Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon

Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon Peng Wang , Zhenxing Liu , Qiangqiang Kang , Chongyu Liao , Luming Zou , Kaikai Mao , Hui Yao , Yiping Li , Yutao Xiao doi: https://doi.org/10.1101/2025.06.05.658017 Peng Wang a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhenxing Liu a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qiangqiang Kang a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China c Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau of Ministry of Agriculture and Rural Affairs, College of Plant Protection, Northwest A&F University , Yangling, Shaanxi, 712100, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chongyu Liao a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Luming Zou a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kaikai Mao d Guangxi Key Laboratory of Agro-Environment and Agric-Products Safety, College of Agriculture, Guangxi University , Nanning, Guangxi, 530004, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hui Yao a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yiping Li c Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau of Ministry of Agriculture and Rural Affairs, College of Plant Protection, Northwest A&F University , Yangling, Shaanxi, 712100, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yutao Xiao a Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Gene Editing Technologies(Hainan), Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences , Shenzhen, 518000, China b National Nanfan Research Institute, Chinese Academy of Agricultural Sciences , Sanya, 572024, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: xiaoyutao{at}caas.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Bacillus thuringiensis (Bt) crops, engineered to produce insecticidal proteins such as Vip3Aa and Cry toxins, have revolutionized pest management by providing a sustainable alternative to chemical pesticides. However, the development of resistance to these toxins has driven the investigation of underlying mechanisms to better understand how pests evade toxicity and to develop more effective resistance management strategies. Recently, a laboratory-selected Spodoptera frugiperda strain exhibited a high level of resistance to Vip3Aa (resistance ratio: 5562-fold), with the mutation of the chitin synthase gene, SfCHS2 , identified as a key factor. In this study, we extend these findings to additional lepidopteran species, including Spodoptera exigua and Agrotis ipsilon . Our results show that CHS2 knockout strains lack the peritrophic matrix (PM), while the resistant Sfru_R3 strain retains its PM. Knockout of this gene resulted in high levels resistance to Vip3Aa. These findings further validate the role of the CHS2 gene in Vip3Aa resistance and highlight its potential as a target for resistance management in lepidopteran pests. This study advances our understanding of the molecular mechanisms behind resistance to Vip3Aa and supports the development of strategies to delay resistance in Bt crop management. 1. Introduction Genetically modified (GM) plants that produce toxins derived from the bacterium Bacillus thuringiensis (Bt) are known as Bt crops and represent one of the most successful applications of genetic engineering in agriculture. These transgenic plants (eg: cotton, maize and soybean) can produce toxic proteins to disrupt the digestive system of many key lepidopteran and coleopteran pests, thus have revolutionized pest management by offering an efficient, environmentally friendly alternative to chemical pesticides. The most widely used are crops expressing Cry toxins, which have been commercialized and widely grown around the world for decades( Hutchison et al., 2010 ; Lu et al., 2012 ). However, intensive planting of these Bt crops has created a growing number of resistance in target insect species ( Dhurua and Gujar, 2011 ; Orozco-Restrepo et al., 2024 ; Tabashnik et al., 2023 ; Yang et al., 2022a ; Zhang et al., 2016 ). The Vip3Aa toxin, which is produced in the vegetative stage of Bt, was introduced in crops alone, or with other Cry toxins together, to confer higher protection and delay insect resistance ( Kurtz et al., 2007 ; Niu et al., 2021 ; Rabelo et al., 2020 ; Yang et al., 2022a ). Understanding