LC₅₀ value in our FAW, which possibly related to the stabilized 4743M on SNP frequency of RyR. These findings documented the genetic background of RyR of FAW and indicated that application of chlorantraniliprole has a high risk of controlling FAW in China.

Conclusion

In brief, our results provide a better understanding of the mechanisms of chlorantraniliprole toxicity and detoxification in FAW, and will aid in monitoring the development of resistant strains for a newly pest to an old insecticide.

Chlorantraniliprole

Spodoptera frugiperda

Detoxification enzymes

RyR

SNPs

Since it was first detected in 2019, the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), has become a serious pest in China [1]. Many strategies were developed to control the outbreak of S. frugiperda, but chemical control remains the preferred measure due to its high efficiency, low cost, and ease of mechanized application. Conventional insecticides recommended for field application include chlorantraniliprole, emamectin benzoate, spinetoram, indoxacarb, and lufenuron. Therefore, pesticide resistance and FAW’s response to pesticides must be closely monitored.

Chlorantraniliprole is an anthranilic diamide insecticide that binds to ryanodine receptors (RyR), resulting in the uncontrolled release of calcium stores from muscle cells. Diamide insecticides are highly selective against lepidopteran and coleopteran insects but have low toxicity to non-target species such as mammals and fish. Chlorantraniliprole was commercialized around 2007 [2] and quickly gained popularity due to its high insecticidal efficacy. However, overuse and incorrect application practices have led to increasing resistance chlorantraniliprole. Indeed, Richardson, et al. (2020) reported that frequent application has resulted in the development of diamide resistance in many destructive lepidopteran species [3]. The main resistance mechanism of insects to chlorantraniliprole appears to be target-site insensitivity rather than the action of detoxification enzymes [4–6]. Mutations identified on insect RyRs have been found to cause resistance in several major agricultural pests across multiple continents [7]. For example, an I4734M mutation of RyR in a Brazilian FAW strain was found to result in target-site resistance to chlorantraniliprole [8]. Interestingly, overexpression of RyR at the mRNA level is also involved in chlorantraniliprole resistance [9].

The patent on core chemical of chlorantraniliprole in China expired in August 2022, and therefore numerous new pesticide varieties based on chlorantraniliprole will likely become commercially available in the near future. Therefore, it is critical to continue monitoring chlorantraniliprole, especially concerning the development of resistance in FAW. RNA-sequencing is a highly efficient technique for high-resolution genome-wide transcriptomic analysis that provides large-scale gene expression data. In this study, we aimed to investigate transcriptomic information and molecular mechanisms of chlorantraniliprole toxicology by screening and identifying gene expressions and enriched pathways related to physiological and biochemical changes of FAW. We hope our findings will help avoid the problems of diamide resistance that have been observed in many destructive lepidopteran species over the past decade from developing in FAW in China.

Insect rearing and toxicity to S. frugiperda larval

FAW was originally collected from maize field in Chenzhou City, Hunan province, China, in 2019 and was maintained in our laboratory for more than 30 generations under a 16:8 h (L:D) photoperiod at 26 ± 1℃ and 80% relative humidity, without any insecticide-induced stress. Larvae were fed a 1.5 kg artificial diet which was prepared with 240 g vitakraft, 225 g wheat germ slice, 15 g yeast, 45 g agar, 12 g vitamin C, 5.7 g Methyl 4-hydroxybenzoate, and 1.5 g Sorbic acid. Adults were fed 10% honey water.

Bioassays of S. frugiperda third instar larvae were performed using the diet incorporation method. Chlorantraniliprole was dissolved in 1 mL DMSO (analytical grade) with 1% (weight/volume, w/v) Tween 80 to make a 10000 mg/L primary solution. Six working-solution concentrations were serially diluted from the primary solution using sterile distilled water containing 1% (weight/volume, w/v) Tween 80. The control group was treated with sterile distilled water containing 1% (w/v) Tween 80 and 1% (v/v) DMSO. For the diet incorporation method, 1 mL of working-solution was mixed into 20g of the artificial diet. Third instar larvae selected for chlorantraniliprole treatments were then reared on the diet incorporation mix. For the experiment, we used six well culture plates and fed one larva per well a different diet. Each treatment had 30 larvae and was replicated three times.

