Dermonecrotic toxins from Pyemotes zhonghuajia induce tissue necrosis and show potent insecticidal activity against Spodoptera frugiperda

Dermonecrotic toxins from Pyemotes zhonghuajia induce tissue necrosis and show potent insecticidal activity against Spodoptera frugiperda | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dermonecrotic toxins from Pyemotes zhonghuajia induce tissue necrosis and show potent insecticidal activity against Spodoptera frugiperda Liang Wang, Junxian Fu, Yong Ruan, Jifeng Hu, Jianfeng Liu, Jiuya He, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9386274/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Resistance to conventional insecticides motivates the search for novel bioactive agents. Dermonecrotic toxins from arthropod venoms but underexplored candidates for crop protection. Here we report the cloning, bacterial expression and identification of three recombinant dermonecrotic toxin proteins (PzDNT2, PzDNT3 and PzDNT8) from the ectoparasitic mite Pyemotes zhonghuajia , and we evaluate their insecticidal activity against the invasive lepidopteran pest Spodoptera frugiperda . Hemocoelic bioassays show that all three proteins are potently toxic when injected into fourth-instar larvae, with PzDNT8 displaying the greatest potency (median lethal dose, LD 50 = 0.354 µg/larva; PzDNT2 and PzDNT3: LD 50 = 1.131 and 1.169 µg/larva, respectively). Histopathology reveals that toxin treatment induces rapid and progressive tissue necrosis—characterized by cuticular blackening, fat-body disintegration, midgut epithelial lysis, and muscle degeneration. Furthermore, biochemical assays show time-dependent perturbation of detoxification enzymes (carboxylesterase (CarE), acetylcholinesterase (AChE), and glutathione S-transferase (GST)), indicating a complex metabolic response. Together, these findings indicate that P. zhonghuajia dermonecrotic toxins cause extensive internal tissue damage and overwhelm larval physiological defenses, identifying them as promising leads for next-generation bioinsecticides. Pyemotes zhonghuajia Dermonecrotic toxin Spodoptera frugiperda Tissue necrosis Bioinsecticide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction The escalating challenges of pest resistance and environmental concerns linked to synthetic chemical insecticides have driven the search for sustainable alternatives in modern agriculture (Kesho 2020 ; Reyes-Ávila et al. 2024 ). Biological control, utilizing agents from natural organisms such as fungi, bacteria, viruses, and arthropods, has emerged an essential component of integrated pest management (IPM) strategies (Chandler et al. 2011 ; Baker et al. 2020 ). These bioinsecticides are valued for their high specificity, low environmental persistence, and reduced risk to non-target organisms (Haddi et al. 2020 ; Qu et al. 2022 ). Among the most successful examples are Bacillus thuringiensis (Bt) insecticidal Cry proteins, widely deployed in transgenic crops and microbial formulations to control major pest orders (Schnepf et al. 1998 ; Kumar et al. 2021 ; Karim et al. 2023 ). However, the extensive application of Bt toxins has inevitably accelerated the evolution of resistance among pest populations, undermining long-term effectiveness and highlighting the urgent need for agents with novel modes of action (Jurat-Fuentes et al. 2021 ; Fabrick and Wu 2023 ; Dong et al. 2025 ). Venom-derived proteins from arthropods—including spiders (Yan et al. 2025 ), scorpions (Froy et al. 2000 ), parasitic wasps (Moreau and Asgari 2015 ), and mites (Tomalski et al. 1988 )—represent a promising frontier for novel biopesticide development. Unlike gut-active toxins such as Bt proteins, many arthropod venom components are neurotoxic or have other distinct physiological targets, potentially circumventing existing resistance mechanisms (Fletcher et al. 1997 ; Zhang et al. 2003 ; Bende et al. 2014 ; Yan et al. 2025 ). Notably, mites of the genus Pyemotes produce potent insecticidal toxins. Early studies on P. tritici identified paralytic neurotoxins (TxP-I/II) with high insect specificity and negligible mammalian toxicity (Tomalski et al. 1988 , 1989 ), a gene ( Tox-34 ) of which has been engineered into baculoviruses to enhance their insecticidal speed (Tomalski and Miller 1991 ). Pyemotes zhonghuajia Yu, Zhang & He (Prostigmata: Pyemotidae), a venomous ectoparasitic mite indigenous to China, has demonstrated remarkable efficacy as a biological control agent against Lepidopteran, Hemiptera and Coleoptera pests (Li et al. 2019b ; Liu et al. 2020 ; Tian et al. 2020 ; Chen et al. 2021 ; Song et al. 2022a ). Its venom induces rapid paralysis and mortality in hosts (Li et al. 2019a ; Song et al. 2022b ; Wang et al. 2025 ). For example, crude venom extract of P. zhonghuajia caused paralysis in fifth-instar larvae of Galleria mellonella and Helicoverpa armigera within 30 minutes after hemocoelic injection (He et al. 2006 ). Genome analysis of P. zhonghuajia further revealed expanded gene families encoding neurotoxins and dermonecrotic toxins, as well as a agatoxin gene cluster potentially involved in host paralysis (Song et al. 2022b ). Members of the phospholipase family (also referred to as dermonecrotic toxins) have been identified in Loxosceles venom (Senff-Ribeiro et al. 2008 ; Gremski et al. 2010 ; Justa et al. 2025 ), with their expressed proteins ranging from 30 to 35 kDa, responsible for dermonecrotic lesions and systemic toxicity (Kalapothakis et al. 2007 ; Binford et al. 2009 ; Gremski et al. 2020 ; Justa et al. 2025 ). The widespread presence and functional conservation of dermonecrotic toxins across venomous arthropods highlight their potential as efficient bioactive tools (Senff-Ribeiro et al. 2008 ; Vuitika et al. 2013 ; Torabi et al. 2017 ). However, despite their genomic identification in P. zhonghuajia , functional studies on dermonecrotic toxin homologs in mite venoms remain largely unexplored, leaving their insecticidal mode of action and toxicological pathways unclear. To address this knowledge gap, this study focused on three dermonecrotic toxin candidates (PzDNT2, PzDNT3, and PzDNT8) from P. zhonghuajia. We cloned and expressed these proteins, then systematically evaluated their insecticidal activity against Spodoptera frugiperda larvae. Through hemocoel injection bioassays, histopathological examination, and biochemical analyses, we investigated the acute toxicity, induced tissue damage, and host detoxification enzyme responses. Our findings provide direct evidence for the insecticidal potential of mite-derived dermonecrotic toxins and expand our understanding of the molecular arsenal of mite venoms. 2. Materials and Methods 2.1 Mites and insects rearing Pyemotes zhonghuajia were originated from the Changli Institute of Pomology, Hebei Academy of Agriculture and Forestry Sciences in October 2019. Mites were then maintained on the mature larvae of Phthorimaea operculella (Zeller) in an artificial climate incubator (25 ± 1°C, 70 ± 5% relative humidity (RH), with a 14:10 h (L:D) photoperiod). Spodoptera frugiperda individuals were collected from corn fields in Guiding County, Guizhou Province, China, on April 22, 2019. They were subsequently fed on artificial diets in an artificial climate chamber (28 ± 1°C, 70 ± 5% RH, and a 14:10 h (L:D) photoperiod). 2.2 Bacterial expression and purification of recombinant proteins Total RNA of P. zhonghuajia was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and complementary DNA (cDNA) was synthesized with the HiScript II 1st Strand cDNA Synthesis kit (Vazyme Biotech Co., Ltd, Nanjing, China) in accordance with the provided instructions. Based on the genome data (Song et al. 2022b), specific primers (Table S1) containing restriction enzyme sites were designed and then used to amplify the toxin gene (such as PzDNT2 , PzDNT3 and PzDNT8 ) by PCR. The PCR products of the genes were purified by SanPrep Column DNA Gel Extraction Kit (Sangon Biotech Co., Ltd, Shanghai, China), by restriction digestion with the restriction endonuclease Eco RI and Xho I (Thermo Fisher Scientific), then ligated into an Eco RI/ Xho I-digested vector pET-28a (Sangon Biotech Co., Ltd). After transforming the construct into Escherichia coli DH5α competent cells (Sangon Biotech Co., Ltd), positive clones identified via PCR were sequenced (Sangon Biotech Co., Ltd). Sequence analysis was performed with DNAMAN software. Following confirmation, the recombinant plasmids were introduced into the E. coli BL21 (DE3) cells (Sangon Biotech Co., Ltd). Subsequently, the transformation mixture was plated on Lauria Bertani (LB) agar plates containing 50 µg/mL kanamycin and incubated overnight at 37°C. A single colony was selected to inoculate 10 mL of LB medium, followed by incubation in a shaker (37°C, 220 rpm, 10-14 h). This starter culture was then used to inoculate 300 mL of fresh LB medium, and the cells were grown until the OD 600 reached 0.6-0.8 (about 8 h). Recombinant protein production was initiated by adding IPTG to a final concentration of 0.5 mM. Cells were harvested after culturing at 37℃ for 5 h and centrifuged 4℃, 8,000 ×g for 10 min. The cells were suspended in PBS and homogenized using an ultrasonic homogenizer (Tuohe, China). The supernatant and the pellets were collected separately after centrifugation (4℃, 12,000 ×g, 15 min). The induced proteins were separated by 4-12% SDS‑PAGE and stained with Coomassie brilliant blue for visualization. Subsequently, the recombinant proteins were affinity-purified using Ni-Charged MagBeads (Genescript, Nanjing, China) as per the instructions. The eluate was analyzed by SDS-PAGE, and the supernatant with single band was selected for dialysis. The samples were renatured gradually by dialysis against buffers with decreasing concentrations of urea (6, 4, 2, and 0 M) and imidazole (400, 300, 100, and 0 mM) in 20 mM of Tris-HCl and 0.5 M of NaCl. Following concentration and purification with Amicon Ultra centrifugal filters (Merck Millipore, Darmstadt, Germany), the final protein samples were subjected to LC-MS/MS analysis, aliquoted and stored at -80°C for future use. 2.3 Bioassays of recombinant proteins against S. frugiperda larvae The recombinant proteins PzDNT2, PzDNT3 and PzDNT8 were injected into fourth-instar S. frugiperda larvae (basically the same size) in the bioassays. These three proteins were prepared at five different concentrations in PBS. For each concentration, a volume of 2 µL was injected into the abdominal segment of 20 individual S. frugiperda larvae, with the experiment replicated three times independently. An equivalent volume of PBS served as the negative control. After injection, the larvae transferred to 24-well plates containing artificial diets and maintained in an artificial climate chamber as described above. Larvae that showed no movement upon gentle prodding with a brush were recorded as dead. Mortality was recorded after injection for 2, 4, 8, 12, 24, 48, 72 h, respectively. Data were analyzed by Probit analysis (IBM SPSS Statistics 27.0) to estimate LD 50 and 95% confidence intervals. 2.4 Sample preparation and histopathological observation Given that the LD 50 dose induced excessively severe phenotypes, the sublethal LD 25 was used for all subsequent histopathological and biochemical analyses. S. frugiperda larvae were injected with toxin proteins (LD 25 ) or PBS (control), and samples were then collected at 2, 6, and 12 h post-injection for each assay. The larvae that survived the above assay were selected randomly and washed with PBS. The larvae were then fixed overnight at 4°C using 4% paraformaldehyde (Biosharp, China). After fixation, dehydration through a graded ethanol series (70%, 80%, 90%, 95%, and 100%) for 1 h each step and infiltration through different grades of xylene (Sangon Biotech Co., Ltd) (50 and 100% in ethanol) for 20 min. Subsequently, the larvae samples were embedded and sectioned at a thickness of 5 μm using Leica © RM 2235 microtome. Sections were mounted on slides and heat-dried at 55°C for 1 h. And they were deparaffinized through successive xylene solutions and rehydrated through a descending ethanol series (100%, 95%, 90%, 80%, and 70%) followed by distilled water. After hematoxylin and eosin H&E staining (Solarbio, Beijing, China), slides were dehydrated with serial ethanol (75%, 85%, 95%, and 100%). Then, transparency was ensured by xylene solutions and sealed with neutral balsam. Histopathological alterations in the S. frugiperda larval tissues were examined and imaged under a microscope (Nikon, Japan). 