the mechanism of Vip3Aa toxicity and resistance is essential for effective pest management ( Wu, 2014 ; Yang et al., 2022b ). The protein sequence of Vip3Aa shows no homology with Cry proteins ( Estruch et al., 1996 ), and current evidence suggests no cross-resistance between the two ( Bachler et al., 2024 ; Chakroun et al., 2016 ; Kurtz et al., 2007 ; Liu et al., 2024 ; Sena et al., 2009 ), indicating a different resistance mechanism for Vip3Aa. Although the resistance to Vip3Aa in field pest populations is rare ( Amaral et al., 2020 ; Yang et al., 2021a ), several laboratory-isolated strains have shown medium-to-high resistance, providing valuable resources for detecting the underlying mechanism ( Bachler et al., 2024 ; Bernardi et al., 2016 ; Carrière et al., 2023 ; Jin et al., 2023 ; Kerns et al., 2023 ; Liu et al., 2024 ; Pickett et al., 2017 ; Roy et al., 2025a , b; Yang et al., 2020 ; Yang et al., 2021b ). Only a few genes have been identified as being associated with Vip3Aa resistance in vivo. In Helicoverpa armigera, HaVipR1 was identified as a crucial determinant of Vip3Aa resistance from two field-derived resistant lines, with its role in mediating resistance confirmed through knock-out experiments ( Bachler et al., 2024 ). The involvement of this gene in Vip3Aa resistance was further validated in Spodoptera frugiperda ( Zhang et al., 2024 ). Recently, two genes associated with Vip3Aa resistance in S. frugiperda were identified from two distinct field-derived populations in our group. The downregulation of SfMyb gene in DH-R strain confers 206-fold resistance ( Jin et al., 2023 ), while a naturally occurring mutation of a chitin synthase gene ( SfCHS2 ) in Sfru_R3 strain confers 5560-fold resistance ( Liu et al., 2024 ). The CHS2 gene is primarily expressed in insect gut epithelial cells and produces chitin for the formation of the peritrophic matrix (PM) ( Merzendorfer and Zimoch, 2003 ). However, the relationship between PM formation and Vip3Aa resistance has not been well defined ( Jiang et al., 2023 ). Following the knock-out of CHS2 genes in S. frugiperda, Spodoptera litura , and Mythimna separata in our last paper ( Liu et al., 2024 ), we here extend the knock-out to two additional lepidopteran species— Spodoptera exigua , and Agrotis ipsilon . The results showed high levels resistance to Vip3Aa and the absence of PM in all knock-out strains, while the PM was retained in the Sfru_R3 strain. These findings provide further evidence of CHS2 gene’s association with Vip3Aa resistance in lepidopteran insects in general. 2. Materials and methods 2.1. Insect strains and rearing The S. frugiperda, S. litura , and M. separata strains were described in our previous work ( Liu et al., 2024 ). The Se-WT strain of S. exigua was first collected in Yantai City, Shandong Province, China, in 2023. The Ai-WT strain of A. ipsilon was purchased from Keyun Biological Co., Ltd. (China) in 2024. The wild-type strains and their corresponding knock-out strains, SeCHS2-KO and AiCHS2-KO, were maintained at 26 ± 2 °C and 70 ± 10% relative humidity (RH) under a 14 h light:10 h dark photoperiod. The larvae were fed with a wheat germ based diet ( Jin et al., 2021 ). Adults were provided with 10% sucrose solution. 2.2. Design and preparation of sgRNAs The SeCHS2 and AiCHS2 genes were identified by using the protein sequence of SfCHS2 (protein_id: WXB24377.1) to perform a BLAST search against the S. exigua and A. ipsilon data base. The sgRNA target sites of SeCHS2 and AiCHS2 were then identified using the sgRNAcas9 (V3.0) software ( www.biootools.com/software ). Two sgRNAs were used for each insect species (Se-sgRNA1 targeting exon21 of SeCHS2 : 5’-TGTCCACCACTAAATTGGATTGG-3’; Se-sgRNA2 targeting exon22 of SeCHS2 : 5’- ACGACTGAACACCGACGACCTGG-3’; Ai-sgRNA1 targeting exon21 of AiCHS2 : 5’- TCTCTACCACGCATCTCAACTGG-3’; Ai-sgRNA2 targeting exon22 of AiCHS2 : 5’- ACGTCTGAACACAGACGACTTGG-3’. PAM sequences are underlined). The GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific, cat. no. A29377) was used to synthesize and purify the sgRNAs according to the manufacturer’s instructions. 