As eusocial insects prevalent in tropical and subtropical regions, termites are characterized by highly organized behaviors and exceptional adaptability, rooted in caste differentiation and chemical communication. These traits make them excellent models for studying insect social structures and ecological interactions. Investigating how termites use chemical signals to perceive and respond to their environment provides insights into their coordination and adaptation within complex ecosystems. This study delved into the chemosensory mechanisms of Reticulitermes aculabialis, examining the interactions of four olfactory proteins with 70 ligands, including host volatiles, cuticular hydrocarbons (CHCs), and free fatty acids (FFAs). Molecular docking simulations revealed varied affinities of the olfactory proteins for long-chain hydrocarbons (n-C to n-C), suggesting a nuanced chemical communication system through specific hydrocarbon detection. RacuCSP1 and RacuCSP2 exhibited specific binding to linoleic acid and undecanoic acid, respectively, highlighting the significance of FFAs in the physiological and behavioral processes of termites. The four olfactory proteins showed a strong affinity for longifolene in fluorescence competitive binding experiments. Notably, RacuOBPs specifically exhibited unique affinities for terpenoid volatiles such as β-lonone and neocembrene, while RacuCSPs specifically bound with terpenoids like 3-carene, myrtenol, α-pinene oxide and β-pinene indicating their critical roles in host detection. Behavioral observations following gene silencing revealed that RacuOBP5 was essential for recognizing longifolene and α-lonone recognition, while RacuCSP1 was key for detecting α-pinene in termites. These findings enhance our understanding of the termite chemosensory system and offer insights for developing precise pest management strategies.

The brown planthopper (Nilaparvata lugens) is an important insect pest of rice, and can rapidly adapt to insect-resistant rice varieties. In our previous studies, alanine aminotransferase in N. lugens (NlALT) was found to play an important role in the adaptation of the brown planthopper to resistant rice IR36. Here, we further identified CCAAT/enhancer binding protein (NlC/EBP) as a vital transcription factor of NlALT. Nlp38b in the MAPKs pathway regulated the expression of NlALT by influencing the phosphorylation level of NlC/EBP. In addition, we found that NlGRL101, a G protein-coupled receptor (GPCR), was significantly higher expressed in the N. lugens population adapted to IR36 (P-IR36). After knockdown of NlGRL101 through RNAi in P-IR36 population, lower expressions of Nlp38b and NlC/EBP, along with reduced phosphorylation levels of Nlp38b and NlC/EBP were observed; moreover, NlALT activity and honeydew amount were decreased by 15.68% and 76.08%, respectively. These results indicated that insect-resistant rice IR36 induced expression of NlGRL101, which enhanced expression of NlALT through Nlp38b and NlC/EBP. These findings are helpful for better understanding of insect adaptation to resistant crop varieties.

Nutrition plays a major role in host immune responses and in pathogen resistance. Understanding the network that modulates the relationship between nutrition and immunity remains a challenge. Several pathways govern the direct effects of nutrition on host immunity and the indirect effects mediated by pathogen populations. We note host microbiota also influence the intricate relationships between nutrition and immunity. The purpose of this review is to discuss recent findings from nutritional research in relation to insect immunology. We outline the relationship between diet, immunity, disease, and microbiota in insects and emphasize the significance of utilizing an integrative, multifaceted approach to grasping the influence of nutrition on immunity.

Insect vectors significantly threaten global agriculture by transmitting numerous plant viruses. Various measures, from conventional insecticides to genetic engineering, are used to mitigate this threat. However, none provide complete resistance. Therefore, researchers are looking for novel control options. In recent years with the advancements in genomic technologies, genomes and transcriptomes of various insect vectors have been generated. However, the lack of knowledge about gene functions hinders the development of novel strategies to restrict virus spread. RNA interference (RNAi) is widely used to elucidate gene functions, but its variable efficacy hampers its use in managing insect vectors and plant viruses. Genome editing has the potential to overcome these challenges and has been extensively used in various insect pest species. This review summarizes the progress and potential of genome editing in plant virus vectors and its application as a functional genomic tool to elucidate virus-vector interactions. We also discuss the major challenges associated with editing genes of interest in insect vectors.