Illumina sequencing and transcriptomic analysis

Thirty larvae samples were exposed to chlorantraniliprole for 48 hs and were collected in 1.5 mL eppendorf tubes and frozen in liquid nitrogen immediately and then stored at − 80°C. And then samples were mailed on dry ice to Novogene Co., Ltd. in Tianjing (China) for transcriptome sequencing within three days. The clustering of the index-coded samples was sequenced on an Illumina Novaseq 6000 platform and 150 bps paired-end reads were generated. Reference genome and gene model annotation files were downloaded directly from the relevant genome website (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/011/064/685/GCF_011064685.1_ZJU_Sfru_1.0/GCF_011064685.1_ZJU_Sfru_1.0_genomic.gff.gz). The mapped reads of each sample were assembled into the transcriptome using String Tie(1.3.3b)software and novel genes were identified by comparing transcriptome and reference genome. The featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. And then fragments per kilobase per million (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene.

Differential expression analysis of two conditions/groups was performed using the DESeq2 R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate Padj < = 0.05 and |log₂(Foldchange)| ≥1 were set as the threshold for significantly differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package (3.8.1), in which gene length bias was corrected. GO terms with corrected P value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies (http://www.genome.jp/kegg/). We used cluster Profiler R package (3.8.1) to test the statistical enrichment of differential expression genes in KEGG pathway. A HeatMap of candidate genes based on FPKM data was drawn using the software TBtools [10]. And gene physical map of candidate genes was drawn with tool of Mapgene2chrom [11].

Real-time PCR for revalidation of the expression levels of candidate genes

2 µg total RNA of each sample was used for qRT PCR cDNA synthesis using a PrimeScript™ RT reagent Kit (with gDNA Eraser) following the manufacturer’s instruction. Primers were designed using the ncbi and synthesized by Tsingke Biotechnology Co., Ltd (Table S1). The 2X SYBR® Green Pro Taq HS Premix was used for qRT-PCR in a 10 µL reaction solution on a Thermal cycler c1000 touch machine. qRT-PCR has proceeded as follows: one cycle of denaturation at 94°C for 30 s, followed with 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, followed by a melting curve analysis. One reference gene β-actin was selected for normalization of qRT-PCR results [12]. mRNA levels were analyzed by the 2−^△△CT method. Each assay was repeated three times.

Data analysis

The DPS statistical analysis software was used to calculate LC₅₀, with corresponding 95% confidence limit (CL) and chi-square (χ²) of the probit regression equation. The statistical significance of differences between chlorantraniliprole treatment and control means were assessed using Student’s t-test. All analyses and figures were prepared using software of GraphPad Prims 7 and Adobe PhotoShop.

Chlorantraniliprole toxicity on S. frugiperda larval growth and mortality

The toxicity of chlorantraniliprole was investigated in 3rd instar FAW larvae fed an artificial diet containing a chlorantraniliprole concentration gradient for 24 h. FAW larvae mortality increased with increasing concentration of chlorantraniliprole, and the mortality data indicated a good fit to the probit model (χ² = 9.33, df = 4, P > 0.05) (Fig. 1). The estimated LC₁₀, LC₃₀, and LC₅₀ values after 24h were 100.95 mg/L (72.43 ~ 125.27), 194.37 mg/L (165.34 ~ 218.42), and 305.96 mg/L (278.00 ~ 338.13), respectively. Treatment with a sub-lethal concentration (LC₁₀ and LC₃₀) of chlorantraniliprole significantly extended larval development time and mortality (Table 1), indicating chlorantraniliprole inhibits development and ultimately has a lethal effect on larvae. In contrast, there was no significant effect of chlorantraniliprole on pupal weight or survival, or the egg production capacity or survival of adult females.