2.3. Embryo collection and microinjection Freshly laid eggs (within 1 hour after oviposition) were collected and fixed onto a glass slide with double-sided tape. A mixture of sgRNAs (200 ng/μl) and Cas9 protein (500 ng/μl) (TrueCut TM Cas9 Protein v2, Thermo Fisher Scientific, cat. no. A36497) was injected into individual eggs using a FemtoJet 4i and InjectMan 4 microinjection system (Eppendorf, Hamburg, Germany) within 1 hour. Injected eggs were placed at 26 ± 2 ° C with 70 ± 10% relative humidity under a 14 h light:10 h dark photoperiod until hatching. 2.4. Mutagenesis detection and homozygous screening Genomic DNA was extracted from the hind legs of adults prior to mating. Primers flanking the target sites were used to detect mutagenesis (Table S1), and the PCR product was sequenced by Sangon Biotech (Shanghai, China). Microinjected G 0 individuals were single paired with wild-type individuals to generate G 1 progeny. Heterozygous G 1 adults with the same mutation were crossed to generate G 2 progeny. The homozygous G 2 individuals were then crossed to establish a knock-out strain. 2.5. Bt toxin and bioassays The Vip3Aa protoxin was purchased from Beijing Generalpest Biotech Research Co., Ltd. (Beijing, China). Diet overlay bioassays were conducted as previously described ( Liu et al., 2024 ). Briefly, diet was dispensed into 24-well plates, and different concentrations of Vip3Aa (0, 200, 400, and 800μg /cm 2 ) were evenly applied to the surface and allowed to dry. A single unfed neonate was transferred to each well. After 7 days, mortality was recorded (larvae were considered dead if they had died or remained in the first or second instar). SPSS18.0 was used to calculate the LC 50 . LC 50 values with non-overlapping 95% confidence intervals (CIs) were considered significantly different. Resistance ratios were calculated by dividing the LC 50 of the tested strain by the LC 50 of the wild-type or susceptible strain (SS). For homozygous knock-out individuals that suffered severe fitness cost and could not reach the adult stage, progeny from heterozygous crosses were used for the overlay bioassay. Survivors were subjected to PCR testing to determine their mutagenesis types. 2.6. Dissection and histological analysis The final instar larvae were used for dissection in all strains. The midgut was fixed with Bouin’s solution to observe the presence of PM. For histological analysis of the midgut, the samples were fixed in 4% paraformaldehyde and sent to Servicebio (Wuhan, China) for paraffin embedding. Briefly, the samples were dehydrated using a serial grades of ethanol, followed by clearing with xylene. The pretreated midgut was embedded in paraffin (Sigma-Aldrich, USA), and the midgut samples were cross-sectioned into 5 μm thick slices. The paraffin sections were then deparaffinized through successive baths of xylene and rehydrated through serial grades of ethanol, stained with hematoxylin–eosin (Servicebio, Wuhan, China), and finally mounted with neutral resin (Servicebio, Wuhan, China). The slides were examined under a digital pathology system (Pannoramic MIDI, 3DHISTECH Int.) to assess the morphological characteristics of the midgut samples. 3. Results 3.1. Knockout of CHS2 resulted in high levels resistance to Vip3Aa toxin in S. exigua and A. ipsilon The CHS2 gene sequences of S. exigua (GenBank ID: DQ912929.1 ) and A. ipsilon (GenBank ID: OL826754.1 ) were used to design sgRNAs to induce mutations at the target sites. Two strains with frameshift mutations were obtained and designated as SeCHS2-KO and AiCHS2-KO (Fig. S1). Each strain carries a deletion of 5 or 146 base pairs, respectively. The altered sequence of SeCHS2 introduced a stop codon in exon 21, yielding a truncated protein consisting of 1350 amino acids. Similarly, the modified AiCHS2 sequence introduced a stop codon in exon 23, producing a truncated protein of 1,396 amino acids. The bioassay results showed that SeCHS2-KO strain survived well on a diet overlaid with 800 μg/cm 2 Vip3Aa toxin, exhibiting a resistance ratio of >33,333-fold compared to the wild-type susceptible strain ( Table 1 ). However, the homozygous mutants of A. ipsilon experienced a very high fitness cost, with only a few individuals reaching the adult stage and no offspring obtained. Alternatively, we used the progeny of heterozygous crosses in the bioassay. Survivors were observed in all treatments, with 13 out of 72 progressing beyond the second instar when exposed to ≥200 μg/cm 2 (maximum: 800 μg/cm 