2.5 Enzyme activity assays Following treatment with recombinant proteins or PBS, fourth-instar S. frugiperda larvae were homogenized in pre-cooled extraction medium at a 1:10 (w/v) ratio. The homogenate was centrifuged (4°C, 8,000 × g, 10 min), and the resulting supernatant was used for enzymatic assays. The enzymatic activity of carboxylesterase (CarE), acetylcholinesterase (AChE) and glutathione S-transferase (GST) were measured using corresponding kits (Sangon Biotech Co., Ltd) as the manufacturer’s instructions. Each treatment included three biological replicates, with each replicate consisting of 0.05 g larval tissue. The activities of the three enzymes were expressed as a U/g protein. Statistical analysis was conducted using IBM SPSS Statistics 27.0. Differences were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons, with a p -value < 0.05 considered statistically significant. 3. Results 3.1 Expression and identification of recombinant proteins In the chromosome-level genome assembly of P. zhonghuajia , the dermonecrotic toxin gene family was identified as one of the significantly expanded families, comprising a total of nine member genes. Based on their genomic proximity and high sequence similarity, we selected PzDNT2 , PzDNT3 , and PzDNT8 as representative candidates for further functional characterization (Song et al. 2022b). Agarose gel electrophoresis and sequencing analysis revealed that the lengths of the 3 dermonecrotic toxin genes were 975, 951 and 846 bp respectively (Fig. S1). The specific information regarding the encoded amino acids, predicted theoretical isoelectric points and molecular weight, etc., can be referred to by Song et al. (Song et al. 2022b). NCBI BLAST searches showed structural similarities between PzDNTs and the LiRecDT family proteins from Loxosceles intermedia spider venoms (Fig. 1), with conserved catalytic residues and cysteine positions evident in the alignment (Fig. 1a). MEME analysis identified a total of 10 conserved motifs among PzDNTs and LiRecDT family members. Among these, motifs 1 to 6 were found to be shared by both PzDNTs and LiRecDT family members (Fig. 1b-c). The mature peptide sequences of the three dermonecrotic toxins were cloned into the pET-28a vector and expressed in E. coli BL21 as inclusion bodies (Fig. S2). Protein purification was performed by Ni-NTA affinity chromatography under denaturing conditions, followed by gradual dialysis to renature the proteins. LC-MS/MS analysis of the band (Fig. 2) corresponding to these three recombinant proteins yielded high Mascot scores and sequence coverage rates, the measured molecular masses matched the theoretical values (Table 1). Additionally, unique peptides spanning N-, middle- and C-terminal regions were respectively identified. Representative MS/MS spectra exhibited complete b/y ion series with high signal-to-noise ratios (Fig. S3). These results confirm that the purified protein band is indeed PzDNT2, PzDNT3 and PzDNT8. Table 1 LC-MS/MS identification of three recombinant proteins Recombinant proteins Score Mass (kDa) Matches Sequences emPAI PzDNT2 9052 35.26 276（252） 40（39） 14846.22 PzDNT3 8762 34.37 257（237） 43（41） 8255.73 PzDNT8 10768 32.97 333（301） 45（42） 8258.37 3.2 Insecticidal activity of dermonecrotic toxins Bioassays revealed that all three recombinant toxins exhibited potent insecticidal activity against S. frugiperda larvae in a time- and dose-dependent manner. Among them, PzDNT8 was the most efficacious, with an LD 50 of 0.354 µg/larva, approximately three times more potent than PzDNT2 (1.131 μg/larva) and PzDNT3 (1.169 μg/larva) (Table 2). Cumulative mortality curves (Fig. 3) and median lethal time (LT 50 ) data (Table 3) further confirmed this superiority. PzDNT8 achieved a significantly faster knock-down effect (LT 50 = 2.956 h) compared to PzDNT2 (20.162 h) and PzDNT3 (20.735 h) at equivalent dosages (Table 3). Additionally, all toxin-treated groups had significantly higher mortality rates than the control (Fig. 3). Accompanying these lethal effects, the toxin treatments induced the appearance of distinctive black pathological lesions on the larval cuticle (Fig. 4). Consistent with the mortality trends, these phenotypic changes were highly dosage-dependent. Specifically, high-dose (LD 50 ) proteins not only accelerated larval mortality but also elicited more pronounced black lesions compared to the LD 25 dosage (Fig. 4b-c). Table 2 Insecticidal activity of three dermonecrotic toxin proteins against fourth-instar S. frugiperda larvae Recombinant proteins LD 50 μg/larva (95% confidence interval) Regression equation R 2 χ 2 PzDNT2 1.131（0.972~1.341） y=2.47x-0.13 0.993 0.971 PzDNT3 1.169（1.002~1.392） y=2.42x-0.16 0.994 0.866 PzDNT8 0.354（0.200~0.533） y=4.08x+1.98 0.911 14.638 Table 3 Median lethal time (LT 50 ) of three dermonecrotic toxin proteins against fourth-instar S. frugiperda larvae Recombinant proteins LT 50 /h (95% confidence interval) Regression equation R 2 χ 2 PzDNT2 20.162 (15.548~28.134) y=1.27x-1.66 0.922 5.424 PzDNT3 20.735 (13.369~41.725) y=1.50x-1.99 0.903 8.240 PzDNT8 2.956 (1.810~4.107) y=1.18x-0.55 0.974 1.645 3.3 Effect of dermonecrotic toxins on the internal tissue structure of S. frugiperda larvae To assess the impact of dermonecrotic toxin proteins (sub-lethal dose, LD 25 ) on S. frugiperda larvae, histopathological changes in the inner tissues were examined via H&E staining. Longitudinal sections revealed that the larvae of the control group had a complete body wall structure, including cuticle and epidermis, the fat body beneath the body wall displayed a banded distribution, the cells tightly arranged and the cell structure was intact (Fig. 5a). The larval inner tissues remained unchanged following the 2 h of protein treatments, but the localized blackening of the PzDNT3 and PzDNT8 cuticle. After the larvae were treated with toxin proteins for 6 h, the internal morphological structure of the larvae was significantly abnormal, with disorganization of cells arrangement, gradual separation of fat body, and loosening of muscle tissue. Meanwhile, the blackened areas of PzDNT3 and PzDNT8 larvae expanded. By 12 h of treatment, the histopathological changes were more drastic, with scattering of cellular debris and enlargement of the lumen of the malpighian tubule (Fig. 5b-d). 3.4 Effect of dermonecrotic toxins on the midgut structure of S. frugiperda larvae The midgut of control larvae exhibited tightly arranged cells, with clearly defined structures of goblet cells, columnar cells, and the epithelial layer. Moreover, the circular and longitudinal muscles were regularly arranged, continuous and tidy (Fig. 6a). The midgut tissue remained unchanged following the 2 h toxin protein treatments. After 6 h of treatments, only some of the cells were disordered. However, the part of the midgut cells separated and lysed, and intestinal walls cracked in the severely damaged part after toxin proteins treatment for 12 h (Fig. 6b-d). 3.5 Biochemical response of S. frugiperda larvae to dermonecrotic toxins-induced stress The major detoxification enzymes (CarE, GST and AChE) activities were further examined after injection of the S. frugiperda larvae to sub-lethal dose (LD 25 ) of toxin proteins for 2, 6, 12 h (Fig. 7). The CarE activity of larvae decreased gradually with increasing time, with the activities of PzDNT8 larvae decreased significantly over time. The CarE activity of PzDNT2 larvae decreased significantly after 12 h. However, there was no significant difference among the larvae of PzDNT3. Relative to control group, CarE activities exhibited significantly higher levels at 2 and 6 h in larvae injected with the three toxin proteins. While the CarE activity of PzDNT8 group larvae was significantly decreased after 12 h of injection, PzDNT3 groups were significantly increased (Fig. 7a). The GST and AChE activities of S. frugiperda larvae increased over time. Fluctuations in both enzyme activities were observed at 2 h, but they were not significantly different between the treatment and control groups (Fig. 7b-c). However, GST activity was significantly enhanced after 6 and 12 h of injection as compared to the control (Fig. 7b). Similarly, AChE activity increased significantly after 6 and 12 h of treatment with PzDNT2 and PzDNT8, and after 12 h following PzDNT3 treatment (Fig. 7c). 4. Discussion The development of bioinsecticides derived from natural organisms has become a crucial strategy in modern agriculture to address the issues of pest resistance and environmental concerns linked to synthetic chemical insecticides (Qu et al. 2022 ; Reyes-Ávila et al. 2024 ). Here, we assessed the insecticidal potential of three dermonecrotic toxin proteins (PzDNT2, PzDNT3, and PzDNT8) from the ectoparasitic mite P. zhonghuajia against fourth-instar S. frugiperda larvae. Our integrated analyses—including bioassays, histopathology and detoxification-enzyme profiling—indicate that all three proteins are potently toxic following hemocoelic delivery, that they provoke rapid and characteristic tissue lesions, and that they trigger complex changes in larval detoxification pathways, highlighting their potential as novel bioinsecticides. Advances in omics technologies are rapidly expanding the repertoire of characterized toxins from spiders and parasitic wasps, offering a rich source of candidate insecticidal agents (Quintero-Hernández et al. 2011 ; Yan et al. 2016 ; Cardoso et al. 2022 ; Megaly et al. 2022 ; Yang et al. 2024 ). In this study, we obtained three recombinant dermonecrotic toxins (PzDNTs) via heterologous expression, all of which exhibited potent insecticidal effects on fourth-instar S. frugiperda larvae. Among them, PzDNT8 demonstrated the highest toxicity with an LD 50 of 0.354 µg/larva, approximately three-fold more potent than PzDNT2 and PzDNT3.These toxins exhibited dose- and time-dependent toxicity in bioassays, accompanied by distinct black pathological changes on the larval cuticle. This insecticidal activity aligns with reports on other arthropod-derived toxins, and many spider neurotoxins have demonstrated high insecticidal activity against agricultural pests, indicating their promising potential as a resource for the development of bioinsecticides (King and Hardy 2013 ; King 2019 ; Yu et al. 2023 ; Yan et al. 2025 ). Histopathological analyses showed multi-tissue injury—including cuticular melanization, fat-body disaggregation, midgut epithelial lysis and muscle degeneration—further indicating that PzDNTs exhibit potent activity on S. frugiperda larvae. PzDNTs share similar structural characteristics with the LiRecDT family from brown spider venoms, containing conserved catalytic and cysteine residues (Vuitika et al. 2013 ). The recombinant dermonecrotic toxin (LiRecDT) from L. intermedia exhibits dermonecrotic and inflammatory effects similar to its crude venom (Chaim et al. 2006 ; da Silveira et al. 2006 ). Similarly, our findings demonstrate that PzDNTs induce severe pathological alterations extending beyond cuticular blackening to include widespread damage across internal tissues. This pattern of injury suggests that the underlying mechanism of PzDNTs likely involves a generalized disruption of cellular membrane integrity—a mode of action analogous to that of established spider dermonecrotic toxins (e.g., from Loxosceles spp.), which cause dermal necrosis in rabbit and rat through membranolytic effects (da Silveira et al. 2007 ; de Oliveira Christoff et al. 2008 ; Chaim et al. 2011 ; Justa et al. 2025 ). Beyond direct cellular injury, we further investigated the physiological response of the insect detoxification system to sublethal envenomation. The insect detoxification machinery, which includes key enzymes such as carboxylesterases (CarE), acetylcholinesterase (AChE) and glutathione S-transferases (GST), plays a central role in enabling insects to tolerate both xenobiotics and diverse environmental stressors (Wei et al. 2020 ; Fan et al. 2023 ; Li et al. 2023 ; Yuan et al. 2024 ). Here, these enzyme systems responded dynamically after sublethal envenomation: CarE activity was transiently elevated at early time points (2–6 h) in several treatment groups but declined markedly by 12 h in PzDNT8-treated larvae, whereas GST and AChE activities showed a progressive increase over the 12 h period (Fig. 7 ). These results indicate that PzDNTs trigger the complex regulation of detoxification enzymes in S. frugiperda larvae. During this process, the initial induction of the detoxification pathway (especially for the most toxic toxins) is followed by depletion or dysfunction of the enzymes. This disruption may lead to a severe imbalance in the detoxification homeostasis, ultimately resulting in the death of the S. frugiperda larvae. 