Table 1 Effects of sublethal concentrations of chlorantraniliprole on the development of Spodoptera frugiperda. ＆, 4~6^th larval stage. Control, treated with DMSO and 1% Tween 80. Data are the means ±SE of the three replications. Means within a column followed by different letters are significantly different according to Duncan’s new multiple range test (P<0.05). ﹟, indicates the pupation rate or emergence rate was calculated from the data of the original 90 larvae.

	Larval stage^＆(d)	Pupal stage (d)	Adult stage (d)	Larval sur- vival (%)	Pupation rate (%)﹟	Pupa weigh (mg)	Emergence rate (%)﹟	Egg numbers per female
Control	9.5±0.6b	8.7±0.1a	8.7±0.3a	98.89±1.1a	95.55±2.9a	194.69±1.9b	91.11±2.9a	885.0±129.8a
LC₁₀	11.5±0.9a	9.0±0.2a	8.4±0.4a	62.66±4.8b	62.22±4.8b	206.35±3.5a	51.1±2.2b	650.7±78.9a
LC₃₀	11.7±0.7a	9.0±1.1a	8.8±0.4a	60.00±5.1b	48.89±2.9c	200.37±3.8ab	41.11±2.9c	565.9±58.0a

Transcriptomic analysis and DEGs identification after chlorantraniliprole exposure

Transcriptomic analysis was performed on the control group and larvae treated with a medium lethal concentration (LC₅₀) of chlorantraniliprole for 48 hr. Compared to the control group, 3309 unigenes were identified as differentially expressed genes (DEGs) in the treatment group, of which 1436 unigenes were upregulated and 1873 were down-regulated (Fig. 2A). According to the log₂Foldchange, more than half the DEGs, whether down- or up-regulated, have 1- to 2-fold changes in their expression level (Fig. 2B). The top down- or up-regulated DEGs with a log₂Foldchange of more than 5 (Table S2) should be focused on, such as cuticle protein 16.5-like (gene ID: 118277907, log₂Foldchange = 6.9), esterase E4-like (118279226, 6.7), lipase 3 (118268348, 5.4), peroxidase-like (118280587, -9.6), esterase FE4-like (118276233, -9.3), vitellogenin-like (118280769, -8.5), and other DEGs whose function was uncharacterized. Carboxylesterases (COEs) are usually involved in mediating insecticide metabolism and resistance [13]. Thus, the significant change in both up- and down-regulated expression levels of the same esterase family following chlorantraniliprole exposure warrants further investigation. The DEG, especially detoxification genes, findings from our study could provide significant insights into the molecular mechanisms of chlorantraniliprole’s toxicity to FAW.

DEGs annotated in the GO database were divided into three categories, of which 1104 were annotated in 389 GO terms of Biological process, 492 were annotated in 77 terms of Cellular component, and 1555 were mapped in 255 GO terms of Molecular function (Fig. 2C). Among these GO terms, 27 were significantly enriched (corrected P-values < 0.05). However, no GO terms of Biological Process were significantly enriched. The enriched GO terms for Cellular component included ‘endoplasmic reticulum’ and ‘extracellular region’. The enriched GO terms for Molecular function included 25 sub-terms, in which the top three were ‘serine-type peptidase activity’, ‘serine hydrolase activity’, and ‘coenzyme binding’.

KEGG analysis revealed that 803 DEGs between the control and treatment groups were assigned to 120 pathways. Among these KEGG pathways, 10 were significantly enriched (corrected P-values < 0.05), and the top three pathways were “One carbon pool by folate” (involves 10 DEGs), “Drug metabolism-other enzymes” (involves 45 DEGs), and “Toll and Imd signaling pathway” (involves 27 DEGs) (Fig. 2D).

