Dinotefuran, a third-generation neonicotinoid insecticide, is widely used in agriculture production due to its excellent insecticidal efficacy. Considering its persistence and high toxicity in soil, it is essential to evaluate its low-dose toxic effects on non-target soil organisms such as the springtail (Folsomia candida). The results revealed that the 7-day half lethal concentration (7d-LC) of dinotefuran contact toxicity to springtails was 0.029 μg cm. Its chronic toxicity in 4 soil types was ranked as: red soil (0.021 mg kg) > fluvo-aquic soil (0.040 mg kg) > artificial soil (0.049 mg kg) > black soil (0.085 mg kg). Soil organic matter (SOC), pH, and total nitrogen (TN) were identified as critical factors affecting dinotefuran toxicity. Biochemical assay results showed that environmental concentrations (0.2-1.6 mg kg) of dinotefuran induced oxidative stress and oxidative damage in springtails. Oxidative stress-related enzymes (including superoxide dismutase (SOD) and catalase (CAT)) and detoxification enzymes were subjected to initial activation at low dinotefuran concentrations, inhibition and re-activation at high concentration. Target enzyme acetylcholinesterase (AChE), malondialdehyde (MDA) content, and total protein content were inhibited with prolonged exposure time and increasing concentrations of dinotefuran. Molecular docking analysis showed that dinotefuran bound to the active sites of related enzymes, thus disrupting their structure and functions, eventually resulting in damages to physiological functions of springtails. In summary, this study deciphers the dinotefuran toxicological mechanism on soil springtails at environmental concentrations. Our findings lay theoretical basis for further assessing its pollution risk and managing its application.

Glyphosate (N-[phosphonomethyl] glycine, GLY) is the active ingredient of the most widely used commercialized herbicide, and its use increases the potential for co-occurrence with flame retardants such as Tetrabromobisphenol A (TBBPA), posing a threat to aquatic systems and food safety. Therefore, it is important to prioritize evaluating these two compounds' combined toxicity. However, only a few studies have analyzed the effects of pollutant mixing on fish from the perspectives of molecular and nutritional components. In this study, the impact of TBBPA and GLY on muscle development and flesh quality was investigated by exposing common carp to water-borne TBBPA and/or GLY for 30 days. The results showed that TBBPA and GLY exposure decreased the anti-oxidant capacity and content of most free amino acids in common carp muscle. Textural analysis suggested that the meat flesh's hardness, cohesiveness, and chewiness were decreased under TBBPA and GLY exposure. In addition, the decreased cross-sectional area of muscle fibers and collagen deposition were observed in the carp muscle exposed to TBBPA and/or GLY. Further analysis of related genes indicated the co-exposure of TBBPA and GLY significantly upregulated the levels of FoxO1 and MuRF-1, and decreased the levels of MyoD1, Collagen I, α-SMA, and TGF-β. Collectively, our results illustrated that exposure to TBBPA and GLY could inhibit muscle growth and decrease nutritional value in common carp.

Amisulbrom, a triazole-based fungicide, is utilized in agriculture to increase agricultural production by controlling fungal infections. The long disappearance time of 50 % (DT50) and potential toxic effects of amisulbrom on nontarget organisms have been reported. However, the toxic effects on the pregnancy process remain unclear. This study aims to determine the cytotoxic responses of human trophoblast cells (HTR-8/SVneo) and human endometrial cells (T HESCs), which are associated with implantation upon amisulbrom exposure. Mitochondrial dysfunction and intracellular Ca overload were determined in both cells that are exposed to amisulbrom. Additionally, amisulbrom arrested the cell cycle progression in the G/M phase, causing apoptosis and reduced survival. Excessive reactive oxygen species (ROS) accumulation and dephosphorylation of PI3K/AKT signaling proteins by amisulbrom exposure mediated these toxic effects. Additionally, spheroid formation was inhibited by amisulbrom treatment in the three-dimensional hanging drop culture model. These results indicate that amisulbrom may pose an adverse effect on the implantation process. Further research is required to identify the toxicity of amisulbrom in vivo. This is the first study to raise concerns about possible toxicity mechanisms of amisulbrom in the implantation process.