5. Conclusion Our study demonstrates that recombinant dermonecrotic toxins (PzDNTs) from P. zhonghuajia are potent bioinsecticides against S. frugiperda larvae, exerting toxicity through systemic tissue disruption and disruption of detoxification homeostasis. The toxins exert primary lethality by triggering extensive injury to internal tissues, including the lysis of the midgut and fat body. Simultaneously, the transition from initial induction to subsequent exhaustion of key detoxification enzymes (CarE, GST, and AChE)—particularly evident in the PzDNT8-treated group—signals a catastrophic failure of larval homeostasis. Ultimately, the synergy between irreversible tissue degradation and the breakdown of the metabolic defense machinery leads to larval death, highlighting the potential of PzDNTs as novel agents. Declarations Author contributions LW performed methodology, validation, visualization, and wrote the original draft, as well as reviewing and editing the manuscript. JXF contributed to methodology, formal analysis, and validation. YR contributed to methodology. JFH reviewed and edited the manuscript. JFL acquired funding, supervised the study, and reviewed and edited the manuscript. JYH and MFY reviewed and edited the manuscript. All the authors have read and approved the final draft of the manuscript. Funding This research was supported by the National Natural Science Foundation of China (32260708 and 32060637), the High-level Talent Innovation and Entrepreneurship Funding Project in Guizhou Province, China [(2021)01], the Guizhou Province Science and Technology Innovation Talent Team Project [Qian Ke He Pingtai Rencai-CXTD (2021)004], the Growth Project of Youth Talent in Ordinary Universities in Guizhou Province [(2021)079], and the Natural Science Special Project in Guizhou University [Special post, (2020)-02]. Data Availability The datasets used and analyzed during the current study can be supplied by the corresponding author upon reasonable request. Conflict of interest The authors declare no conflicts of interest. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. References Baker BP, Green TA, Loker AJ (2020) Biological control and integrated pest management in organic and conventional systems. Biol Control 140:104095. https://doi.org/10.1016/j.biocontrol.2019.104095 Bende NS, Dziemborowicz S, Mobli M, Herzig V, Gilchrist J, Wagner J, et al (2014) A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nat Commun 5:4350. https://doi.org/10.1038/ncomms5350 Binford GJ, Bodner MR, Cordes MHJ, Baldwin KL, Rynerson MR, Burns SN, et al (2009) Molecular evolution, functional variation, and proposed nomenclature of the gene family that includes sphingomyelinase D in sicariid spider venoms. Mol Biol Evol 26:547–566. https://doi.org/10.1093/molbev/msn274 Cardoso FC, Pineda SS, Herzig V, Sunagar K, Shaikh NY, Jin AH, et al (2022) The deadly toxin arsenal of the tree-dwelling Australian funnel-web spiders. Int J Mol Sci 23:13077. https://doi.org/10.3390/ijms232113077 Chaim OM, da Silveira RB, Trevisan-silva D, Ferrer VP, Sade YB, Bóia-ferreira M, et al (2011) Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin : Endothelial cell membrane phospholipids as targets for toxicity. Biochim Biophys Acta 1811:84–96. https://doi.org/10.1016/j.bbalip.2010.11.005 Chaim OM, Sade YB, da Silveira RB, Toma L, Kalapothakis E, Chávez-Olórtegui C, et al (2006) Brown spider dermonecrotic toxin directly induces nephrotoxicity. Toxicol Appl Pharmacol 211:64–77. https://doi.org/10.1016/j.taap.2005.05.015 Chandler D, Bailey AS, Mark Tatchell G, Davidson G, Greaves J, Grant WP (2011) The development, regulation and use of biopesticides for integrated pest management. Philos Trans R Soc Lond B Biol Sci 366:1987–1998. https://doi.org/10.1098/rstb.2010.0390 Chen YC, Tian TA, Chen YH, Yu LC, Hu JF, Yu XF, et al (2021) The biocontrol agent pyemotes zhonghuajia has the highest lethal weight ratio compared with its prey and the most dramatic body weight change during pregnancy. Insects 12:490. https://doi.org/10.3390/insects12060490 da Silveira RB, Pigozzo RB, Chaim OM, Appel MH, Dreyfuss JL, Toma L, et al (2006) Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (Brown spider) venom gland. Biochimie 88:1241–1253. https://doi.org/10.1016/j.biochi.2006.02.008 da Silveira RB, Pigozzo RB, Chaim OM, Appel MH, Silva DT, Dreyfuss JL, et al (2007) Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from Brown spider ( Loxosceles intermedia ) venom : From cloning to functional characterization. Biochimie 89:289–300. https://doi.org/10.1016/j.biochi.2006.12.002 de Oliveira Christoff A, de Oliveira A, Chaim OM, Lugarini D, Bastos Pereira AL, Paludo KS, et al (2008) Effects of the venom and the dermonecrotic toxin LiRecDT1 of Loxosceles intermedia in the rat liver. Toxicon 52:695–704. https://doi.org/10.1016/j.toxicon.2008.08.001 Dong LN, Zhu LH, Cheng ZQ, Bai Y, Li PX, Yang HC, et al (2025) Characterization of an ecdysone oxidase from Plutella xylostella (L.) and its role in Bt Cry1Ac resistance. J Agric Food Chem 73:1193–1202. https://doi.org/10.1021/acs.jafc.4c10298 Fabrick JA, Wu YD (2023) Mechanisms and molecular genetics of insect resistance to insecticidal proteins from Bacillus thuringiensis . Adv In Insect Phys 65:123–183. https://doi.org/10.1016/bs.aiip.2023.09.005 Fan R, Fan ZF, Sun ZX, Chen YP, Gui FR (2023) Insecticide susceptibility and detoxification enzyme activity of Frankliniella occidentalis under three habitat conditions. Insects 14:643. https://doi.org/10.3390/insects14070643 Fletcher JI, Smith R, O’Donoghue SI, Nilges M, Connor M, Howden MEH, et al (1997) The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nat Struct Biol 4:559–566. https://doi.org/10.1038/nsb0797-559 Froy O, Zilberberg N, Chejanovsky N, Anglister J, Loret E, Shaanan B, et al (2000) Scorpion neurotoxins: Structure/function relationships and application in agriculture. Pest Manag Sci 56:472–474. https://doi.org/10.1002/(SICI)1526-4998(200005)56:53.3.CO;2-6 Gremski LH, da Justa HC, da Silva TP, Polli NLC, Antunes BC, Minozzo JC, et al (2020) Forty years of the description of Brown Spider venom Phospholipases-D. Toxins 12:164. https://doi.org/10.3390/toxins12030164 Gremski LH, da Silveira RB, Chaim OM, Probst CM, Ferrer VP, Nowatzki J, et al (2010) A novel expression profile of the Loxosceles intermedia spider venomous gland revealed by transcriptome analysis. Mol Biosyst 6:2403–2416. https://doi.org/10.1039/c004118a Haddi K, Turchen LM, Viteri Jumbo LO, Guedes RNC, Pereira EJG, Aguiar RWS, et al (2020) Rethinking biorational insecticides for pest management: unintended effects and consequences. Pest Manag Sci 76:2286–2293. https://doi.org/10.1002/ps.5837 He LM, Jiao R, Han JC, Yu LC, Hao BF, Xu CX (2006) Preliminary detection of toxicity of the crude toxin extract of Pyemotes spp. to insects and the screening of bioassay target insects. In: 2006 Annual Meeting of Chinese Society of Plant Protection, pp 419–422 Jurat-Fuentes JL, Heckel DG, Ferré J (2021) Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis . Annu Rev Entomol 66:121–140. https://doi.org/10.1146/annurev-ento-052620-073348 Justa HC De, Baldissera AB, Machado MI, Souza SH, Polli NLC, Boia-ferreira M, et al (2025) Induction of ectosome formation by binding of phospholipases D from Loxosceles venoms to endothelial cell surface : Mechanism of interaction. Biochim Biophys Acta Mol Cell Biol Lipids 1870:159579. https://doi.org/10.1016/j.bbalip.2024.159579 Kalapothakis E, Chatzaki M, Gonçalves-Dornelas H, de Castro CS, Silvestre FG, Laborne F V, et al (2007) The Loxtox protein family in Loxosceles intermedia (Mello-Leitão) venom. Toxicon 50:938–946. https://doi.org/10.1016/j.toxicon.2007.07.001 Karim AA, Idris AB, Yilmaz S (2023) Bacillus thuringiensis pesticidal toxins: A global analysis based on a scientometric study (1980–2021). Heliyon 9:e18730. https://doi.org/10.1016/j.heliyon.2023.e18730 Kesho A (2020) Microbial bio-pesticides and their use in integrated pest management. Chem Biomol Eng 5:26. https://doi.org/10.11648/j.cbe.20200501.15 King GF (2019) Tying pest insects in knots: The deployment of spider-venom-derived knottins as bioinsecticides. Pest Manag Sci 75:2437–2445. https://doi.org/10.1002/ps.5452 King GF, Hardy MC (2013) Spider-venom peptides: Structure, pharmacology, and potential for control of insect pests. Annu Rev Entomol 58:475–496. https://doi.org/10.1146/annurev-ento-120811-153650 Kumar P, Kamle M, Borah R, Mahato DK, Sharma B (2021) Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture. Egypt J Biol Pest Control 31:95. https://doi.org/10.1186/s41938-021-00440-3 Li LT, He LM, Yu LC, He XZ, Xu CX, Jiao R, et al (2019a) Preliminary study on the potential of Pyemotes zhonghuajia (Acari: Pyemotidae) in biological control of Aphis citricola (Hemiptera: Aphididae). Syst Appl Acarol 24:1116–1120. https://doi.org/10.11158/saa.24.6.12 Li M, Wei XM, Li J, Wei SM, Zhang JL, Chen GH, et al (2023) Effect of short-term exposure to high temperatures on the reproductive behavior and physiological enzyme activities in the fruit fly Zeugodacus tau (Walker). Front Physiol 14:1036397. https://doi.org/10.3389/fphys.2023.1036397 Li YC, Huang CY, Xia ZR, Zhou JS, Li JS, Sun YZ, et al (2019b) Preliminary study on biocontrol potential of Pyemotes zhonghuajia on Monochamus alternatus Hope. Genomics Appl Biol 38:2516–2521. https://doi.org/10.13417/j.gab.038.002516 Liu JF, Tian TA, Li XL, Chen YC, Yu XF, Tan XF, et al (2020) Is Pyemotes zhonghuajia (Acari: Pyemotidae) a suitable biological control agent against the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)? Syst Appl Acarol 25:649–657. https://doi.org/10.11158/saa.25.4.5 Megaly AMA, Miyashita M, Abdel-Wahab M, Nakagawa Y, Miyagawa H (2022) Molecular diversity of linear peptides revealed by transcriptomic analysis of the venom gland of the spider Lycosa poonaensis . Toxins 14:854. https://doi.org/10.3390/toxins14120854 Moreau SJM, Asgari S (2015) Venom proteins from parasitoid wasps and their biological functions. Toxins 7:2385–2412. https://doi.org/10.3390/toxins7072385 Qu MB, Merzendorfer H, Moussian B, Yang Q (2022) Bioinsecticides as future mainstream pest control agents: Opportunities and challenges. Front Agric Sci Eng 9:82–97. https://doi.org/10.15302/J-FASE-2021404 Quintero-Hernández V, Ortiz E, Rendón-Anaya M, Schwartz EF, Becerril B, Corzo G, et al (2011) Scorpion and spider venom peptides: Gene cloning and peptide expression. Toxicon 58:644–663. https://doi.org/10.1016/j.toxicon.2011.09.015 Reyes-Ávila A, López-Ruiz R, Egea González FJ, Romero-González R, Garrido Frenich A (2024) Chemistry and development of bioinsecticides for safe and sustainable use. Curr Opin Environ Sci Heal 41:100568. https://doi.org/10.1016/j.coesh.2024.100568 Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806. https://doi.org/10.1128/mmbr.62.3.775-806.1998 Senff-Ribeiro A, Henrique da Silva P, Chaim OM, Gremski LH, Paludo KS, Bertoni da Silveira R, et al (2008) Biotechnological applications of brown spider ( Loxosceles genus ) venom toxins. Biotechnol Adv 26:210–218. https://doi.org/10.1016/j.biotechadv.2007.12.003 Song YF, Du HL, Chen YC, Ye S, Yang MF, Liu JF (2022a) Effect of different densities on the parasitic ability of Pyemotes zhonghuajia on Myzus persicae . J Mt Agric Biol 41:15–20. https://doi.org/10.15958/j.cnki.sdnyswxb.2022.05.003 Song YF, Yu LC, Yang MF, Ye S, Yan B, Li LT, et al (2022b) A long-read genome assembly of a native mite in China Pyemotes zhonghuajia Yu, Zhang & He (Prostigmata: Pyemotidae) reveals gene expansion in toxin-related gene families. Toxins 14:571. https://doi.org/10.3390/toxins14080571 Tian TA, Yu LC, Sun GJ, Yu XF, Li LT, Wu CX, et al (2020) Biological control efficiency of an ectoparasitic mite Pyemotes zhonghuajia on oriental armyworm Mythimna separata . Syst Appl Acarol 25:1683–1692. https://doi.org/10.11158/saa.25.9.13 Tomalski MD, Bruce WA, Travis J, Blum MS (1988) Preliminary characterization of toxins from the straw itch mite, Pyemotes tritici , which induce paralysis in the larvae of a moth. Toxicon 26:127–132. https://doi.org/10.1016/0041-0101(88)90164-X Tomalski MD, Kutney R, Bruce WA, Brown MR, Blum MS, Travis J (1989) Purification and characterization of insect toxins derived from the mite, Pyemotes tritici . Toxicon 27:1151–1167. https://doi.org/10.1016/0041-0101(89)90009-3 Tomalski MD, Miller LK (1991) Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene. Nature 352:82–85. https://doi.org/10.1038/352082a0 Torabi E, Behdani M, Chafi MH, Moazzami R, Sabatier JM, Khalaj V, et al (2017) Characteristics and lethality of a novel recombinant dermonecrotic venom phospholipase D from Hemiscorpius lepturus . Toxins 9:102. https://doi.org/10.3390/toxins9030102 Vuitika L, Gremski LH, Belisário-Ferrari MR, Chaves-Moreira D, Ferrer VP, Senff-Ribeiro A, et al (2013) Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. J Cell Biochem 