Functional and distribution analysis of chlorantraniliprole-responsive DEGs

Many DEGs were found to be involved in detoxification, including genes encoding cytochrome monooxygenases (P450s), glutathione S-transferases (GSTs), carboxylesterases (COEs), UDP glucosyltransferases (UGTs), and ATP-binding cassette transporters (ABCs) (Fig. 3A). Additionally, 137 detoxification genes were differentially expressed between the chlorantraniliprole-treated and control groups. Chlorantraniliprole treatment had a significant effect on 47 P450s, with 15 genes up-regulated in the chlorantraniliprole-treated larvae, including one gene in clan CYP2, seven genes in clan CYP3, seven genes in clan CYP4, and one mitochondrial CYP gene. The P450 members related to insecticides resistance are mainly concentrated in the CYP3 and CYP4 families. In 18 genes belonging to GSTs, only two genes encoding GSTs were up-regulated by chlorantraniliprole. Of the 18 UGTs identified as DEGs, eight of them were up-regulated by chlorantraniliprole. Furthermore, chlorantraniliprole had a significant effect on 40 COEs, with 11 up-regulated, and 14 genes encoding ABC transporters, with seven up-regulated.

To determine if the 137 DEGs correlated in chromosomal position, we obtained their distribution on the 32 chromosomes of FAW (Fig. S1). We found the distance between most of the DEGs was reasonably large. The shortest spacing between two DEGs was found on chromosome 3, for example, 118261876 (COEs), 118282316 (P450s), and 118261999 (P450s) (Fig. 3B). When we analyzed the intergenic space of adjacent DGEs, we found that the smallest distances were usually over 4 Kbs (Fig. 3C). Our findings indicate that the possibility of co-transcriptional regulation of different members from the above 137 DEGs is relatively small. So far, we have not been able to identify the upstream or downstream relationships between the detoxification related genes in the catabolism pathway of chlorantraniliprole.

Chitin is the main component of insect epidermis, midgut peritrophic membrane, and tracheal system. Given the decline in pupation and emergence rates of FAW under chlorantraniliprole-induced stress we also looked at genes involved in chitin metabolism. Previous studies identified eight key enzymes involved in the chitin biosynthesis pathway including trehalase (TRE), hexokinase (HK), glucose-6-phosphate isomerase (G6PI), glutamine–fructose-6-phosphate aminotransferase (GFAT), glucosamine 6-phosphate N-acetyltransferase (GNA), acetylglucosamine mutase (AGM), UDP-N-acetylhexosamine pyrophosphorylase (UAP), and chitin synthase (CHS). Our results showed that when exposed to chlorantraniliprole, seven of the enzymes were down-regulated DEGs, including 6 TREs, 1 HK, 1 G6PI, 1 GFAT, 1 GNA, 2 AGM, and 1 UAPs (Fig. 3D). However, none of CHS enzymes, which play a crucial role in the final step of the chitin biosynthesis pathway, were DEGs. Chitinase (CHT) is hydrolytic enzymes that degrade chitin chains to chitooligosaccharides by interrupting the glycosidic bonds to form chiobiose. Chitin deacetylase (CDA) are essential enzymes involved in chitin deposition via N-deacetylation of chitin to form chitosan. Both CHTs and CDAs were down-regulated in larvae under chlorantraniliprole-induced stress (Fig. 3D).

Cuticle proteins (CPs) are critical structural components for insect tissues and influence the penetration efficiency of insecticides into the insect body. Of the 252 CPs in the FAW genome, six (2.4%) displayed up-regulated profiles, including two interesting candidates, bursicon-like (118276337) and extensin-like (118282269). However, 23 (9.1%) CPs were down-regulated, nearly four times the number of up-regulated CPs (Fig. 3D). Moreover, the expressions of 18 of 23 CPs were down-regulated fourfold compared to the control group. The down-regulation of genes involved in chitin synthesis and genes encoding CPs under chlorantraniliprole-induced stress was consistent with the chemical’s effect on larval growth inhibition and pupation rate decline.