The cotton-melon aphid Aphis gossypii Glover is a severe pest worldwide. Interhaplotype genomic variation can be used as a starting point to analyze the adaptability of Ap. gossypii. In this study, we utilized long-read PacBio HiFi sequencing and HiC scaffolding techniques to assemble a near telomere-to-telomere gap-free genome assembly of Hap4. The assembly had two gaps totaling 321.24 Mb. We characterized five telomeric repetitive regions (GGTTA), including the four found at the 3' end of the chromosomes, and obtained new structural information about the telomeres. Due to the improved sequencing technology, we also identified more than 55.03 Mb of repetitive DNA in the genome assembly of Hap4, which contributed significantly to the increase in genome size compared to that of Hap1 and Hap3. Most of the additional repetitive DNA content was located on the X chromosome, and the tandem repeat sequence occupied 16.8% of the X chromosome length. The Hap4 assembly showed that the X chromosome exhibited a greater abundance of AT-rich satDNA arrays (11 satDNA arrays longer than 100 kb) than that observed in the autosomes (A1 and A2 harboured 3 and 1 satDNA arrays). We detected presence-absence variations, insertions, and deletions events between Hap1, Hap3, and Hap4 Ap. gossypii, which had significant effects on gene expression. Additionally, we identified a male-specific glyceraldehyde-3-phosphate dehydrogenase of fungal origin in all strains of Ap. gossypii. This comprehensive genome assembly provides valuable insights into the structural characteristics of highly repetitive regions and allows comparative genomic analyses that facilitate our understanding of Ap. gossypii's adaptation and diversification.

The conservative post-transcriptional modification in mammals and Drosophila is adenosine-to-inosine (A-to-I) deamination in double-stranded RNA, catalyzed by RNA-editing enzymes known as adenosine deaminases acting on RNA (ADARs). The traditional nuclear import pathway for ADARs involves the recognition of a putative classical nuclear localization sequence (NLS) by importin α4 and α5. In our previous research, ADAR in silkworm, Bombyx mori (BmADARa) was confirmed predominantly located in the nucleus. However, the location of the NLS in BmADARa and its impact on nuclear import and self-dimerization remained unclear. Utilizing NLS prediction software, we predicted the presence of a bipartite NLS within the amino-terminal, 85 amino acids of BmADARa (N85). This prediction was validated through point mutation, which demonstrated that the bipartite NLS could directly mediate nuclear import of BmADARa. Co-immunoprecipitation analysis revealed that BmADARa is mainly dependent on BmKaryopherin α3 (homologous to mammalian importin α4) for nuclear import, although both BmKaryopherin α3 and BmImportin α5 could recognize bipartite NLS. The N-terminal truncated mutants and the bipartite NLS mutants of BmADARa suggest that the bipartite NLS is the major nuclear import site and a crucial structure for self-dimerization of BmADARa. In conclusion, the N-terminal bipartite NLS of BmADARa is recognized by BmKaryopherin α3 and BmImportin α5, facilitating its nuclear import. This promotes BmADARa self-dimerization and maintains the stability of dimerization, thereby enhancing its editing efficiency on target substrates. The results of this research demonstrate the role of bipartite NLS in BmADARa editing and laying a foundation for further research on the regulation of BmADARa in the growth and development in B. mori.

Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.