114:2479–2492. https://doi.org/10.1002/jcb.24594 Wang L, Li XL, Fan D, Chen YC, Song YF, Hu JF, et al (2025) Multi-omics analyses provide molecular insights into host immune responses and metabolic disruption in Spodoptera frugiperda parasitized by Pyemotes zhonghuajia . Pest Manag Sci 81:6303–6315. https://doi.org/10.1002/ps.8969 Wei ZH, Wang XQ, Li PR, Tan XL, Yang XQ (2020) Diet-mediated effects of cadmium on fitness-related traits and detoxification and antioxidative enzymes in the oriental armyworm, Mythimna separata . Entomol Gen 40:407–419. https://doi.org/10.1127/entomologia/2020/0869 Yan YY, Wang K, Wang JT, Han QQ, Zhang Z, Yu N, et al (2025) Peptide neurotoxins affecting insect voltage-gated calcium channels and possessing insecticidal toxicity: Two ω-Atypitoxins from Calommata signata . Pestic Biochem Physiol 208:106279. https://doi.org/10.1016/j.pestbp.2024.106279 Yan ZC, Fang Q, Wang L, Liu JD, Zhu Y, Wang F, et al (2016) Insights into the venom composition and evolution of an endoparasitoid wasp by combining proteomic and transcriptomic analyses. Sci Rep 6:19604. https://doi.org/10.1038/srep19604 Yang HY, Lu JY, Wang K, Wu CY, Yang B, Zhu JY (2024) Transcriptome analysis reveals the venom genes of the ectoparasitoid Habrobracon hebetor (Hymenoptera: Braconidae). Insects 15:426. https://doi.org/10.3390/insects15060426 Yu N, Yan YY, Han QQ, Zhang LC, Liu ZW (2023) Insecticidal toxicity of ω-Atypitoxin-Cs1a and its inhibitory effects on insect voltage-gated calcium channels. Pest Manag Sci 79:4879–4885. https://doi.org/10.1002/ps.7689 Yuan LS, Li T, Huang Y, Zhang AY, Yan SC, Jiang D (2024) Identification and potential application of key insecticidal metabolites in Tilia amurensis , a low-preference host of Hyphantria cunea . Pestic Biochem Physiol 199:105796. https://doi.org/10.1016/j.pestbp.2024.105796 Zhang PF, Chen P, Hu WJ, Liang SP (2003) Huwentoxin-V, a novel insecticidal peptide toxin from the spider Selenocosmia huwena , and a natural mutant of the toxin: indicates the key amino acid residues related to the biological activity. Toxicon 42:15–20. https://doi.org/10.1016/S0041-0101(03)00095-3 Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 06 May, 2026 Reviewers agreed at journal 28 Apr, 2026 Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 13 Apr, 2026 Submission checks completed at journal 13 Apr, 2026 First submitted to journal 11 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9386274","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633954312,"identity":"ac593536-c972-412c-9547-350ab2bfbd76","order_by":0,"name":"Liang Wang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Wang","suffix":""},{"id":633954314,"identity":"b722cf91-aa5f-4ad4-8a9f-06344ea58cfd","order_by":1,"name":"Junxian Fu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Junxian","middleName":"","lastName":"Fu","suffix":""},{"id":633954316,"identity":"435e96f5-c5a7-445a-b261-892e2f18f9f7","order_by":2,"name":"Yong Ruan","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Ruan","suffix":""},{"id":633954318,"identity":"af1d5f7a-ba73-4907-83c4-1333f27649f4","order_by":3,"name":"Jifeng Hu","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Jifeng","middleName":"","lastName":"Hu","suffix":""},{"id":633954320,"identity":"f0cfedd8-012e-4e34-aa49-384bf9638182","order_by":4,"name":"Jianfeng Liu","email":"data:image/png;base64,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","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Jianfeng","middleName":"","lastName":"Liu","suffix":""},{"id":633954322,"identity":"31afb1ea-1883-4577-a73a-c2b387f290b5","order_by":5,"name":"Jiuya He","email":"","orcid":"","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Jiuya","middleName":"","lastName":"He","suffix":""},{"id":633954323,"identity":"f065a4c9-7b76-4cb8-80c7-46a483e9620b","order_by":6,"name":"Maofa Yang","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Maofa","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-04-11 08:55:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9386274/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9386274/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108805304,"identity":"e070226b-912f-44e7-bf3f-62d249ff4d11","added_by":"auto","created_at":"2026-05-08 15:25:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1075766,"visible":true,"origin":"","legend":"\u003cp\u003eComparative sequence alignment of PzDNTs with LiRecDT protein family members. (a) Multiple alignment analysis of the PzDNTs protein sequences with \u0026nbsp;phospholipase‐D toxin family members from brown spider venoms. LiRecDT1-7 sequences were from \u003cem\u003eL. intermedia\u003c/em\u003e (GenBank accession number ABA62021, ABB69098, ABB71184, ABD91846, ABD91847, ABO87656 and AGN52903, respectively). The sequences were aligned using the MEGA 7.0 software and ESPript 3.2 program (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). \u0026nbsp;The red shaded regions show amino acid identity, and the red character show conservative substitutions. The arrows point to amino acid residues involved in catalysis, and the asterisks show cysteine residues. (b) Motif distribution of PzDNTs and LiRecDT protein family members. (c) Motif logs of PzDNTs and LiRecDT protein family members. Motifs were detected using the MEME suite (https://meme-suite.org/meme/), and then visualized using the TBtools-II software\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/668f7e48503d47bfb5abcd62.png"},{"id":108586642,"identity":"fb53594d-8f2e-4f9a-bfd9-1348ac834843","added_by":"auto","created_at":"2026-05-06 09:03:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156323,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of the recombinant proteins. (a) PzDNT2; (b) PzDNT3; (c) PzDNT8; M, Molecular weight marker; Lane 1 and 2, protein profiles of total bacterial BL21 (DE3) lysate expressing the recombinant proteins without IPTG and with IPTG induction, respectively; Lane 3, purified target protein profiles after renaturing\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/f203b30cd3f2b62123a96491.png"},{"id":108805981,"identity":"e20c444d-94e7-42cb-84ce-a7bcd5df446b","added_by":"auto","created_at":"2026-05-08 15:27:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13598,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative mortality curves of fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae after treatment three dermonecrotic toxin proteins and PBS. This bioassay included 60 larvae\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/cbefd3f4d0ae126b0ce77193.png"},{"id":108805498,"identity":"7f0a84cf-c4ef-490c-9cfd-66b2fab6a000","added_by":"auto","created_at":"2026-05-08 15:26:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":637843,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype changes in \u003cem\u003eS. frugiperda \u003c/em\u003elarvae after treatment with two doses (LD\u003csub\u003e25\u003c/sub\u003e, LD\u003csub\u003e50\u003c/sub\u003e) of dermonecrotic toxin protein. (a) PBS; (b) LD\u003csub\u003e25\u003c/sub\u003e; (c) LD\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/691ce3ff5e06e99993e81160.png"},{"id":108586646,"identity":"3a3d3d9e-1f26-4289-89cb-103ecd583f3c","added_by":"auto","created_at":"2026-05-06 09:03:44","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":455930,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological observation of on the internal tissue structure in \u003cem\u003eS. frugiperda \u003c/em\u003elarvae after different durations with toxin protein treatments. (a) PBS; (b) PzDNT2; (c) PzDNT3; (d) PzDNT8. Cu, Cuticle; Ep, Epidermis; FB, Fat body; MT, Malpighian tubules; Mu, Muscle\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/8aa43260a35ff08a4487a66d.jpeg"},{"id":108805291,"identity":"42091ec3-f3f8-4f01-bc31-a6599cd3cb8a","added_by":"auto","created_at":"2026-05-08 15:25:30","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":410042,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological observation of the midgut in \u003cem\u003eS. frugiperda \u003c/em\u003elarvae after different durations with toxin protein treatments. (a) PBS; (b) PzDNT2; (c) PzDNT3; (d) PzDNT8. BB, Brush border; CC, Columnar cells; CM, Circular muscle; Cu, Cuticle; EL, Epithelial layer; Ep, Epidermis; FB, Fat body; GC, Goblet cells; LM, Longitudinal muscle; Lu, Lumen; MT, Malpighian tubules; Mu, Muscle; PM, Peritrophic membrane\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/812135b54956a1dd9d6c45a3.jpeg"},{"id":108586648,"identity":"b6200ec4-19a3-4c9d-a69e-9299dcb4d583","added_by":"auto","created_at":"2026-05-06 09:03:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":18409,"visible":true,"origin":"","legend":"\u003cp\u003eEnzymes activities of CarE, GST and AChE of the control (PBS) and treated \u003cem\u003eS. frugiperda \u003c/em\u003elarvae. (a) CarE enzyme activity; (b) GST enzyme activity; (c) AChE enzyme activity. Different capital letters above the bars indicate significant difference in the same treatment at different times, and different lowercase letters above the bars indicate significant difference in different treatments at the same time (Tukey’s test, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/de4eed2dd1f39ffd1f90bbd0.png"},{"id":108809989,"identity":"479774c0-6d43-46a5-b18d-c97a4c5ad9d9","added_by":"auto","created_at":"2026-05-08 15:56:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3160326,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/3e9cd000-8116-4a0d-a64a-b517613f5e58.pdf"},{"id":108586641,"identity":"aacf9686-df55-4623-a65f-31301c8dc79f","added_by":"auto","created_at":"2026-05-06 09:03:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":482716,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9386274/v1/91a715d70d7025a46132d420.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dermonecrotic toxins from Pyemotes zhonghuajia induce tissue necrosis and show potent insecticidal activity against Spodoptera frugiperda","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe escalating challenges of pest resistance and environmental concerns linked to synthetic chemical insecticides have driven the search for sustainable alternatives in modern agriculture (Kesho \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Reyes-\u0026Aacute;vila et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biological control, utilizing agents from natural organisms such as fungi, bacteria, viruses, and arthropods, has emerged an essential component of integrated pest management (IPM) strategies (Chandler et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Baker et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These bioinsecticides are valued for their high specificity, low environmental persistence, and reduced risk to non-target organisms (Haddi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among the most successful examples are \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) insecticidal Cry proteins, widely deployed in transgenic crops and microbial formulations to control major pest orders (Schnepf et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Karim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the extensive application of Bt toxins has inevitably accelerated the evolution of resistance among pest populations, undermining long-term effectiveness and highlighting the urgent need for agents with novel modes of action (Jurat-Fuentes et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fabrick and Wu \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Dong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVenom-derived proteins from arthropods\u0026mdash;including spiders (Yan et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), scorpions (Froy et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), parasitic wasps (Moreau and Asgari \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and mites (Tomalski et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1988\u003c/span\u003e)\u0026mdash;represent a promising frontier for novel biopesticide development. Unlike gut-active toxins such as Bt proteins, many arthropod venom components are neurotoxic or have other distinct physiological targets, potentially circumventing existing resistance mechanisms (Fletcher et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Bende et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notably, mites of the genus \u003cem\u003ePyemotes\u003c/em\u003e produce potent insecticidal toxins. Early studies on \u003cem\u003eP. tritici\u003c/em\u003e identified paralytic neurotoxins (TxP-I/II) with high insect specificity and negligible mammalian toxicity (Tomalski et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1988\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), a