Analysis the expression level and SNPs of RYR genes

RyR has previously been identified as the target protein of chlorantraniliprole. According to the reference genome data [14], there are only two RyR genes in the FAW genome. We found that the expression levels of the two RyR genes did not differ significantly between the chlorantraniliprole-treated and control groups, despite having relatively high fragments per kilobase per million (FPKM) values. Additionally, we obtained data on the single nucleotide polymorphisms (SNPs) of the two RyR genes based on the transcriptomic sequences of six biological samples from the chlorantraniliprole-treated and control groups (Table S3, S4). There were several genotypes for the above SNPs, including 0/1, 1/1, and 1/2. Type 0/1 refers to one copy of the sequence being identical to the known sequence of the reference genome and another copy being a new point mutation from the DNA sample of this study (Fig. 4A). Type 1/1 and 1/2 indicate homozygous and heterozygous mutation types, respectively, with point mutations found in the DNA sample from this study but not in FAW’s reference genome. Evidently, 0/1 had the most genotypes, with values of 75.9% and 96.8% in two RyR genes, respectively (Fig. 4B). SNPs can cause synonymous mutations, missense mutations, frameshift, and other effects. We found that synonymous variants accounted for the largest proportion of mutation type of SNPs, accounting for 91.4% in 118277034 and 88.3% in 118276673, respectively (Fig. 4C).

In fact, an I4734M amino acid substitution of RyR (GenBank MK226188) has been reported to confer target-site resistance to chlorantraniliprole in a Brazilian FAW strain [8]. The MK226188 was just the same gene to RyR (118277034) in this study. We found that there were no SNPs on the site of amino acid 4743 of RyR (118277034) in our FAW samples. Moreover, the amino acid on site of 4743 was already M, a short form of methionine (Fig. 4D). It indicated that application of chlorantraniliprole has a high risk of controlling FAW in China. Additionally, a G4946E amino acid substitution (corresponding to site of amino acid 4891 of 118277034) was also reported to confer target-site resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella [15]. There were no SNPs on the site of amino acid 4891 either in our FAW samples. And the amino acid on 4891 is S (a short form of serine), not the E (a short form of glutamicacid) which confer target-site resistance to chlorantraniliprole in P. xylostella.

qRT-PCR validation

To validate the transcriptomic analysis results, we selected DEGs based on RNA-seq data for qRT-PCR validation. DEGs involved in detoxification and chitin catabolism were the first candidates to be selected, including six up-regulated and six down-regulated members (Fig. 5). All these 12 DEGs differed significantly between the cyproflanilide and control treatment groups and followed the same trend, indicating that the changes in gene expression levels based on qRT-PCR were largely consistent with the transcriptomic data. We also validated the mRNA level of two RyRs, the target protein of cyproflanilide, and found that these two RyRs indeed did not change significantly between the cyproflanilide and control treatment groups.

According to our findings, the estimated 24 h LC₅₀ value of chlorantraniliprole for FAW is 305.96 mg/L (278.00 ~ 338.13) (Fig. 1). Previous studies have shown that the estimated 48 h LC₅₀ value of chlorantraniliprole for Spodoptera cosmioides (Lepidoptera: Noctuidae) is only 54 µg/L [16], and at 72 h the LC₅₀ value of chlorantraniliprole for Spodoptera exigua (Lepidoptera:Noctuidae) is only 6.7 µg/L [17], and at 72 h the LC₅₀ value of chlorantraniliprole for Chilo suppressalis (Lepidoptera:Crambidae) is 210 µg/L [18]. These LC₅₀ values are much smaller than that of our study. In addition, it was reported that the Puerto Rico population of FAW exhibited a remarkable field-evolved resistance to chlorantraniliprole within six years, with the resistance ratio reaching to 160-fold [19]. The newly invaded FAW population in China is directly derived from Southeast Asia in 2019. However, FAW in Southeast Asia is derived from West and Central Africa which was originated from the tropical-subtropical regions of the American continent in 2016 [20]. The high LC₅₀ chlorantraniliprole value for our FAW population could be related to the experience on insecticides stress in American continent. Thus, when talking about the word "newly invaded", it is necessary to go back to the pest history of the invasion and of the exposure to pesticides.