Flusulfinam is a novel chiral amide herbicide widely used for controlling annual weeds in rice paddies. However, the mechanism underlying their enantioselective herbicidal activity remain unclear. Herein, it was found that flusulfinam enantiomers, similar to typical HPPD inhibitor mesotrione, reduced chlorophyll and carotenoid levels, decreased HPPD enzyme activity, and upregulated gene expression. Additionally, homogentisate supplementation alleviated the bleaching symptoms caused by flusulfinam and all these results validate that flusulfinam is indeed an HPPD inhibitor. To further investigate the mechanism of enantioselectivity, molecular docking was used and showed that R-flusulfinam (-6.55 kcal/mol) had higher binding energy than S-flusulfinam (-5.60 kcal/mol), due to more stable hydrogen bonds with Gln293. After mutating Gln293 to His, the IC values for R-flusulfinam and S-flusulfinam on MutQ293H were 0.73 mg/L and 0.11 mg/L, respectively, indicating swapped enantioselective inhibition compared to AtHPPD, with IC values of 0.52 mg/L and 1.93 mg/L, respectively. The Microscale Thermophoresis assay further revealed that the dissociation constant (Kd) for MutQ293H with R-flusulfinam was 20.40 ± 4.19 μM, similar to the Kd value of 15.63 ± 4.51 μM for S-flusulfinam. The findings reveal that mutation of the Gln293 residue in the AtHPPD enzyme significantly altered its enantioselective inhibition by flusulfinam. This study is the first to verify the mode of action of flusulfinam and identifies that Gln293 may play a key role in flusulfinam enantioselectivity in the AtHPPD, laying the foundation for future HPPD inhibitor development based on flusulfinam.

Organisms exposed to environmental stimuli can develop "memory" of those experiences, a phenomenon known as priming, which allows them to better adapt to subsequent stimuli. Growing evidence has shown that fungi can "remember" past encounters, but the priming effect remains poorly understood in phytopathogenic fungi. In this study, we examined the priming effect in Fusarium graminearum, the causative agent of Fusarium head blight (FHB), by culturing its conidia in the presence of a median effective concentration (EC) of triazole fungicide. We observed that primed conidia exhibited significantly higher germination rates and longer hyphal length than unprimed conidia when exposed to double EC concentration of triazole. The triazole priming effect in F. graminearum was retained in conidia for an extended period but was not stably heritable. Further investigations revealed that this priming effect was linked to increased over-expression of the fungicide target genes (FgCYP51s) above the level seen in non-primed F. graminearum. This study reveals that F. graminearum develops adaptive resistance following treatment with triazole fungicides, and elucidates the mechanism behind priming, which is regulated by the transcription factor FgSR. This regulation leads to the upregulation of FgCYP51 expression, thereby mediating the observed adaptive resistance. This provides a theoretical basis for understanding the development of resistance in pathogens and offers relevant guidance for the use of triazole fungicides in the control of FHB.

The instability of double-stranded RNA (dsRNA) restricts the application of RNA interference (RNAi) technology in agricultural pest management. Various types of nanocarriers have been developed and employed for the stable delivery of dsRNA. Nonetheless, it remains unclear which type of nanomaterial could deliver dsRNA stably and efficiently for gene knockdown in Locusta migratoria. In this study, we evaluated the ability of three biocompatible and low-toxicity inorganic nanomaterials-polyethylenimine (PEI)-functionalized single-walled carbon nanotube (PEI-SWNT), polyethylenimine-functionalized carbon quantum dots (PEI-CQDs), and layered double hydroxide (LDH)-to bind and stabilize dsRNA. The results revealed that, compared to PEI-CQDs and LDH, PEI-SWNT more effectively protected dsRNA from degradation in locust gut fluids, across various temperatures, and under different pH conditions. Furthermore, we investigated the efficacy of PEI-SWNT/dsRNA complexes in suppressing endogenous genes in locusts through both injection and oral administration methods. Compared to bare dsRNA, PEI-SWNT/dsRNA complexes enhanced RNAi efficiency by up to 46.0 % and increased mortality by up to 39.0 %. Moderate levels of PEI-SWNT could improve the germination rate of wheat, while not affecting leaf growth in the short term. To our knowledge, this study is the first to apply PEI-SWNT inorganic nanomaterials in insects, which provides a foundational basis and compelling evidence for the development of nanomaterial-based nucleic acid pesticides.