gene (\u003cem\u003eTox-34\u003c/em\u003e) of which has been engineered into baculoviruses to enhance their insecticidal speed (Tomalski and Miller \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e Yu, Zhang \u0026amp; He (Prostigmata: Pyemotidae), a venomous ectoparasitic mite indigenous to China, has demonstrated remarkable efficacy as a biological control agent against Lepidopteran, Hemiptera and Coleoptera pests (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Its venom induces rapid paralysis and mortality in hosts (Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). For example, crude venom extract of \u003cem\u003eP. zhonghuajia\u003c/em\u003e caused paralysis in fifth-instar larvae of \u003cem\u003eGalleria mellonella\u003c/em\u003e and \u003cem\u003eHelicoverpa armigera\u003c/em\u003e within 30 minutes after hemocoelic injection (He et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Genome analysis of \u003cem\u003eP. zhonghuajia\u003c/em\u003e further revealed expanded gene families encoding neurotoxins and dermonecrotic toxins, as well as a agatoxin gene cluster potentially involved in host paralysis (Song et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Members of the phospholipase family (also referred to as dermonecrotic toxins) have been identified in \u003cem\u003eLoxosceles\u003c/em\u003e venom (Senff-Ribeiro et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gremski et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Justa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), with their expressed proteins ranging from 30 to 35 kDa, responsible for dermonecrotic lesions and systemic toxicity (Kalapothakis et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Binford et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gremski et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Justa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The widespread presence and functional conservation of dermonecrotic toxins across venomous arthropods highlight their potential as efficient bioactive tools (Senff-Ribeiro et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vuitika et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Torabi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, despite their genomic identification in \u003cem\u003eP. zhonghuajia\u003c/em\u003e, functional studies on dermonecrotic toxin homologs in mite venoms remain largely unexplored, leaving their insecticidal mode of action and toxicological pathways unclear.\u003c/p\u003e \u003cp\u003eTo address this knowledge gap, this study focused on three dermonecrotic toxin candidates (PzDNT2, PzDNT3, and PzDNT8) from \u003cem\u003eP. zhonghuajia.\u003c/em\u003e We cloned and expressed these proteins, then systematically evaluated their insecticidal activity against \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e larvae. Through hemocoel injection bioassays, histopathological examination, and biochemical analyses, we investigated the acute toxicity, induced tissue damage, and host detoxification enzyme responses. Our findings provide direct evidence for the insecticidal potential of mite-derived dermonecrotic toxins and expand our understanding of the molecular arsenal of mite venoms.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Mites and insects rearing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e were originated from the Changli Institute of Pomology, Hebei Academy of Agriculture and Forestry Sciences in October 2019. Mites were then maintained on the mature larvae of \u003cem\u003ePhthorimaea operculella\u003c/em\u003e (Zeller) in an artificial climate incubator (25 \u0026plusmn; 1\u0026deg;C, 70 \u0026plusmn; 5% relative humidity (RH), with a 14:10 h (L:D) photoperiod).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpodoptera frugiperda\u003c/em\u003e individuals were collected from corn fields in Guiding County, Guizhou Province, China, on April 22, 2019. They were subsequently fed on artificial diets in an artificial climate chamber (28 \u0026plusmn; 1\u0026deg;C, 70 \u0026plusmn; 5% RH, and a 14:10 h (L:D) photoperiod).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Bacterial expression and purification of recombinant proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA of \u003cem\u003eP. zhonghuajia\u003c/em\u003e was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and complementary DNA (cDNA) was synthesized with the HiScript II 1st Strand cDNA Synthesis kit (Vazyme Biotech Co., Ltd, Nanjing, China) in accordance with the provided instructions. Based on the genome data (Song et al. 2022b), specific primers (Table S1) containing restriction enzyme sites were designed and then used to amplify the toxin gene (such as \u003cem\u003ePzDNT2\u003c/em\u003e, \u003cem\u003ePzDNT3\u003c/em\u003e and\u003cem\u003e\u0026nbsp;PzDNT8\u003c/em\u003e) by PCR. The PCR products of the genes were purified by SanPrep Column DNA Gel Extraction Kit (Sangon Biotech Co., Ltd, Shanghai, China), by restriction digestion with the restriction endonuclease \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eXho\u003c/em\u003eI (Thermo Fisher Scientific), then ligated into an \u003cem\u003eEco\u003c/em\u003eRI/\u003cem\u003eXho\u003c/em\u003eI-digested vector pET-28a (Sangon Biotech Co., Ltd). After transforming the construct into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5\u0026alpha; competent cells (Sangon Biotech Co., Ltd), positive clones identified via PCR were sequenced (Sangon Biotech Co., Ltd). Sequence analysis was performed with DNAMAN software. Following confirmation, the recombinant plasmids were introduced into the \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) cells (Sangon Biotech Co., Ltd). Subsequently, the transformation mixture was plated on Lauria Bertani (LB) agar plates containing 50 \u0026micro;g/mL kanamycin and incubated overnight at 37\u0026deg;C. A single colony was selected to inoculate 10 mL of LB medium, followed by incubation in a shaker (37\u0026deg;C, 220 rpm, 10-14 h). \u0026nbsp;This starter culture was then used to inoculate 300 mL of fresh LB medium, and the cells were grown until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6-0.8 (about 8 h). Recombinant protein production was initiated by adding IPTG to a final concentration of 0.5 mM. Cells were harvested after culturing at 37℃ for 5 h and centrifuged 4℃, 8,000 \u0026times;g for 10 min. The cells were suspended in PBS and homogenized using an ultrasonic homogenizer (Tuohe, China). The supernatant and the pellets were collected separately after centrifugation (4℃, 12,000 \u0026times;g, 15 min). The induced proteins were separated by 4-12% SDS‑PAGE and stained with Coomassie brilliant blue for visualization. Subsequently, the recombinant proteins were affinity-purified using Ni-Charged MagBeads (Genescript, Nanjing, China) as per the instructions. The eluate was analyzed by SDS-PAGE, and the supernatant with single band was selected for dialysis. The samples were renatured gradually by dialysis against buffers with decreasing concentrations of urea (6, 4, 2, and 0 M) and imidazole (400, 300, 100, and 0 mM) in 20 mM of Tris-HCl and 0.5 M of NaCl. Following concentration and purification with Amicon Ultra centrifugal filters (Merck Millipore, Darmstadt, Germany), the final protein samples were subjected to LC-MS/MS analysis, aliquoted and stored at -80\u0026deg;C for future use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Bioassays of recombinant proteins against \u003cem\u003eS. frugiperda\u003c/em\u003e larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recombinant proteins PzDNT2, PzDNT3 and PzDNT8 were injected into fourth-instar\u003cem\u003e\u0026nbsp;S. frugiperda\u003c/em\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003elarvae (basically the same size) in the bioassays. These three proteins were prepared at five different concentrations in PBS. For each concentration, a volume of 2 \u0026micro;L was injected into the abdominal segment of 20 individual \u003cem\u003eS. frugiperda\u003c/em\u003e larvae, with the experiment replicated three times independently. An equivalent volume of PBS served as the negative control. After injection, the larvae transferred to 24-well plates containing artificial diets and maintained in an artificial climate chamber as described above. Larvae that showed no movement upon gentle prodding with a brush were recorded as dead. Mortality was recorded after injection for 2, 4, 8, 12, 24, 48, 72 h, respectively. Data were analyzed by Probit analysis (IBM SPSS Statistics 27.0) to estimate LD\u003csub\u003e50\u003c/sub\u003e and 95% confidence intervals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Sample preparation and histopathological observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that the LD\u003csub\u003e50\u003c/sub\u003e dose induced excessively severe phenotypes, the sublethal LD\u003csub\u003e25\u003c/sub\u003e was used for all subsequent histopathological and biochemical analyses. \u003cem\u003eS. frugiperda\u003c/em\u003e larvae were injected with toxin proteins (LD\u003csub\u003e25\u003c/sub\u003e) or PBS (control), and samples were then collected at 2, 6, and 12 h post-injection for each assay. The larvae that survived the above assay were selected randomly and washed with PBS. The larvae were then fixed overnight at 4\u0026deg;C using 4% paraformaldehyde (Biosharp, China). After fixation, dehydration through a graded ethanol series\u0026nbsp;(70%, 80%, 90%, 95%, and 100%) for 1 h each step and infiltration through different grades of xylene (Sangon Biotech Co., Ltd) (50 and 100% in ethanol) for 20 min. Subsequently, the larvae samples were embedded and sectioned at a thickness of 5 \u0026mu;m using Leica\u003csup\u003e\u0026copy;\u003c/sup\u003e RM 2235 microtome. Sections were mounted on slides and heat-dried at 55\u0026deg;C for 1 h. And they were deparaffinized through successive xylene solutions and rehydrated through a descending ethanol series (100%, 95%, 90%, 80%, and 70%) followed by distilled water. After hematoxylin and eosin H\u0026amp;E staining (Solarbio, Beijing, China), slides were dehydrated with serial ethanol (75%, 85%, 95%, and 100%). Then, transparency was ensured by xylene solutions and sealed with neutral balsam. Histopathological alterations in the \u003cem\u003eS. frugiperda\u003c/em\u003e larval tissues were examined and imaged under a microscope (Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Enzyme activity assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing treatment with recombinant proteins or PBS, fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae were homogenized in pre-cooled extraction medium at a 1:10 (w/v) ratio. The homogenate was centrifuged (4\u0026deg;C, 8,000 \u0026times; g, 10 min), and the resulting supernatant was used for enzymatic assays. \u0026nbsp;The enzymatic activity of carboxylesterase (CarE), acetylcholinesterase (AChE) and glutathione S-transferase (GST) were measured using corresponding kits (Sangon Biotech Co., Ltd) as the manufacturer\u0026rsquo;s instructions. Each treatment included three biological replicates, with each replicate consisting of 0.05 g larval tissue. The activities of the three enzymes were expressed as a U/g protein. Statistical analysis was conducted using IBM SPSS Statistics 27.0. Differences were evaluated by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons, with a \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 considered statistically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Expression and identification of recombinant proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the chromosome-level genome assembly of \u003cem\u003eP. zhonghuajia\u003c/em\u003e, the dermonecrotic toxin gene family was identified as one of the significantly expanded families, comprising a total of nine member genes. Based on their genomic proximity and high sequence similarity, we selected \u003cem\u003ePzDNT2\u003c/em\u003e, \u003cem\u003ePzDNT3\u003c/em\u003e, and \u003cem\u003ePzDNT8\u003c/em\u003e as representative candidates for further functional characterization (Song et al. 2022b). Agarose gel electrophoresis and sequencing analysis revealed that the lengths of the 3 dermonecrotic toxin genes were 975, 951 and 846 bp respectively (Fig. S1). The specific information regarding the encoded amino acids, predicted theoretical isoelectric points and molecular weight, etc., can be referred to by Song et al. (Song et al. 2022b). NCBI BLAST searches showed structural similarities between PzDNTs and the LiRecDT family proteins from \u003cem\u003eLoxosceles intermedia\u003c/em\u003e spider venoms (Fig. 1), with conserved catalytic residues and cysteine positions evident in the alignment (Fig. 1a). MEME analysis identified a total of 10 conserved motifs among PzDNTs and LiRecDT family members. \u0026nbsp;Among these, motifs 1 to 6 were found to be shared by both PzDNTs and LiRecDT family members (Fig. 1b-c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mature peptide sequences of the three dermonecrotic toxins were cloned into the pET-28a vector and expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 as inclusion bodies (Fig. S2). Protein purification was performed by Ni-NTA affinity chromatography under denaturing conditions, followed by gradual dialysis to renature the proteins. LC-MS/MS analysis of the band (Fig. 2) corresponding to these three recombinant proteins yielded high Mascot scores and sequence coverage rates, the measured molecular masses matched the theoretical values (Table 1). \u0026nbsp;Additionally, unique peptides spanning N-, middle- and C-terminal regions were respectively identified. Representative MS/MS spectra exhibited complete b/y ion series with high signal-to-noise ratios (Fig. S3). These results confirm that the purified protein band is indeed PzDNT2, PzDNT3 and PzDNT8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e LC-MS/MS identification of three recombinant proteins\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"586\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003eRecombinant proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003eScore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eMass (kDa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003eMatches\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003eSequences\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eemPAI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePzDNT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e9052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e35.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e276（252）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e40（39）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e14846.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePzDNT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e8762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e34.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e257（237）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e43（41）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e8255.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003ePzDNT8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 60px;\"\u003e\n \u003cp\u003e10768\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e32.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e333（301）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e45（42）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e8258.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Insecticidal activity of dermonecrotic toxins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioassays revealed that all three recombinant toxins exhibited potent insecticidal activity against \u003cem\u003eS. frugiperda\u003c/em\u003e larvae in a time- and dose-dependent manner. Among them, PzDNT8 was the most efficacious, with an LD\u003csub\u003e50\u003c/sub\u003e of 0.354 \u0026micro;g/larva, approximately three times more potent than PzDNT2 (1.131 \u0026mu;g/larva) and PzDNT3 (1.169 \u0026mu;g/larva) (Table 2). Cumulative mortality curves (Fig. 3) and median lethal time (LT\u003csub\u003e50\u003c/sub\u003e) data (Table 3) further confirmed this superiority. PzDNT8 achieved a significantly faster knock-down effect (LT\u003csub\u003e50\u003c/sub\u003e = 2.956 h) compared to PzDNT2 (20.162 h) and PzDNT3 (20.735 h) at equivalent dosages (Table 3). Additionally, all toxin-treated groups had significantly higher mortality rates than the control (Fig. 3). Accompanying these lethal effects, the toxin treatments induced the appearance of distinctive black pathological lesions on the larval cuticle (Fig. 4). Consistent with the mortality trends, these phenotypic changes were highly dosage-dependent. Specifically, high-dose (LD\u003csub\u003e50\u003c/sub\u003e) proteins not only accelerated larval mortality but also elicited more pronounced black lesions compared to the LD\u003csub\u003e25\u003c/sub\u003e dosage (Fig. 4b-c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Insecticidal activity of three dermonecrotic toxin proteins against fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eRecombinant proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eLD\u003csub\u003e50\u0026nbsp;\u003c/sub\u003e\u0026mu;g/larva (95% confidence interval)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eRegression equation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;\u003c/em\u003e\u003cem\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e1.131（0.972~1.341）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=2.47x-0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.993\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0.971\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e1.169（1.002~1.392）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=2.42x-0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.994\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e0.354（0.200~0.533）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=4.08x+1.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.911\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e14.638\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Median lethal time (LT\u003csub\u003e50\u003c/sub\u003e) of three dermonecrotic toxin proteins against fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"548\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003eRecombinant proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003eLT\u003csub\u003e50\u003c/sub\u003e/h (95% confidence interval)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003eRegression equation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026chi;\u003c/em\u003e\u003cem\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e20.162 (15.548~28.134)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=1.27x-1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e5.424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e20.735 (13.369~41.725)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=1.50x-1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.903\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e8.240\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 141px;\"\u003e\n \u003cp\u003ePzDNT8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 152px;\"\u003e\n \u003cp\u003e2.956 (1.810~4.107)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 124px;\"\u003e\n \u003cp\u003ey=1.18x-0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e1.645\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Effect of dermonecrotic toxins on the internal tissue structure of \u003cem\u003eS. frugiperda\u0026nbsp;\u003c/em\u003elarvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of dermonecrotic toxin proteins (sub-lethal dose, LD\u003csub\u003e25\u003c/sub\u003e) on \u003cem\u003eS. frugiperda\u003c/em\u003e larvae, histopathological changes in the inner tissues were examined via H\u0026amp;E staining. Longitudinal sections revealed that the larvae of the control group had a complete body wall structure, including cuticle and epidermis, the fat body beneath the body wall displayed a banded distribution, the cells tightly arranged and the cell structure was intact (Fig. 5a). \u0026nbsp;The larval inner tissues remained unchanged following the 2 h of protein treatments, but the localized blackening of the PzDNT3 and PzDNT8 cuticle. After the larvae were treated with toxin proteins for 6 h, the internal morphological structure of the larvae was significantly abnormal, with disorganization of cells arrangement, gradual separation of fat body, and loosening of muscle tissue. Meanwhile, the blackened areas of PzDNT3 and PzDNT8 larvae expanded. By 12 h of treatment, the histopathological changes were more drastic, with scattering of cellular debris and enlargement of the lumen of the malpighian tubule (Fig. 5b-d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effect of dermonecrotic toxins on the midgut structure of \u003cem\u003eS. frugiperda\u003c/em\u003e larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe midgut of control larvae exhibited tightly arranged cells, with clearly defined structures of goblet cells, columnar cells, and the epithelial layer. Moreover, the circular and longitudinal muscles were regularly arranged, continuous and tidy (Fig. 6a). The midgut tissue remained unchanged following the 2 h toxin protein treatments. After 6 h of treatments, only some of the cells were disordered. However, the part of the midgut cells separated and lysed, and intestinal walls cracked in the severely damaged part after toxin proteins treatment for 12 h (Fig. 6b-d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Biochemical response of \u003cem\u003eS. frugiperda\u003c/em\u003e larvae to dermonecrotic toxins-induced stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe major detoxification enzymes (CarE, GST and AChE) activities were further examined after injection of the \u003cem\u003eS. frugiperda\u003c/em\u003e larvae to sub-lethal dose (LD\u003csub\u003e25\u003c/sub\u003e) of toxin proteins for 2, 6, 12 h (Fig. 7). The CarE activity of larvae decreased gradually with increasing time, with the activities of PzDNT8 larvae decreased significantly over time. The CarE activity of PzDNT2 larvae decreased significantly after 12 h. However, there was no significant difference among the larvae of PzDNT3. Relative to control group, CarE activities exhibited significantly higher levels at 2 and 6 h in larvae injected with the three toxin proteins. While the CarE activity of PzDNT8 group larvae was significantly decreased after 12 h of injection, PzDNT3 groups were significantly increased (Fig. 7a). The GST and AChE activities of \u003cem\u003eS. frugiperda\u003c/em\u003e larvae increased over time. Fluctuations in both enzyme activities were observed at 2 h, but they were not significantly different between the treatment and control groups (Fig. 7b-c). However, GST activity was significantly enhanced after 6 and 12 h of injection as compared to the control (Fig. 7b). Similarly, AChE activity increased significantly after 6 and 12 h of treatment with PzDNT2 and PzDNT8, and after 12 h following PzDNT3 treatment (Fig. 7c).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe development of bioinsecticides derived from natural organisms has become a crucial strategy in modern agriculture to address the issues of pest resistance and environmental concerns linked to synthetic chemical insecticides (Qu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reyes-\u0026Aacute;vila et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Here, we assessed the insecticidal potential of three dermonecrotic toxin proteins (PzDNT2, PzDNT3, and PzDNT8) from the ectoparasitic mite \u003cem\u003eP. zhonghuajia\u003c/em\u003e against fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. Our integrated analyses\u0026mdash;including bioassays, histopathology and detoxification-enzyme profiling\u0026mdash;indicate that all three proteins are potently toxic following hemocoelic delivery, that they provoke rapid and characteristic tissue lesions, and that they trigger complex changes in larval detoxification pathways, highlighting their potential as novel bioinsecticides.