2 ) Vip3Aa toxin ( Table 2 ). PCR analysis showed that all these survivors were homozygotes (Fig. S2). The proportion of survivors did not significantly differ from the expected 25% homozygous ratio, as determined by a one-sample z-test for proportions ( p = 0.174). These results suggest that the resistance ratio of homozygous mutants of A. ipsilon is >11,268-fold compared to the wild-type susceptible strain. View this table: View inline View popup Download powerpoint Table 1. Responses of CHS2-knockout strains to Vip3Aa View this table: View inline View popup Download powerpoint Table 2. Bioassay with offspring from heterozygous crosses of Agrotis ipsilon 3.2. The peritrophic matrix was absent in the CHS2 knockout strains Since CHS2 is responsible for chitin synthesis in the midgut of lepidopterans, it’s important to determine whether knockout of this gene affects midgut structure. Knockout strains and their corresponding wild-type strains were dissected at their final instar of larval stage. The midgut tissues were excised and fixed in Bouin’s solution, which imparts a bright yellow coloration to enhance visibility. Because the midgut and PM have different contraction ratios, the PM protrudes from the midgut when excised, providing a clear indicator of its presence. The results showed the PM were absent in all knockout strains but present in wild-type strains ( Fig. 1 ). Download figure Open in new tab Fig. 1. The peritrophic matrix was absent in the CHS2 knockout strains. Dissection of final instar larvae clearly show the presence of the PM in wild-type strains of S. exigua (a) and A. ipsilon (c), but it is absent in their corresponding knockout strains (b and d). MG, midgut; PM, peritrophic matrix. The CHS2 gene was initially associated with Vip3Aa resistance in a laboratory-selected S. frugiperda strain (Sfru_R3). To further investigate this, we also dissected the Sfru_R3 strain along with the S. frugiperda knockout strain (SfCHS2-KO-A), and the M. separata (MsCHS2-KO) and S. litura (SlCHS2-KO) knockout strains described in our previous study ( Liu et al., 2024 ). The results confirmed that the PM was absent in all knockout strains, but present in Sfru_R3 and the corresponding wild-type strains (Fig. S3). 3.3. Histological structure analysis further confirmed the absence of PM in knockout strains of two species The A. ipsilon strains were selected for histological structure analysis. Transverse sections of the midgut showed that the PM was absent in the knockout strain AiCHS2-KO, but present in wild-type susceptible strains ( Fig. 2 ). Download figure Open in new tab Fig. 2. Histological analysis of transverse midgut sections in A. ipsilon . The results show that the PM is absent in the A. ipsilon knockout strain (AiCHS2-KO), but present in the wild-type strain (Ai-WT). MG, midgut; PM, peritrophic matrix. 4. Discussion In our previous work, we identified a retrotransposon (Yaoer)-mediated disruption of CHS2 in S. frugiperda , which conferred high resistance to Vip3Aa ( Liu et al., 2024 ). Knockout of this gene in S. frugiperda , as well as in two other lepidopteran species, S. litura and M. separata , resulted very high resistance to Vip3Aa. In this study, we generated knockout strains for two additional species, S. exigua and A. ipsilon , both of which also exhibited high resistance to Vip3Aa ( Table 1 and Table 2 ). The role of CHS2 in determining Vip3Aa susceptibility across five species from three different noctuid tribes suggests a conserved function in Lepidoptera. The outcomes of CHS2 knockout, however, differed markedly across species. In A. ipsilon , only a few homozygous mutants survived to adulthood, and none produced viable offspring. Similarly, our attempts to establish a knockout strain of H. armigera failed due to rapid colony collapse. By contrast, a homozygous knockout strain of S. exigua could be obtained, although many individuals died during larval development (data not shown), indicating a significant but less absolute fitness cost. Together with our previous findings in three other species( Liu et al., 2024 ), these results demonstrate that CHS2 disruption severely compromises survival and reproduction, but the magnitude of this effect varies among species. Comparing these species-level outcomes provides critical insight