Neonicotinoid insecticides, such as imidacloprid, have been used for over three decades to control major agricultural pests, including brown planthopper (Nilaparvata lugens), aphids, and other sap-feeding insects. However, the extensive use of imidacloprid has led to widespread resistance across Asia in many pest populations. Bioassay results demonstrated significant resistance in N. lugens, with a resistance ratio of 32. RNA-seq analysis revealed that the overexpression of detoxification enzyme genes plays a central role in resistance, with CYP6ER1 being the most significantly upregulated gene, showing a 28-fold increase in resistant strain compared to susceptible strain. Among detoxification enzymes, carboxylesterases and glutathione-S-transferases exhibited slightly resistance-specific overexpression, while others remained unchanged. To date, six main variants of CYP6ER1 have been reported. In the current study, CYP6ER1vA has been identified as the predominant variant across all resistant strains, field populations collected in 2024, and museum samples from 1975. This suggests that the vA variant predated the widespread use of imidacloprid and that its expression level, rather than its mere presence, is the key factor driving resistance. A LAMP-PCR diagnostic method was developed to detect CYP6ER1vA, offering a rapid and reliable tool for field-based resistance monitoring. These findings support systematic imidacloprid resistance management in N. lugens, integrating molecular diagnostic with sustainable pest control strategies.

Tyramine (TA) is an important biogenic amine present in the central nervous system of insects, and mediates a variety of physiological and behavioral functions via tyramine receptors (TARs). However, TARs have not yet been characterized in planthoppers, and their physiological functions remain poorly understood in rice pests. Here, we cloned a tyramine receptor gene (LsTAR2) from the small brown planthopper Laodelphax striatellus, one of the most destructive rice pests. LsTAR2 shares high sequence identity with its orthologous receptors, and is closely related to its corresponding receptor groups. LsTAR2 transcript was expressed mostly in the egg stage and brain. RNAi-mediated knockdown of LsTAR2 significantly prolonged the preoviposition period and decreased the fecundity in females. Furthermore, LsTAR2 knockdown reduced the expression levels of vitellogenin (LsVg) in the fat body and ovary of L. striatellus, and changed the expressions of juvenile hormone (JH) and 20-hydroxyecdysone (20E) pathway genes. In addition, LsTAR2 knockdown significantly decreased the honeydew excretion of the adults, and affected the transcript levels of feeding-related neuropeptide signaling genes. These results provide critical information concerning the role of LsTAR2 in reproduction and feeding behavior in L. striatellus, and open the way for further investigations into novel strategies targeting TARs for pest control.

Bee health is influenced by multiple factors, including nutrition, immunity, and parasitic pressures. Since the spread of Varroa destructor, overwintering survival has significantly declined, making it one of the most serious threats to honey bee (Apis mellifera L.) populations worldwide. Natural acaricides, such as oxalic acid (OA), are widely employed for managing Varroa mites; however, their pharmacodynamics, particularly their impacts on honey bee physiology and immunity, remain insufficiently understood. We studied effects of oxalic acid on honey bee workers. The study compared three treatments: flumethrin, OA-glycerine strips (OA-G), and OA trickling (OA-T). Twelve colonies were divided into four groups, with samples collected at five time points (0, 24, 48, 72, and 192 h). Physiological changes were assessed through markers of oxidative stress, longevity, and immune parameters. Exposure to oxalic acid via glycerine strips induced a humoral immune response in adult bees. The antimicrobial activity of hemolymph and levels of antimicrobial peptides (abaecin, apidaecin, defensin, and hymenoptaecin) were elevated between 48 and 192 h after OA-G treatment compared to the control group. In contrast, these parameters were not influenced by OA-T or flumethrin treatment. These findings suggest that OA-G strips activate the honey bee's immune system, providing insights into broader implications of OA use in beekeeping. It is crucial to determine whether the activation of humoral immune systems has positive or negative effects, as well as to develop standardized and reliable treatment protocols that ensure both - health of colonies and their effectiveness in controlling Varroa mite infestations.

Mosquitoes as vectors of life-threatening diseases pose significant risks to human health. While essential oils (EOs) are increasingly utilized as potential mosquitocides, their specific biochemical, genotoxic, and molecular impacts on mosquitoes are not well-documented. This study evaluates the biochemical, genotoxic, and molecular effects of five EOs on Culex pipiens larvae. Late third-instar larvae were treated for 24 h with LC of orange, black pepper, camphor, lemon, and sandalwood EOs. Biochemical studies revealed that these EOs enhanced α-esterase, glutathione S-transferase, and peroxidase activities while reducing total protein, lipid, and carbohydrate contents and amylase, invertase, and trehalase activities. Acetylcholinesterase, protease, catalase, acid phosphatase, and alkaline phosphatase activities were EO-dependent. The comet assay revealed a slight increase in comet parameters, including tailed %, tail length, % DNA in the tail, tail moment, and olive tail moment, indicating low to mild DNA damage. Gene expression studies using orange and black pepper EOs demonstrated significant upregulation of genes related to immunity, detoxification, metabolism, and sensory perception. Molecular docking analysis revealed significant binding affinities between d-Limonene, the major component of orange oil, and its target proteins, with binding energies of -4.87, -6.33, -5.82, and - 3.38 kcal/mol for inhibitor of apoptosis protein, autophagy, juvenile hormone protein, and octopamine receptor, respectively. These findings highlight the potential of d-Limonene as an effective inhibitor, with favorable interactions at the receptor's active sites. This study provides insights into the possible mechanism of EO toxicity, offering promising directions for developing eco-friendly mosquito control strategies.