Metabolic resistance plays an important role in the early development of pesticide resistance in insects. Lv et al. (2021) found that Chinese FAW population still shows low levels of resistance to diamide insecticides [21]. It seems a little inconsistent with the inference we made according to the high LC₅₀ chlorantraniliprole value mentioned above. Additionally, Lv et al. (2021) also reported that the differences in relative resistance among many FAW populations in China are not caused by RyR mutations or RyR expression [21]. There are numerous other reports on metabolic resistance in FAW. For example, knockout of the ATP-binding cassette transporter B1 (ABCB1) gene has been shown to increase FAW susceptibility to chlorantraniliprole [22]. Overexpression of cytochrome P450 CYP6BG1 [23] and flavin-dependent monooxgenase [24] may contribute to chlorantraniliprole resistance in Plutella xylostella, and Zhang et al. (2020) found that five P450s, including CYP321A8, CYP321A9, CYP321B1, CYP337B5, and CYP6AE44, could be strongly induced by chlorantraniliprole [25]. Another recent study showed that adipokinetic hormone signaling regulates cytochrome P450-mediated chlorantraniliprole sensitivity in FAW [26]. They also reported that 10 P450s, including SpfCYP321A10, SpfCYP321A7, SpfCYP321A9, SpfCYP4G75, SpfCYP337B5, SpfCYP6AE44, SpfCYP9A60, SpfCYP9A59, SpfCYP9A58, and SpfCY321A8, were significantly up-regulated in FAW exposed to chlorantraniliprole. However, there are no published and available DNA sequences for any of the above fifteen P450s. Hence, we were unable to obtain sequences from the reference genome using their gene names alone, and unable to analyze the correlation or difference between above 15 P450s and our 15 up-regulated P450 genes in this research (Fig. 3A). Previous reports also found that miRNA could confer chlorantraniliprole resistance by regulating P450 in FAW [27]. In addition to the P450 genes, we also obtained approximately 100 genes encoding detoxification enzymes or other unknown important proteins that work against chlorantraniliprole, such as GSTs, COEs, UGTs, and ABCs. Identifying these detoxification enzymes and their roles is crucial to understanding the development of insecticide resistance and the insecticidal mechanisms of chlorantraniliprole in FAW larvae before resistant strains of FAW emerge in China.

Insect resistance to insecticides involves not only metabolic resistance, but also cuticular resistance and target-protein resistance. Chitin is the primary component of insect epidermis, midgut peritrophic membrane, and tracheal system, and it is closely related to insect cuticular resistance. Validamycin, a biological pesticide, has been shown to disrupt the chitin biosynthesis pathways, resulting in abnormal phenotypes or even death in the rice brown planthopper [28]. We found that seven of the eight key enzymes in the chitin biosynthesis pathway were down-regulated in FAW following chlorantraniliprole exposure (Fig. 3D). Furthermore, two genes encoding enzymes related to the chitin catabolism pathway were also down-regulated DEGs. During chlorantraniliprole-induced stress, the catalytic capacity of key enzymes in both the chitin biosynthesis and degradation pathways decreased. There was a similar trend in the cuticle proteins (CPs) gene family, with six CPs up-regulated and 23 CPs down-regulated of the 252 CPs in the FAW genome (Fig. 3D). The presence of direct or indirect chlorantraniliprole target proteins in the above genes warrants further investigation in the development of resistance in FAW.

There are different voices and evidences suggesting that the primary mechanism of insect resistance to chlorantraniliprole appears to be target-site insensitivity rather than the action of detoxification enzymes [4–6]. Hence, we analyzed the SNPs of RyR, the target protein of chlorantraniliprole. While FAW has only recently invaded China and thus has had a relatively short exposure period to chlorantraniliprole, we cannot rule out that it had been exposed to the pesticide prior to its invasion. However, recent reports indicate that FAW populations in China still display low resistance levels to other diamide insecticides [21]. Hence, it is important to understand the DNA polymorphism of target genes, especially RyR, in the genome of the current FAW strain. We did not obtain chlorantraniliprole-resistant FAW strains for transcriptomic analysis in this study due to the short stress period. Thus, based on the SNPs data from two RyR genes, we could not identify a definitive mutation site to determine the pesticide-resistance of FAW to chlorantraniliprole. The most important mechanism of resistance in the diamondback moth, Plutella xylostella, has been functionally linked to two target-site mutations in the RyR transmembrane domain, including the amino acid substitutions I4790M (corresponding to substitution of I4743M in FAW RyR) and G4946E (corresponding to site of amino acid 4891 in FAW RyR) (Fig. 4D) [15, 29]. Interestingly, both amino acid residues have been shown to be mutated in diamide resistant tomato leafminers [30] and rice stem borers [31, 32]. Additionally, an I4734M amino acid substitution of RyR (GenBank MK226188) has been shown to confer target-site resistance to chlorantraniliprole in a Brazilian FAW strain [8]. MK226188 is identical to the 118277034 mentioned in this study (Fig. 4D). We did not obtain any SNPs at the site of amino acids 4743 and 4891 from our FAW samples. However, we discovered that the amino acid on site 4743 was already ascribed to ‘M’ in the reference genome of FAW in China and our FAW populations in this study. It indicated that application of chlorantraniliprole probably has a high risk of controlling FAW in China. We obtained information on approximately 300 SNPs for two RyR genes, but none of the SNPs were associated with resistance to chlorantraniliprole. Whether it is inevitable that the FAW population in China evolves resistant strains due to the 4734M within a short time period, similar to what occurred in Brazil [8], or if the population gradually evolves to produce other novel strains resistant to chlorantraniliprole requires further investigation. Nonetheless, our SNP data can be utilized to record the history and genetic background of current FAW populations, and monitor the development of novel target-site chlorantraniliprole-resistant strains in the future.