into the likelihood of CHS2-associated resistance persisting in natural populations. The PM in insects functions as a permeable barrier between the food bolus and the midgut epithelium, facilitating digestive processes while protecting the brush border from mechanical damage and attacks by toxins and pathogens ( Tellam, 1996 ). The PM is composed of a complex mixture of chitin, proteins, glycoproteins and proteoglycans ( Wang and Granados, 2001 ). Our study demonstrates that knocking out the CHS2 gene resulted in the absence of the PM in all five species during the larval stage, as shown in both dissection results ( Fig 1 and Fig S3) and histological structure analysis ( Fig 2 ). Many homozygous individuals died during the larval stage, suggesting impaired digestive capability. The PM in the Sfru_R3 strain, on the other hand, remained intact ( Fig 1 ), likely due to the reduced yet persistent presence of wild-type SfCHS2 transcripts ( Liu et al., 2024 ). It appears that the level of wild-type SfCHS2 transcripts is negative correlated with the resistance ratio to Vip3Aa and, consequently, with the chitin content in the PM. Recent research has shown that the binding of Domain V of Vip3Aa toxin to the PM via glycan-binding activity contributes to Vip3Aa’s insecticidal activity, while domains II-III of Vip3Aa were involved in binding to brush border membrane vesicles (BBMV) proteins ( Infante et al., 2024 ; Jiang et al., 2023 ). Understanding how Vip3Aa interacts with its receptor and functions as an efficient insecticide is crucial for countering resistance in the field. It has been proposed that proteins such as scavenger receptor-C (Sf-SR-C), fibroblast growth factor receptor-like protein (Sf-FGFR), and Prohibitin II serve as Vip3Aa receptors in Sf9 cell line ( An et al., 2022 ; Jiang et al., 2018a ; Jiang et al., 2018b ). However, knockout experiments using CRISPR/Cas9 in S. frugiperda failed to confirm Sf-SR-C and Sf-FGFR as functional receptors (Sh an et al., 2022 ). To date, three genes have been reported as being associated with Vip3Aa resistance in S. frugiperda : SfMyb, SfCHS2 and SfVipR1 ( Jin et al., 2023 ; Liu et al., 2024 ; Zhang et al., 2024 ). Additionally, a recent study indicates that three distinct genetic loci are involved in Vip3Aa resistance in five resistant H. zea strains ( Yang et al., 2024 ). Collectively, these findings indicate that resistance to Vip3Aa is likely determined by multiple factors, with mutations in CHS2 having greater potential to confer high levels of resistance. In summary, our findings demonstrate here that knocking out CHS2 in multiple lepidopteran species confers high levels resistance to Vip3Aa toxin, with the phenotype closely linked to the absence of the PM. These results highlight the essential role of CHS2 in maintaining PM integrity and its association with Vip3Aa susceptibility. Further investigation into the involvement of the PM in Vip3Aa resistance presents an intriguing research avenue. A deeper understanding of this mechanism could provide valuable insights into the mode of action of Vip3Aa and contribute to the development of innovative strategies for sustainable pest management. Data availability All data generated and analyzed in this study are available within the article and the supplementary information. Declaration of competing Interests The authors declare no competing interests. Acknowledgements The authors acknowledge the financial support by Biological Breeding-National Science and Technology Major Project (2022ZD04021), theAgricultural Science and Technology Innovation Program (CAAS-ZDRW202412), Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSCB-202303), Shenzhen Science and Technology Program (KQTD20180411143628272), the National Natural Science Foundation of China (32202352), and The Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences. Funder Information Declared Biological Breeding-National Science and Technology Major Project , 2022ZD04021 Agricultural Science and Technology Innovation Program , CAAS-ZDRW202412 Innovation Program of Chinese Academy of Agricultural Sciences , CAAS-CSCB-202303 Shenzhen Science and Technology Program , KQTD20180411143628272 National Natural Science Foundation of China , 32202352 The Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences Footnotes We found that the fitness cost of Spodoptera frugiperda strains reared on corn leaves has not been adequately evaluated; therefore, this part has been omitted. References ↵ Amaral , F.S.A. , Guidolin , A.S. , Salmeron , E. , Kanno , R.H. , Padovez , F.E.O. , Fatoretto , J.C. , Omoto , C. , 2020 . Geographical distribution of Vip3Aa20 resistance allele frequencies in Spodoptera frugiperda (Lepidoptera: Noctuidae) populations in Brazil . Pest Manage. Sci . 