Black-grass (Alopecurus myosuroides), one of the most economically destructive herbicide-resistant weeds in Europe, is rapidly expanding in winter wheat regions of China. In recent years, the recommended application rate of fenoxaprop-P-ethyl in the field has failed to effectively control Alopecurus myosuroides populations, thereby threatening wheat yields at risk. In this study, we collected a suspected herbicide-resistant population (R-HB) of Alopecurus myosuroides from a wheat field in Hebei Province and confirmed its resistance to fenoxaprop-P-ethyl, with a resistance index of 26.73-fold. Sensitivity analyses of other ACCase-inhibiting herbicides revealed cross-resistance in the R-HB population to clethodim and pinoxaden. Molecular analysis indicated that the resistance phenotype in this population was not due to alterations in the target site. Pretreatment with the cytochrome P450 (P450) inhibitor malathion partially reversed fenoxaprop-P-ethyl resistance in the R-HB population. RNA-seq and RT-qPCR validation revealed the constitutive overexpression of the P450 gene CYP71AF43 in the R-HB population. Molecular docking predictions suggest that the CYP71AF43 protein may have metabolic activity toward fenoxaprop-P-ethyl. In genetically modified yeast, overexpression of AmCYP71AF43 was found to enhance tolerance to fenoxaprop-P-ethyl, but not to clethodim and pinoxaden. Additionally, rice calli overexpressing the AmCYP71AF43 gene exhibited resistance to fenoxaprop-P-ethyl, but not to clethodim or pinoxaden. Collectively, the increased expression of CYP71AF43 may enhance P450-mediated metabolism, conferring resistance to fenoxaprop-P-ethyl in the R-HB population. This is the first report of this mechanism in Alopecurus myosuroides. This discovery provides a novel perspective for the in-depth analysis of resistance mechanisms in weeds against the ACCase-inhibiting herbicide fenoxaprop-P-ethyl.

Multiple pesticide residues in the diet can be ingested through the intestine; however, the interactions affecting intestinal health remain unclear. Histopathological analysis of mouse intestines revealed synergistic damage in those exposed to a binary mixture of abamectin, fluazinam, and spirodiclofen. The combined exposure to fluazinam and spirodiclofen resulted in a 73.35 % reduction in the expression level of the tight junction protein claudin-1 in Caco-2 cells. The studies on the transport in Caco-2 cells revealed that the combined exposure to abamectin and spirodiclofen resulted in transport amounts that were 5.37 and 19.98 times greater than those observed with individual exposures, respectively. The transporter inhibitors and molecular docking analysis indicated that competitive inhibition of the breast cancer resistance protein (BCRP) led to decreased pesticide efflux. Therefore, the disruption of the intestinal barrier caused by the interaction of pesticide mixtures warrants attention when evaluating the safety of various pesticide residues.

The oriental fruit fly, Bactrocera dorsalis (Hendel) is an invasive pest threatening global fruit industries. Field populations of B. dorsalis exhibit complex insecticide resistance, hindering pest control efforts and exacerbating damage. Long non-coding RNAs (lncRNAs) are critical regulators of multiple bioprocess in insects, including insecticide resistance, and have potentials as novel target for pest management. Here, the candidate lncRNAs associated with malathion resistance in B. dorsalis were identified through RNA-seq. One of the isoforms of lnc37707, designated as lnc37707.10, was significantly enriched in the detoxification tissues of malathion-resistant (MR) strain. A specific fragment of lnc37707.10 (sflnc37707) was strongly associated with malathion resistance, and silencing sflnc37707 increased the susceptibility, whereas overexpressing it decreased susceptibility to malathion. Silencing sflnc37707 resulted in the down-regulation of 248 genes, but none of them included the four adjacent genes as its potential target. Instead, pathway analysis revealed significant enrichment of down-regulated genes involved in drug and xenobiotics metabolism, including P450s and GSTs. Bioinformatic analysis suggested a potential regulatory role of miRNA in the function of lnc37707. Further combining silence or overexpression sflnc37707 with miRNA mimic treatment identified that BdGSTd10 (an important gene involved in malathion resistance) and miR-1000 was strongly linked to lnc37707.10. Finally, a ceRNA (competing endogenous RNA) regulatory axis was proposed, where lnc37707.10 might indirectly modulate BdGSTd10 by sponging miR-1000 to regulate the malathion resistance in B. dorsalis. These findings provide a new insight into insecticide resistance and a potential lncRNA target for the sustainable pest management.