\u003c/p\u003e \u003cp\u003eAdvances in omics technologies are rapidly expanding the repertoire of characterized toxins from spiders and parasitic wasps, offering a rich source of candidate insecticidal agents (Quintero-Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cardoso et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Megaly et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we obtained three recombinant dermonecrotic toxins (PzDNTs) via heterologous expression, all of which exhibited potent insecticidal effects on fourth-instar \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. Among them, PzDNT8 demonstrated the highest toxicity with an LD\u003csub\u003e50\u003c/sub\u003e of 0.354 \u0026micro;g/larva, approximately three-fold more potent than PzDNT2 and PzDNT3.These toxins exhibited dose- and time-dependent toxicity in bioassays, accompanied by distinct black pathological changes on the larval cuticle. This insecticidal activity aligns with reports on other arthropod-derived toxins, and many spider neurotoxins have demonstrated high insecticidal activity against agricultural pests, indicating their promising potential as a resource for the development of bioinsecticides (King and Hardy \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; King \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistopathological analyses showed multi-tissue injury\u0026mdash;including cuticular melanization, fat-body disaggregation, midgut epithelial lysis and muscle degeneration\u0026mdash;further indicating that PzDNTs exhibit potent activity on \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. PzDNTs share similar structural characteristics with the LiRecDT family from brown spider venoms, containing conserved catalytic and cysteine residues (Vuitika et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The recombinant dermonecrotic toxin (LiRecDT) from \u003cem\u003eL. intermedia\u003c/em\u003e exhibits dermonecrotic and inflammatory effects similar to its crude venom (Chaim et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; da Silveira et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Similarly, our findings demonstrate that PzDNTs induce severe pathological alterations extending beyond cuticular blackening to include widespread damage across internal tissues. This pattern of injury suggests that the underlying mechanism of PzDNTs likely involves a generalized disruption of cellular membrane integrity\u0026mdash;a mode of action analogous to that of established spider dermonecrotic toxins (e.g., from \u003cem\u003eLoxosceles\u003c/em\u003e spp.), which cause dermal necrosis in rabbit and rat through membranolytic effects (da Silveira et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; de Oliveira Christoff et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Chaim et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Justa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond direct cellular injury, we further investigated the physiological response of the insect detoxification system to sublethal envenomation. The insect detoxification machinery, which includes key enzymes such as carboxylesterases (CarE), acetylcholinesterase (AChE) and glutathione S-transferases (GST), plays a central role in enabling insects to tolerate both xenobiotics and diverse environmental stressors (Wei et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yuan et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Here, these enzyme systems responded dynamically after sublethal envenomation: CarE activity was transiently elevated at early time points (2\u0026ndash;6 h) in several treatment groups but declined markedly by 12 h in PzDNT8-treated larvae, whereas GST and AChE activities showed a progressive increase over the 12 h period (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicate that PzDNTs trigger the complex regulation of detoxification enzymes in \u003cem\u003eS. frugiperda\u003c/em\u003e larvae. During this process, the initial induction of the detoxification pathway (especially for the most toxic toxins) is followed by depletion or dysfunction of the enzymes. This disruption may lead to a severe imbalance in the detoxification homeostasis, ultimately resulting in the death of the \u003cem\u003eS. frugiperda\u003c/em\u003e larvae.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study demonstrates that recombinant dermonecrotic toxins (PzDNTs) from \u003cem\u003eP. zhonghuajia\u003c/em\u003e are potent bioinsecticides against \u003cem\u003eS. frugiperda\u003c/em\u003e larvae, exerting toxicity through systemic tissue disruption and disruption of detoxification homeostasis. The toxins exert primary lethality by triggering extensive injury to internal tissues, including the lysis of the midgut and fat body. Simultaneously, the transition from initial induction to subsequent exhaustion of key detoxification enzymes (CarE, GST, and AChE)\u0026mdash;particularly evident in the PzDNT8-treated group\u0026mdash;signals a catastrophic failure of larval homeostasis. Ultimately, the synergy between irreversible tissue degradation and the breakdown of the metabolic defense machinery leads to larval death, highlighting the potential of PzDNTs as novel agents.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLW performed methodology, validation, visualization, and wrote the original draft, as well as reviewing and editing the manuscript. JXF contributed to methodology, formal analysis, and validation. YR contributed to methodology. JFH reviewed and edited the manuscript. JFL acquired funding, supervised the study, and reviewed and edited the manuscript. \u0026nbsp;JYH and MFY reviewed and edited the manuscript. All the authors have read and approved the final draft of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (32260708 and 32060637), the High-level Talent Innovation and Entrepreneurship Funding Project in Guizhou Province, China [(2021)01], the Guizhou Province Science and Technology Innovation Talent Team Project [Qian Ke He Pingtai Rencai-CXTD (2021)004], the Growth Project of Youth Talent in Ordinary Universities in Guizhou Province [(2021)079], and the Natural Science Special Project in Guizhou University [Special post, (2020)-02].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study can be supplied by the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBaker BP, Green TA, Loker AJ (2020) Biological control and integrated pest management in organic and conventional systems. Biol Control 140:104095. https://doi.org/10.1016/j.biocontrol.2019.104095\u003c/li\u003e\n\u003cli\u003eBende NS, Dziemborowicz S, Mobli M, Herzig V, Gilchrist J, Wagner J, et al (2014) A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a. Nat Commun 5:4350. https://doi.org/10.1038/ncomms5350\u003c/li\u003e\n\u003cli\u003eBinford GJ, Bodner MR, Cordes MHJ, Baldwin KL, Rynerson MR, Burns SN, et al (2009) Molecular evolution, functional variation, and proposed nomenclature of the gene family that includes sphingomyelinase D in sicariid spider venoms. Mol Biol Evol 26:547\u0026ndash;566. https://doi.org/10.1093/molbev/msn274\u003c/li\u003e\n\u003cli\u003eCardoso FC, Pineda SS, Herzig V, Sunagar K, Shaikh NY, Jin AH, et al (2022) The deadly toxin arsenal of the tree-dwelling Australian funnel-web spiders. Int J Mol Sci 23:13077. https://doi.org/10.3390/ijms232113077\u003c/li\u003e\n\u003cli\u003eChaim OM, da Silveira RB, Trevisan-silva D, Ferrer VP, Sade YB, B\u0026oacute;ia-ferreira M, et al (2011) Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin : Endothelial cell membrane phospholipids as targets for toxicity. Biochim Biophys Acta 1811:84\u0026ndash;96. https://doi.org/10.1016/j.bbalip.2010.11.005\u003c/li\u003e\n\u003cli\u003eChaim OM, Sade YB, da Silveira RB, Toma L, Kalapothakis E, Ch\u0026aacute;vez-Ol\u0026oacute;rtegui C, et al (2006) Brown spider dermonecrotic toxin directly induces nephrotoxicity. Toxicol Appl Pharmacol 211:64\u0026ndash;77. https://doi.org/10.1016/j.taap.2005.05.015\u003c/li\u003e\n\u003cli\u003eChandler D, Bailey AS, Mark Tatchell G, Davidson G, Greaves J, Grant WP (2011) The development, regulation and use of biopesticides for integrated pest management. Philos Trans R Soc Lond B Biol Sci 366:1987\u0026ndash;1998. https://doi.org/10.1098/rstb.2010.0390\u003c/li\u003e\n\u003cli\u003eChen YC, Tian TA, Chen YH, Yu LC, Hu JF, Yu XF, et al (2021) The biocontrol agent \u003cem\u003epyemotes zhonghuajia \u003c/em\u003ehas the highest lethal weight ratio compared with its prey and the most dramatic body weight change during pregnancy. Insects 12:490. https://doi.org/10.3390/insects12060490\u003c/li\u003e\n\u003cli\u003eda Silveira RB, Pigozzo RB, Chaim OM, Appel MH, Dreyfuss JL, Toma L, et al (2006) Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from \u003cem\u003eLoxosceles intermedia\u003c/em\u003e (Brown spider) venom gland. Biochimie 88:1241\u0026ndash;1253. https://doi.org/10.1016/j.biochi.2006.02.008\u003c/li\u003e\n\u003cli\u003eda Silveira RB, Pigozzo RB, Chaim OM, Appel MH, Silva DT, Dreyfuss JL, et al (2007) Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from Brown spider (\u003cem\u003eLoxosceles intermedia\u003c/em\u003e) venom : From cloning to functional characterization. Biochimie 89:289\u0026ndash;300. https://doi.org/10.1016/j.biochi.2006.12.002\u003c/li\u003e\n\u003cli\u003ede Oliveira Christoff A, de Oliveira A, Chaim OM, Lugarini D, Bastos Pereira AL, Paludo KS, et al (2008) Effects of the venom and the dermonecrotic toxin LiRecDT1 of \u003cem\u003eLoxosceles intermedia\u003c/em\u003e in the rat liver. Toxicon 52:695\u0026ndash;704. https://doi.org/10.1016/j.toxicon.2008.08.001\u003c/li\u003e\n\u003cli\u003eDong LN, Zhu LH, Cheng ZQ, Bai Y, Li PX, Yang HC, et al (2025) Characterization of an ecdysone oxidase from \u003cem\u003ePlutella xylostella\u003c/em\u003e (L.) and its role in Bt Cry1Ac resistance. J Agric Food Chem 73:1193\u0026ndash;1202. https://doi.org/10.1021/acs.jafc.4c10298\u003c/li\u003e\n\u003cli\u003eFabrick JA, Wu YD (2023) Mechanisms and molecular genetics of insect resistance to insecticidal proteins from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e. Adv In Insect Phys 65:123\u0026ndash;183. https://doi.org/10.1016/bs.aiip.2023.09.005\u003c/li\u003e\n\u003cli\u003eFan R, Fan ZF, Sun ZX, Chen YP, Gui FR (2023) Insecticide susceptibility and detoxification enzyme activity of \u003cem\u003eFrankliniella occidentalis\u003c/em\u003e under three habitat conditions. Insects 14:643. https://doi.org/10.3390/insects14070643\u003c/li\u003e\n\u003cli\u003eFletcher JI, Smith R, O\u0026rsquo;Donoghue SI, Nilges M, Connor M, Howden MEH, et al (1997) The structure of a novel insecticidal neurotoxin, \u0026omega;-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nat Struct Biol 4:559\u0026ndash;566. https://doi.org/10.1038/nsb0797-559\u003c/li\u003e\n\u003cli\u003eFroy O, Zilberberg N, Chejanovsky N, Anglister J, Loret E, Shaanan B, et al (2000) Scorpion neurotoxins: Structure/function relationships and application in agriculture. Pest Manag Sci 56:472\u0026ndash;474. https://doi.org/10.1002/(SICI)1526-4998(200005)56:5\u0026lt;472::AID-PS148\u0026gt;3.3.CO;2-6\u003c/li\u003e\n\u003cli\u003eGremski LH, da Justa HC, da Silva TP, Polli NLC, Antunes BC, Minozzo JC, et al (2020) Forty years of the description of Brown Spider venom Phospholipases-D. Toxins 12:164. https://doi.org/10.3390/toxins12030164\u003c/li\u003e\n\u003cli\u003eGremski LH, da Silveira RB, Chaim OM, Probst CM, Ferrer VP, Nowatzki J, et al (2010) A novel expression profile of the \u003cem\u003eLoxosceles intermedia \u003c/em\u003espider venomous gland revealed by transcriptome analysis. Mol Biosyst 6:2403\u0026ndash;2416. https://doi.org/10.1039/c004118a\u003c/li\u003e\n\u003cli\u003eHaddi K, Turchen LM, Viteri Jumbo LO, Guedes RNC, Pereira EJG, Aguiar RWS, et al (2020) Rethinking biorational insecticides for pest management: unintended effects and consequences. Pest Manag Sci 76:2286\u0026ndash;2293. https://doi.org/10.1002/ps.5837\u003c/li\u003e\n\u003cli\u003eHe LM, Jiao R, Han JC, Yu LC, Hao BF, Xu CX (2006) Preliminary detection of toxicity of the crude toxin extract of \u003cem\u003ePyemotes\u003c/em\u003e spp. to insects and the screening of bioassay target insects. In: 2006 Annual Meeting of Chinese Society of Plant Protection, pp 419\u0026ndash;422\u003c/li\u003e\n\u003cli\u003eJurat-Fuentes JL, Heckel DG, Ferr\u0026eacute; J (2021) Mechanisms of resistance to insecticidal proteins from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e. Annu Rev Entomol 66:121\u0026ndash;140. https://doi.org/10.1146/annurev-ento-052620-073348\u003c/li\u003e\n\u003cli\u003eJusta HC De, Baldissera AB, Machado MI, Souza SH, Polli NLC, Boia-ferreira M, et al (2025) Induction of ectosome formation by binding of phospholipases D from \u003cem\u003eLoxosceles \u003c/em\u003evenoms to endothelial cell surface : Mechanism of interaction. Biochim Biophys Acta Mol Cell Biol Lipids 1870:159579. https://doi.org/10.1016/j.bbalip.2024.159579\u003c/li\u003e\n\u003cli\u003eKalapothakis E, Chatzaki M, Gon\u0026ccedil;alves-Dornelas H, de Castro CS, Silvestre FG, Laborne F V, et al (2007) The Loxtox protein family in \u003cem\u003eLoxosceles intermedia\u003c/em\u003e (Mello-Leit\u0026atilde;o) venom. Toxicon 50:938\u0026ndash;946. https://doi.org/10.1016/j.toxicon.2007.07.001\u003c/li\u003e\n\u003cli\u003eKarim AA, Idris AB, Yilmaz S (2023) \u003cem\u003eBacillus thuringiensis\u003c/em\u003e pesticidal toxins: A global analysis based on a scientometric study (1980\u0026ndash;2021). Heliyon 9:e18730. https://doi.org/10.1016/j.heliyon.2023.e18730\u003c/li\u003e\n\u003cli\u003eKesho A (2020) Microbial bio-pesticides and their use in integrated pest management. Chem Biomol Eng 