76 , 169 – 178 . doi: 10.1002/ps.5490 . OpenUrl CrossRef ↵ An , B. , Zhang , Y. , Li , X. , Hou , X. , Yan , B. , Cai , J. , 2022 . PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability . Virulence 13 , 684 – 697 . doi: 10.1080/21505594.2022.2064596 . OpenUrl CrossRef PubMed ↵ Bachler , A. , Padovan , A. , Anderson , C.J. , Wei , Y. , Wu , Y. , Pearce , S. , Downes , S. , James , B. , Tessnow , A. , Sword , G.A. , Williams , M. , Tay , W.T. , Gordon , K.H.J. , Walsh , T.K. , 2024 . Disruption of HaVipR1 confers Vip3Aa resistance in the moth crop pest Helicoverpa armigera . PLoS Biol 23 , e3003165 . doi: 10.1101/2024.08.11.607516 . OpenUrl Abstract / FREE Full Text ↵ Bernardi , O. , Bernardi , D. , Horikoshi , R.J. , Okuma , D.M. , Miraldo , L.L. , Fatoretto , J. , Medeiros , F.C. , Burd , T. , Omoto , C. , 2016 . Selection and characterization of resistance to the Vip3Aa20 protein from Bacillus thuringiensis in Spodoptera frugiperda . Pest Manage. Sci . 72 , 1794 – 1802 . doi: 10.1002/ps.4223 . OpenUrl CrossRef PubMed ↵ Carrière , Y. , Degain , B. , Unnithan , G.C. , Tabashnik , B.E. , 2023 . Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae) . J. Econ. Entomol . 116 , 1804 – 1811 . doi: 10.1093/jee/toad145 . OpenUrl CrossRef PubMed ↵ Chakroun , M. , Banyuls , N. , Walsh , T. , Downes , S. , James , B. , Ferré , J. , 2016 . Characterization of the resistance to Vip3Aa in Helicoverpa armigera from Australia and the role of midgut processing and receptor binding . Sci Rep 6 , 24311 . doi: 10.1038/srep24311 . OpenUrl CrossRef PubMed ↵ Dhurua , S. , Gujar , G.T. , 2011 . Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India . Pest Manage. Sci . 67 , 898 – 903 . doi: 10.1002/ps.2127 . OpenUrl CrossRef PubMed Web of Science ↵ Estruch , J.J. , Warren , G.W. , Mullins , M.A. , Nye , G.J. , Craig , J.A. , Koziel , M.G. , 1996 . Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects . Proc Natl Acad Sci U S A 93 , 5389 – 5394 . doi: 10.1073/pnas.93.11.5389 . OpenUrl Abstract / FREE Full Text ↵ Hutchison , W.D. , Burkness , E.C. , Mitchell , P.D. , Moon , R.D. , Leslie , T.W. , Fleischer , S.J. , Abrahamson , M. , Hamilton , K.L. , Steffey , K.L. , Gray , M.E. , Hellmich , R.L. , Kaster , L.V. , Hunt , T.E. , Wright , R.J. , Pecinovsky , K. , Rabaey , T.L. , Flood , B.R. , Raun , E.S. , 2010 . Areawide suppression of European corn borer with Bt maize reaps savings to Non-Bt maize growers . Science 330 , 222 – 225 . doi: 10.1126/science.1190242 . OpenUrl Abstract / FREE Full Text ↵ Infante , O. , Gómez , I. , Pélaez-Aguilar , A.E. , Verduzco-Rosas , L.A. , García-Suárez , R. , García-Gómez , B.I. , Wang , Z. , Zhang , J. , Guerrero , A. , Bravo , A. , Soberón , M. , 2024 . Insights into the structural changes that trigger receptor binding upon proteolytic activation of Bacillus thuringiensis Vip3Aa insecticidal protein . PLOS Pathog 20 , e1012765 . doi: 10.1371/journal.ppat.1012765 . OpenUrl CrossRef ↵ Jiang , K. , Chen , Z. , Zang , Y. , Shi , Y. , Shang , C. , Jiao , X. , Cai , J. , Gao , X. , 2023 . Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity . J. Biol. Chem . 299 , 103000 . doi: 10.1016/j.jbc.2023.103000 . OpenUrl CrossRef PubMed ↵ Jiang , K. , Hou , X.-y. , Tan , T.-t. , Cao , Z.-l. , Mei , S.-q. , Yan , B. , Chang , J. , Han , L. , Zhao , D. , Cai , J. , 2018a . Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis . PLOS Pathog 14 , e1007347 . doi: 10.1371/journal.ppat.1007347 . OpenUrl CrossRef PubMed ↵ Jiang , K. , Hou , X. , Han , L. , Tan , T. , Cao , Z. , Cai , J. , 2018b . Fibroblast growth factor receptor, a novel receptor for vegetative insecticidal protein Vip3Aa . Toxins 10 , 546 . doi: 10.3390/toxins10120546 . OpenUrl CrossRef PubMed ↵ Jin , M.-h. , Tao , J.-h. , Li , Q. , Cheng , Y. , Sun , X.-x. , Wu , K.-m. , Xiao , Y.