The red imported fire ant (RIFA), Solenopsis invicta Buren, poses threats to biodiversity, public safety, agriculture, and the economy, especially as global trade expands its reach into China. To address this, researchers screened fungal isolates from soil in Dongguan City, Guangdong Province, aiming to develop a biopesticide against RIFA. Metarhizium anisopliae, known for its biocontrol potential, was identified as a candidate. This insect pathogenic fungus parasitizes Lepidoptera pest larvae and Hemiptera stinkbugs, causing green rigidity and repeated infestations. Microscopic, morphological, and molecular analyses were conducted on the fungal isolates, with ZHKUJGZ1, a strain of M. anisopliae, showing promise. Tests revealed that inoculating RIFA workers with 1 × 10 cfu/mL of ZHKUJGZ1 resulted in an 83.33 ± 1.57 % mortality rate, with an LC50 of 8.36 × 10 cfu/mL. Untargeted metabolomics suggested that ZHKUJGZ1 enhances insecticidal activity by disrupting the nervous system, signaling, digestive system, amino acid metabolism, and biosynthesis in RIFA. This study highlights the potential of using entomopathogenic fungi like M. anisopliae isolated from Dongguan as an effective strategy for controlling RIFA, offering a promising biocontrol option for agricultural pests.

Frankliniella occidentalis (Pergande) is a horticultural pest known for its overwhelming destructive power. Our previous study showed that dinotefuran significantly inhibited the feeding behavior of F. occidentalis on kidney bean plants. However, why dinotefuran suppressed feeding in F. occidentalis is unknown. Here, we analyzed differences in gene expression in the head (containing salivary glands) of F. occidentalis with or without dinotefuran LC stress using transcriptome sequencing. Eventually, a salivary protein gene belonging to the carbonic anhydrase (CA) family was obtained and named as FoCA2. The full-length cDNA of FoCA2 was obtained by cloning, and the role of FoCA2 in the F. occidentalis antidefense toward the kidney bean plant was investigated using RNA interference. The results showed that FoCA2 was highly expressed in the head of F. occidentalis and at the feeding stages (nymph and adult). Silencing FoCA2 significantly inhibited F. occidentalis feeding and decreased its fecundity and survival; activated jasmonic and salicylic acid signaling pathway-related genes and callose synthase genes; and induced callose accumulation. However, dinotefuran down-regulated the expression of FoCA2 thereby attenuating the suppression of plant defense responses by FoCA2, which ultimately reduced the adaptability of F. occidentalis. Taken together, our findings suggest that FoCA2 is a key effector protein involved in F. occidentalis feeding and host adaptation, providing a foundation for studying the interaction between F. occidentalis and its host plants, and providing insights into the role of dinotefuran on pests.

Uridine diphosphate glycosyltransferases (UGTs) play critical roles in xenobiotic detoxification and are involved in insecticide resistance. In this study, UGT inhibitors, sulfinpyrazone and 5-nitrouracil, exhibited significant synergistic effects on clothianidin in the clothianidin-resistant strain (CL-R) of Bradysia odoriphaga. UGT enzyme content was significantly higher in the CL-R strain than in the susceptible strain (SS), and both the SS and CL-R strains showed a significant upregulation of UGT content after exposure to clothianidin. Two UGT genes, UGT36M1 and UGT306K1, were significantly overexpressed in the CL-R strain. UGT36M1 was predominantly expressed in the fat body, and UGT306K1 exhibited high levels of expression in the Malpighian tubules and midgut. UGT36M1 and UGT306K1 in the SS strain were significantly upregulated in response to clothianidin exposure. The silencing of UGT36M1 and UGT306K1 significantly enhanced the susceptibility of B. odoriphaga larvae to clothianidin. Furthermore, transgenic overexpression of UGT36M1 and UGT306K1 in Drosophila melanogaster significantly increased the tolerance of fruit flies to clothianidin. These findings provide evidence of the crucial role of UGT36M1 and UGT306K1 in conferring resistance to clothianidin in B. odoriphaga.