5:26. https://doi.org/10.11648/j.cbe.20200501.15\u003c/li\u003e\n\u003cli\u003eKing GF (2019) Tying pest insects in knots: The deployment of spider-venom-derived knottins as bioinsecticides. Pest Manag Sci 75:2437\u0026ndash;2445. https://doi.org/10.1002/ps.5452\u003c/li\u003e\n\u003cli\u003eKing GF, Hardy MC (2013) Spider-venom peptides: Structure, pharmacology, and potential for control of insect pests. Annu Rev Entomol 58:475\u0026ndash;496. https://doi.org/10.1146/annurev-ento-120811-153650\u003c/li\u003e\n\u003cli\u003eKumar P, Kamle M, Borah R, Mahato DK, Sharma B (2021) \u003cem\u003eBacillus thuringiensis\u003c/em\u003e as microbial biopesticide: uses and application for sustainable agriculture. Egypt J Biol Pest Control 31:95. https://doi.org/10.1186/s41938-021-00440-3\u003c/li\u003e\n\u003cli\u003eLi LT, He LM, Yu LC, He XZ, Xu CX, Jiao R, et al (2019a) Preliminary study on the potential of \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e (Acari: Pyemotidae) in biological control of \u003cem\u003eAphis citricola\u003c/em\u003e (Hemiptera: Aphididae). Syst Appl Acarol 24:1116\u0026ndash;1120. https://doi.org/10.11158/saa.24.6.12\u003c/li\u003e\n\u003cli\u003eLi M, Wei XM, Li J, Wei SM, Zhang JL, Chen GH, et al (2023) Effect of short-term exposure to high temperatures on the reproductive behavior and physiological enzyme activities in the fruit fly \u003cem\u003eZeugodacus tau\u003c/em\u003e (Walker). Front Physiol 14:1036397. https://doi.org/10.3389/fphys.2023.1036397\u003c/li\u003e\n\u003cli\u003eLi YC, Huang CY, Xia ZR, Zhou JS, Li JS, Sun YZ, et al (2019b) Preliminary study on biocontrol potential of \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e on \u003cem\u003eMonochamus alternatus\u003c/em\u003e Hope. Genomics Appl Biol 38:2516\u0026ndash;2521. https://doi.org/10.13417/j.gab.038.002516\u003c/li\u003e\n\u003cli\u003eLiu JF, Tian TA, Li XL, Chen YC, Yu XF, Tan XF, et al (2020) Is \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e (Acari: Pyemotidae) a suitable biological control agent against the fall armyworm \u003cem\u003eSpodoptera frugiperda \u003c/em\u003e(Lepidoptera: Noctuidae)? Syst Appl Acarol 25:649\u0026ndash;657. https://doi.org/10.11158/saa.25.4.5\u003c/li\u003e\n\u003cli\u003eMegaly AMA, Miyashita M, Abdel-Wahab M, Nakagawa Y, Miyagawa H (2022) Molecular diversity of linear peptides revealed by transcriptomic analysis of the venom gland of the spider \u003cem\u003eLycosa poonaensis\u003c/em\u003e. Toxins 14:854. https://doi.org/10.3390/toxins14120854\u003c/li\u003e\n\u003cli\u003eMoreau SJM, Asgari S (2015) Venom proteins from parasitoid wasps and their biological functions. Toxins 7:2385\u0026ndash;2412. https://doi.org/10.3390/toxins7072385\u003c/li\u003e\n\u003cli\u003eQu MB, Merzendorfer H, Moussian B, Yang Q (2022) Bioinsecticides as future mainstream pest control agents: Opportunities and challenges. Front Agric Sci Eng 9:82\u0026ndash;97. https://doi.org/10.15302/J-FASE-2021404\u003c/li\u003e\n\u003cli\u003eQuintero-Hern\u0026aacute;ndez V, Ortiz E, Rend\u0026oacute;n-Anaya M, Schwartz EF, Becerril B, Corzo G, et al (2011) Scorpion and spider venom peptides: Gene cloning and peptide expression. Toxicon 58:644\u0026ndash;663. https://doi.org/10.1016/j.toxicon.2011.09.015\u003c/li\u003e\n\u003cli\u003eReyes-\u0026Aacute;vila A, L\u0026oacute;pez-Ruiz R, Egea Gonz\u0026aacute;lez FJ, Romero-Gonz\u0026aacute;lez R, Garrido Frenich A (2024) Chemistry and development of bioinsecticides for safe and sustainable use. Curr Opin Environ Sci Heal 41:100568. https://doi.org/10.1016/j.coesh.2024.100568\u003c/li\u003e\n\u003cli\u003eSchnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, et al (1998) \u003cem\u003eBacillus thuringiensis \u003c/em\u003eand its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775\u0026ndash;806. https://doi.org/10.1128/mmbr.62.3.775-806.1998\u003c/li\u003e\n\u003cli\u003eSenff-Ribeiro A, Henrique da Silva P, Chaim OM, Gremski LH, Paludo KS, Bertoni da Silveira R, et al (2008) Biotechnological applications of brown spider (\u003cem\u003eLoxosceles genus\u003c/em\u003e) venom toxins. Biotechnol Adv 26:210\u0026ndash;218. https://doi.org/10.1016/j.biotechadv.2007.12.003\u003c/li\u003e\n\u003cli\u003eSong YF, Du HL, Chen YC, Ye S, Yang MF, Liu JF (2022a) Effect of different densities on the parasitic ability of \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e on \u003cem\u003eMyzus persicae\u003c/em\u003e. J Mt Agric Biol 41:15\u0026ndash;20. https://doi.org/10.15958/j.cnki.sdnyswxb.2022.05.003\u003c/li\u003e\n\u003cli\u003eSong YF, Yu LC, Yang MF, Ye S, Yan B, Li LT, et al (2022b) A long-read genome assembly of a native mite in China \u003cem\u003ePyemotes zhonghuajia \u003c/em\u003eYu, Zhang \u0026amp; He (Prostigmata: Pyemotidae) reveals gene expansion in toxin-related gene families. Toxins 14:571. https://doi.org/10.3390/toxins14080571\u003c/li\u003e\n\u003cli\u003eTian TA, Yu LC, Sun GJ, Yu XF, Li LT, Wu CX, et al (2020) Biological control efficiency of an ectoparasitic mite \u003cem\u003ePyemotes zhonghuajia \u003c/em\u003eon oriental armyworm \u003cem\u003eMythimna separata\u003c/em\u003e. Syst Appl Acarol 25:1683\u0026ndash;1692. https://doi.org/10.11158/saa.25.9.13\u003c/li\u003e\n\u003cli\u003eTomalski MD, Bruce WA, Travis J, Blum MS (1988) Preliminary characterization of toxins from the straw itch mite, \u003cem\u003ePyemotes tritici\u003c/em\u003e, which induce paralysis in the larvae of a moth. Toxicon 26:127\u0026ndash;132. https://doi.org/10.1016/0041-0101(88)90164-X\u003c/li\u003e\n\u003cli\u003eTomalski MD, Kutney R, Bruce WA, Brown MR, Blum MS, Travis J (1989) Purification and characterization of insect toxins derived from the mite, \u003cem\u003ePyemotes tritici\u003c/em\u003e. Toxicon 27:1151\u0026ndash;1167. https://doi.org/10.1016/0041-0101(89)90009-3\u003c/li\u003e\n\u003cli\u003eTomalski MD, Miller LK (1991) Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene. Nature 352:82\u0026ndash;85. https://doi.org/10.1038/352082a0\u003c/li\u003e\n\u003cli\u003eTorabi E, Behdani M, Chafi MH, Moazzami R, Sabatier JM, Khalaj V, et al (2017) Characteristics and lethality of a novel recombinant dermonecrotic venom phospholipase D from \u003cem\u003eHemiscorpius lepturus\u003c/em\u003e. Toxins 9:102. https://doi.org/10.3390/toxins9030102\u003c/li\u003e\n\u003cli\u003eVuitika L, Gremski LH, Belis\u0026aacute;rio-Ferrari MR, Chaves-Moreira D, Ferrer VP, Senff-Ribeiro A, et al (2013) Brown spider phospholipase-D containing a conservative mutation (D233E) in the catalytic site: Identification and functional characterization. J Cell Biochem 114:2479\u0026ndash;2492. https://doi.org/10.1002/jcb.24594\u003c/li\u003e\n\u003cli\u003eWang L, Li XL, Fan D, Chen YC, Song YF, Hu JF, et al (2025) Multi-omics analyses provide molecular insights into host immune responses and metabolic disruption in \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e parasitized by \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e. Pest Manag Sci 81:6303\u0026ndash;6315. https://doi.org/10.1002/ps.8969\u003c/li\u003e\n\u003cli\u003eWei ZH, Wang XQ, Li PR, Tan XL, Yang XQ (2020) Diet-mediated effects of cadmium on fitness-related traits and detoxification and antioxidative enzymes in the oriental armyworm, \u003cem\u003eMythimna separata\u003c/em\u003e. Entomol Gen 40:407\u0026ndash;419. https://doi.org/10.1127/entomologia/2020/0869\u003c/li\u003e\n\u003cli\u003eYan YY, Wang K, Wang JT, Han QQ, Zhang Z, Yu N, et al (2025) Peptide neurotoxins affecting insect voltage-gated calcium channels and possessing insecticidal toxicity: Two \u0026omega;-Atypitoxins from \u003cem\u003eCalommata signata\u003c/em\u003e. Pestic Biochem Physiol 208:106279. https://doi.org/10.1016/j.pestbp.2024.106279\u003c/li\u003e\n\u003cli\u003eYan ZC, Fang Q, Wang L, Liu JD, Zhu Y, Wang F, et al (2016) Insights into the venom composition and evolution of an endoparasitoid wasp by combining proteomic and transcriptomic analyses. Sci Rep 6:19604. https://doi.org/10.1038/srep19604\u003c/li\u003e\n\u003cli\u003eYang HY, Lu JY, Wang K, Wu CY, Yang B, Zhu JY (2024) Transcriptome analysis reveals the venom genes of the ectoparasitoid \u003cem\u003eHabrobracon hebetor\u003c/em\u003e (Hymenoptera: Braconidae). Insects 15:426. https://doi.org/10.3390/insects15060426\u003c/li\u003e\n\u003cli\u003eYu N, Yan YY, Han QQ, Zhang LC, Liu ZW (2023) Insecticidal toxicity of \u0026omega;-Atypitoxin-Cs1a and its inhibitory effects on insect voltage-gated calcium channels. Pest Manag Sci 79:4879\u0026ndash;4885. https://doi.org/10.1002/ps.7689\u003c/li\u003e\n\u003cli\u003eYuan LS, Li T, Huang Y, Zhang AY, Yan SC, Jiang D (2024) Identification and potential application of key insecticidal metabolites in \u003cem\u003eTilia amurensis\u003c/em\u003e, a low-preference host of \u003cem\u003eHyphantria cunea\u003c/em\u003e. Pestic Biochem Physiol 199:105796. https://doi.org/10.1016/j.pestbp.2024.105796\u003c/li\u003e\n\u003cli\u003eZhang PF, Chen P, Hu WJ, Liang SP (2003) Huwentoxin-V, a novel insecticidal peptide toxin from the spider \u003cem\u003eSelenocosmia huwena\u003c/em\u003e, and a natural mutant of the toxin: indicates the key amino acid residues related to the biological activity. Toxicon 42:15\u0026ndash;20. https://doi.org/10.1016/S0041-0101(03)00095-3\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyemotes zhonghuajia, Dermonecrotic toxin, Spodoptera frugiperda, Tissue necrosis, Bioinsecticide","lastPublishedDoi":"10.21203/rs.3.rs-9386274/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9386274/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResistance to conventional insecticides motivates the search for novel bioactive agents. Dermonecrotic toxins from arthropod venoms but underexplored candidates for crop protection. Here we report the cloning, bacterial expression and identification of three recombinant dermonecrotic toxin proteins (PzDNT2, PzDNT3 and PzDNT8) from the ectoparasitic mite \u003cem\u003ePyemotes zhonghuajia\u003c/em\u003e, and we evaluate their insecticidal activity against the invasive lepidopteran pest \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e. Hemocoelic bioassays show that all three proteins are potently toxic when injected into fourth-instar larvae, with PzDNT8 displaying the greatest potency (median lethal dose, LD\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.354 \u0026micro;g/larva; PzDNT2 and PzDNT3: LD\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.131 and 1.169 \u0026micro;g/larva, respectively). Histopathology reveals that toxin treatment induces rapid and progressive tissue necrosis\u0026mdash;characterized by cuticular blackening, fat-body disintegration, midgut epithelial lysis, and muscle degeneration. Furthermore, biochemical assays show time-dependent perturbation of detoxification enzymes (carboxylesterase (CarE), acetylcholinesterase (AChE), and glutathione S-transferase (GST)), indicating a complex metabolic response. Together, these findings indicate that \u003cem\u003eP. zhonghuajia\u003c/em\u003e dermonecrotic toxins cause extensive internal tissue damage and overwhelm larval physiological defenses, identifying them as promising leads for next-generation bioinsecticides.\u003c/p\u003e","manuscriptTitle":"Dermonecrotic toxins from Pyemotes zhonghuajia induce tissue necrosis and show potent insecticidal activity against Spodoptera frugiperda","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 09:03:40","doi":"10.21203/rs.3.rs-9386274/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-06T10:31:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176164408862041211896905805035390414799","date":"2026-04-28T08:56:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-27T09:22:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T12:30:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-13T12:30:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Pest Science","date":"2026-04-11T08:46:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-pest-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pest","sideBox":"Learn more about [Journal of Pest Science](https://www.springer.com/journal/10340)","snPcode":"10340","submissionUrl":"https://submission.nature.com/new-submission/10340/3","title":"Journal of Pest Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1fdd7a6d-86a2-4254-8212-8e4e86681393","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-06T10:31:24+00:00","index":17,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T09:03:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 09:03:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9386274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9386274","identity":"rs-9386274","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}