-t. , 2021 . Genome editing of the SfABCC2 gene confers resistance to Cry1F toxin from Bacillus thuringiensis in Spodoptera frugiperda . J Integr Agric 20 , 815 – 820 . doi: 10.1016/S2095-3119(19)62772-3 . OpenUrl CrossRef ↵ Jin , M. , Shan , Y. , Peng , Y. , Wang , W. , Zhang , H. , Liu , K. , Heckel , D.G. , Wu , K. , Tabashnik , B.E. , Xiao , Y. , 2023 . Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm . Proc Natl Acad Sci U S A 120 , e2306932120 . doi: 10.1073/pnas.2306932120 . OpenUrl CrossRef PubMed ↵ Kerns , D.D. , Yang , F. , Kerns , D.L. , Stewart , S.D. , Jurat-Fuentes , J.L. , 2023 . Reduced toxin binding associated with resistance to Vip3Aa in the corn earworm (Helicoverpa zea) . Appl Environ Microbiol 89 , e01644 – 01623 . doi: 10.1128/aem.01644-23 . OpenUrl CrossRef PubMed ↵ Kurtz , R.W. , McCaffery , A. , O’Reilly , D. , 2007 . Insect resistance management for Syngenta’s VipCot™ transgenic cotton . J Invertebr Pathol 95 , 227 – 230 . doi: 10.1016/j.jip.2007.03.014 . OpenUrl CrossRef PubMed ↵ Liu , Z. , Liao , C. , Zou , L. , Jin , M. , Shan , Y. , Quan , Y. , Yao , H. , Zhang , L. , Wang , P. , Liu , Z. , Wang , N. , Li , A. , Liu , K. , Tabashnik , B.E. , Heckel , D.G. , Wu , K. , Xiao , Y. , 2024 . Retrotransposon-mediated disruption of a chitin synthase gene confers insect resistance to Bacillus thuringiensis Vip3Aa toxin . PLoS Biol 22 , e3002704 . doi: 10.1371/journal.pbio.3002704 . OpenUrl CrossRef PubMed ↵ Lu , Y. , Wu , K. , Jiang , Y. , Guo , Y. , Desneux , N. , 2012 . Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services . Nature 487 , 362 – 365 . doi: 10.1038/nature11153 . OpenUrl CrossRef PubMed Web of Science ↵ Merzendorfer , H. , Zimoch , L. , 2003 . Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases . J Exp Biol 206 , 4393 – 4412 . doi: 10.1242/jeb.00709 . OpenUrl Abstract / FREE Full Text ↵ Niu , Y. , Oyediran , I. , Yu , W. , Lin , S. , Dimase , M. , Brown , S. , Reay-Jones , F.P.F. , Cook , D. , Reisig , D. , Thrash , B. , Ni , X. , Paula-Moraes , S.V. , Zhang , Y. , Chen , J.S. , Wen , Z. , Huang , F. , 2021 . Populations of Helicoverpa zea (Boddie) in the southeastern United States are commonly resistant to Cry1Ab, but still susceptible to Vip3Aa20 expressed in MIR 162 corn . Toxins 13 , 63 . doi: 10.3390/toxins13010063 . OpenUrl CrossRef PubMed ↵ Orozco-Restrepo , S.M. , Santos-Amaya , O.F. , Miranda , M.d.S. , Tavares , C.S. , Pereira , E.J.G. , 2024 . Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105 + Cry2Ab and Cry1F Bt maize . J. Econ. Entomol . 117 , 1095 – 1105 . doi: 10.1093/jee/toae082 . OpenUrl CrossRef PubMed ↵ Pickett , B.R. , Gulzar , A. , Ferré , J. , Wright , D.J. , 2017 . Bacillus thuringiensis Vip3Aa toxin resistance in Heliothis virescens (Lepidoptera: Noctuidae) . Appl Environ Microbiol 83 , e03506 – 03516 . doi: 10.1128/AEM.03506-16 . OpenUrl Abstract / FREE Full Text ↵ Rabelo , M.M. , Paula-Moraes , S.V. , Pereira , E.J.G. , Siegfried , B.D. , 2020 . Demographic performance of Helicoverpa zea populations on dual and triple-gene Bt cotton . Toxins 12 , 551 . doi: 10.3390/toxins12090551 . OpenUrl CrossRef PubMed ↵ Roy , R. , Abdelgaffar , H. , Kerns , D. , Huff , M. , Staton , M. , Yang , F. , Huang , F. , Jurat-Fuentes , J.L. , 2025a . Reduced processing and toxin binding associated with resistance to Vip3Aa in a strain of fall armyworm (Spodoptera frugiperda) from Louisiana . Pest Manage. Sci . 81 , 4097 – 4107 . doi: 10.1002/ps.8775 . OpenUrl CrossRef Roy , R. , Abdelgaffar , H. , Kerns , D. , Huff , M. , Staton , M. , Yang , F. , Huang , F. , Jurat-Fuentes , J.L. , 2025b . Reduced processing and toxin binding associated with resistance to Vip3Aa in a strain of fall armyworm (Spodoptera frugiperda) from Louisiana . Pest Manage. Sci . 81 . doi: 10.1002/ps.8775 . OpenUrl CrossRef ↵ Sena , J.A.D. , Hernández-Rodríguez , C.S. , Ferré , J. , 2009 . Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites . Appl Environ Microbiol 75 , 2236 – 2237 . doi: 10.1128/AEM.02342-08 . OpenUrl Abstract / FREE Full Text Shan , Y. , Jin , M. , Chakrabarty , S. , Yang , B. , Li , Q. , Cheng , Y. , Zhang , L. , Xiao , Y. , 2022 . Sf-FGFR and Sf-SR-C are not the receptors for Vip3Aa to exert insecticidal toxicity in Spodoptera frugiperda . Insects 13 , 547 . doi: 10.3390/insects13060547 . OpenUrl CrossRef PubMed ↵ Tabashnik , B.E. , Fabrick , J.A. , Carrière , Y. , 2023 . Global patterns of insect resistance to transgenic Bt crops: The first 25 years . J. Econ. Entomol . 