Our previous study demonstrated that the expression level of circRNA circEgg, which is encoded by histone-lysine N-methyltransferase eggless (BmEgg), is responsive to Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection in the silkworm. However, the precise relationship between BmCPV infection and circEgg remains unclear. In this study, we observed that the expression level of circEgg in both the midguts and cultured BmN cells significantly increased after BmCPV infection, while the expression of its host gene, BmEgg, exhibited an opposite trend. Transient expression experiments revealed that circEgg acts to inhibit BmCPV infection. Additionally, Western blot analyses indicated that BmCPV infection leads to a downregulation of histone 3 lysine 9 trimethylation (H3K9me3) and an upregulation of histone 3 lysine 9 acetylation (H3K9ac). Notably, the levels of H3K9ac and H3K9me3 were found to be positively and negatively correlated with circEgg expression, respectively, suggesting that circEgg may regulate the transition between H3K9me3 and H3K9ac. Mechanistically, we discovered that circEgg inhibits BmCPV infection by enhancing the H3K9ac level through the circEgg-bmo-miR-3391-5p-histone deacetylase Rpd3 network, while simultaneously reducing the H3K9me3 level via the circEgg-encoded protein circEgg-P122. Collectively, these findings indicate that circEgg plays a crucial role in inhibiting BmCPV infection by modulating the balance between H3K9me3 and H3K9ac.

BLTY is a pepper cultivar that has resistance to young larvae of Spodoptera litura Fabricius, whereas FXBX is a pepper cultivar suitable for the growth of S. litura larvae. The obtained microbiomics data suggest that the BLTY leaves fed S. litura larval guts inhabited more Brucella. In this study, two Brucella spp. (2FC-4 and 2FC-15) and one Microbacterium sp. (2FC-22) were isolated from the guts of BLTY and FXBX leaf-fed S. litura, respectively. The loading of 2FC-4 and 2FC-15 reduced the survival rate, average larval weight, diet consumption, pupation rate, and emergence rate of S. litura, whereas the efficacy of 2FC-22 was in contrast with that of the two Brucella spp. By constructing the germ-free (GF) and 2FC-4, 2FC-15, or 2FC-22 mono-associated gnotobiotic S. litura larvae, we found that the loading of 2FC-4 and 2FC-15 disrupted the peritrophic membrane and midgut walls of the tested larvae. In addition, the loading of 2FC-4 or 2FC-15 increased the juvenile hormone content and decreased the ecdysone content in S. litura. The loading of 2FC-4 or 2FC-15 decreased the activities of protease, trehalose, amylase, and invertase and increased the activities of SOD, CAT, and POD. In this study, we analyzed the resistance of BLTY to S. litura from the perspective of the gut microbiota; the selected Brucella sp. can be used for the biological control of Spodoptera pests and Microbacterium sp. can be used as an additive in the daily maintenance of Spodoptera populations.

Serine protease inhibitors (SPIs) have been identified as main common components in the venom of the predatory bugs, while their functional roles remain unexplored. In this study, we identified 35 SPI genes belonging to three subfamilies of serpin, canonical SPI, and A2M in genome of the assassin bug, Sycanus croceovittatus. The amino acid sequences of these SPI genes reveal conserved functional regions, albeit with mutations or deletions at certain active site residues. Transcriptomic and qPCR analyses of gene expression patterns in various tissues across developmental stages indicate that most SPI genes exhibit high expression levels in venom apparatus, suggesting their role as venom proteins. Notably, the ScSPI5 gene from the serpin class was found to be most abundantly expressed in all three distinct venom glands, indicating its significant role as a venomous protein. Functional characterization demonstrated that this venom serpin effectively inhibits trypsin activity in vitro and suppresses phenoloxidase activity, thereby blocking hemolymph melanization in preys, including Spodoptera frugiperda, Achelura yunnanensis, and Tenebrio molitor. When ingested, it reduces the larval and pupal weight of the fall armyworm by impeding trypsin activity in the midgut. Upon injection, ScSPI5 exhibits a dose-dependent insecticidal effect against T. molitor, with an LD of 5.6 ± 1.1 μg/g. These findings elucidate the specific functions of SPIs in the venom of predatory bugs, enhancing our understanding of their predation efficiency, and highlighting the potential application of venomous SPIs as protease inhibitors in pest management strategies.