In conclusion, growth inhibition and lethal effects were observed in FAW larvae treated with chlorantraniliprole, a relatively old pesticide that has been widely used in China for about 15 years. However, the LC₅₀ value of chlorantraniliprole was relatively high compared to previous findings in other pests. Transcriptomic analyses identified numerous DEGs triggered by chlorantraniliprole. Our research analyzed the ranges of mRNA levels, functional categories, and correlations of multiple DEGs genes, focusing on the P450 superfamily, key enzymes coding genes involved chitin metabolism, CPs, and RyR which was the target protein of chlorantraniliprole. Importantly, we obtained the SNPs of two RyR genes and investigated the possible causes of the high LC₅₀ value in China’s recently invaded FAW population. Our findings document the current response and genetic background of the FAW population in China, and provide the foundation for future research on the basic mechanism of cyproflanilide against FAW, as well as aid in monitoring the development of target-site chlorantraniliprole-resistant strains in the future.

Acknowledgments

This work was funded by the key research and development program of Hunan Province (China) (2020NK2034), Science and Technology Program of Changsha (kq2202226). The authors would like to thank Yi Zhou, Qiyao Liang, and Laili Deng for their help in S. frugiperda breeding and insecticide treatments to larvae.

Authors’ contributions

Hualiang He, Yi LI, Youzhi Li and conceived and designed research. Yufeng Lin, Zhengbing Zhang, Lin Qiu, and Wenbing Ding helped with the laboratory and field efficacy trial. Haijuan Shu, Qiao Gao, Jin Xue helped with the data analysis. Hualiang He and Youzhi Li wrote the manuscript. All authors read and approved the manuscript.

Disclosure declaration

The authors declare no conflict of interests.

Compliance with Ethical Standards

Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Availability of Data and Materials

The six datasets generated and analyzed during the current study are available in the NCBI SRA database with the accession number of SRA: SRS15030502, SRS15030503, SRS15030520 (chlorantraniliprole-induced groups), and the accession number of SRA:SRS15030504, SRS15030505, SRS15030506 (control groups) (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA877689).

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The effect of chlorantraniliprole on the transcriptomic profile of Spodoptera frugiperda: a typical case analysis for the response of a newly invaded pest to an old insecticide

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Abstract

Background

Methods and results

Conclusion

Figures

Introduction

Experimental Procedures

Insect rearing and toxicity to S. frugiperda larval

Illumina sequencing and transcriptomic analysis

Real-time PCR for revalidation of the expression levels of candidate genes

Data analysis

Results

Chlorantraniliprole toxicity on S. frugiperda larval growth and mortality

Transcriptomic analysis and DEGs identification after chlorantraniliprole exposure

Functional and distribution analysis of chlorantraniliprole-responsive DEGs

Analysis the expression level and SNPs of RYR genes

qRT-PCR validation

Discussion

Declarations

References

Supplementary Files

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Journal Publication

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