116 , 297 – 309 . doi: 10.1093/jee/toac183 . OpenUrl CrossRef PubMed ↵ Lehane , M.J. , Billingsley , P.F. Tellam , R.L. , 1996 . The peritrophic matrix , in: Lehane , M.J. , Billingsley , P.F. (Eds.), Biology of the Insect Midgut . Springer Netherlands , Dordrecht , pp. 86 – 114 . ↵ Wang , P. , Granados , R.R. , 2001 . Molecular structure of the peritrophic membrane (PM): Identification of potential PM target sites for insect control . Arch Insect Biochem Physiol 47 , 110 – 118 . doi: 10.1002/arch.1041 . OpenUrl CrossRef PubMed Web of Science ↵ Dhadialla , T.S. , Gill , S.S. Wu , Y. , 2014 . Detection and mechanisms of resistance evolved in Insects to Cry toxins from Bacillus thuringiensis , in: Dhadialla , T.S. , Gill , S.S. (Eds.), Advances in Insect Physiology . Academic Press , pp. 297 – 342 . ↵ Yang , F. , González , J.C.S. , Little , N. , Reisig , D. , Payne , G. , Dos Santos , R.F. , Jurat-Fuentes , J.L. , Kurtz , R. , Kerns , D.L. , 2020 . First documentation of major Vip3Aa resistance alleles in field populations of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Texas, USA . Sci Rep 10 , 5867 . doi: 10.1038/s41598-020-62748-8 . OpenUrl CrossRef PubMed ↵ Yang , F. , Head , G.P. , Kerns , D.D. , Jurat-Fuentes , J.L. , Santiago-González , J.C. , Kerns , D.L. , 2024 . Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of in the US . Pest Manage. Sci . 80 , 2796 – 2803 . doi: 10.1002/ps.7988 . OpenUrl CrossRef ↵ Yang , F. , Kerns , D.L. , Little , N. , Brown , S.A. , Stewart , S.D. , Catchot , A.L. , Cook , D.R. , Gore , J. , Crow , W.D. , Lorenz , G.M. , Towles , T. , Tabashnik , B.E. , 2022a . Practical resistance to Cry toxins and efficacy of Vip3Aa in Bt cotton against Helicoverpa zea . Pest Manage. Sci . 78 , 5234 – 5242 . doi: 10.1002/ps.7142 . OpenUrl CrossRef PubMed ↵ Yang , F. , Kerns , D.L. , Little , N.S. , Santiago González , J.C. , Tabashnik , B.E. , 2021a . Early warning of resistance to Bt toxin Vip3Aa in Helicoverpa zea . Toxins 13 , 618 . OpenUrl CrossRef PubMed ↵ Yang , F. , Santiago González , J.C. , Sword , G.A. , Kerns , D.L. , 2021b . Genetic basis of resistance to the Vip3Aa Bt protein in Helicoverpa zea . Pest Manage. Sci . 77 , 1530 – 1535 . doi: 10.1002/ps.6176 . OpenUrl CrossRef PubMed ↵ Yang , F. , Wang , Z. , Kerns , D.L. , 2022b . Resistance of Spodoptera frugiperda to Cry1, Cry2, and Vip3Aa proteins in Bt corn and cotton in the Americas: Implications for the rest of the world . J. Econ. Entomol . 115 , 1752 – 1760 , 1759. doi: 10.1093/jee/toac099 . OpenUrl CrossRef PubMed ↵ Zhang , S. , Zhang , X. , Shen , J. , Mao , K. , You , H. , Li , J. , 2016 . Susceptibility of field populations of the diamondback moth, Plutella xylostella, to a selection of insecticides in Central China . Pestic. Biochem. Physiol . 132 , 38 – 46 . doi: 10.1016/j.pestbp.2016.01.007 . OpenUrl CrossRef PubMed ↵ Zhang , Z. , Wang , L. , Pang , X. , Tay , W.T. , Gordon , K.H.J. , Walsh , T.K. , Yang , Y. , Wu , Y. , 2024 . Knockout of the SfVipR1 gene confers high-level resistance to Bacillus thuringiensis Vip3Aa toxin in Spodoptera frugiperda . bioRxiv , 2024.2009.2026.615236. doi: 10.1101/2024.09.26.615236 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted August 29, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon Peng Wang , Zhenxing Liu , Qiangqiang Kang , Chongyu Liao , Luming Zou , Kaikai Mao , Hui Yao , Yiping Li , Yutao Xiao bioRxiv 2025.06.05.658017; doi: https://doi.org/10.1101/2025.06.05.658017 Share This Article: Copy Citation Tools Functional loss of CHS2 confers high levels resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon Peng Wang , Zhenxing Liu , Qiangqiang Kang , Chongyu Liao , Luming Zou , Kaikai Mao , Hui Yao , Yiping Li , Yutao Xiao bioRxiv 2025.06.05.658017; doi: https://doi.org/10.1101/2025.06.05.658017 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genetics Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41953) Biophysics (21456) Cancer Biology (18595) Cell Biology (25521) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22511) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88623) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)