Salivary proteins in the oral secretion (OS) of chewing insects play a crucial role in insect-plant interactions during feeding. The rice leaf folder Cnaphalocrocis medinalis, a notorious pest in global rice production, triggers defense responses during feeding, but little is known about its salivary proteins. In this study, we confirmed that C. medinalis releases OS during feeding. By employing transcriptomic analysis and liquid chromatography-tandem mass spectroscopy (LC-MS/MS), we examined the salivary proteins from labial salivary glands and OS from C. medinalis. A total of 14,397 genes were expressed at the RNA level and 229 salivary proteins were identified. Comparative analysis with other 25 arthropod species revealed that 43 proteins were unique to C. medinalis. Expression pattern analysis revealed that most of the selected genes were highly expressed in the gut and the larval stages (4th-5th instar). These findings provide a comprehensive resource for future functional studies of salivary proteins, offering new insights into the molecular mechanisms by which C. medinalis modulates plant defenses and potential applications in pest management.

Chemical detection is vital for animal survival, aiding in avoiding toxins and selecting nutritious foods. While Drosophila larvae exhibit appetitive feeding behavior toward ribose, an important sugar for RNA, nucleotide, and nucleoside synthesis, how adult Drosophila perceives ribose remains unclear. Through behavioral and electrophysiological investigations, we unexpectedly discovered that adult flies actively avoid ribose. Our external electrophysiological analysis revealed that ribose is detected through bitter-sensing gustatory receptor neurons in S-type sensilla, suggesting its perception as a bitter compound. Additionally, we identify painless as crucial for both ribose aversion and the neuronal response to ribose.

Manduca sexta hemolymph protease-6 (HP6) plays a central role in coordinating antimicrobial responses, such as prophenoloxidase (PPO) activation and Toll signaling. Our previous studies indicated that HP5 and GP6 activate proHP6 in larval hemolymph and extraembryonic tissues, respectively. Here, we report the characterization of HP17b as another HP6 activating enzyme and its regulation by multiple serpins in hemolymph. The precursor of HP17b expressed in baculovirus infected Sf9 cells became spontaneously cleaved at two sites, and these products were purified together in one preparation named HP17b', a mixture of proHP17b, a 35 kDa intermediate, and HP17b. HP17b' converted proHP6 to HP6. As reported before, HP6 converted precursors of PPO activating protease-1 (PAP1) and HP8 to their active forms. HP8 activates proSpӓtzle-1 to turn on Toll signaling. We found HP17b' directly activated proSPHI and II to form a cofactor for PPO activation by PAP1. Supplementation of larval hemolymph with HP17b', HP17b, or proHP17b significantly increased PPO activation. Adding Micrococcus luteus to the reactions did not enhance PPO activation in the reactions containing HP17b', HP17b, or proHP17b. Using HP17b antibodies, we isolated from induced plasma HP17b fragments and associated proteins (e.g., serpin-4). Serpin-1A, 1J, 1J', 4, 5, or 6 reduced the activation of proHP6 by HP17b' through formation of covalent complexes with active HP17b. We detected an activity for proHP17b cleavage in hemolymph from bar-stage pharate pupae but failed to purify the protease due to its high instability. Other known HPs did not activate proHP17b in vitro. Together, these results suggest that HP17b is a clip-domain protease activated by an unknown endopeptidase in response to a danger signal and regulated by multiple serpins.

Pyrethroid insecticides exert their toxic action by prolonging the opening of insect voltage-gated sodium channels, resulting in the characteristic tail current during membrane repolarization in voltage clamp experiments. Permethrin (PMT) and deltamethrin (DMT), representative type I and type II pyrethroids, respectively, are predicted to bind to two lipid-exposed pyrethroid receptor sites, PyR1 and PyR2, at the lipid-exposed interfaces of repeats II/III and I/II, respectively. Transfluthrin (TF), a volatile type I pyrethroid and mosquito repellent, has received increased attention in the global combat of vector-borne human diseases. However, the electrophysiological and molecular bases of TF action on insect sodium channels remain unexplored. In this study we discovered that, unlike DMT and PMT, TF barely induces the characteristic tail current of the Aedes aegypti mosquito sodium channel (AaNa1-1) expressed in Xenopus oocytes. Instead, TF induces a unique persistent current. We docked TF into the AlphaFold2 model of AaNa1-1 and found that the tetrafluorophenyl ring of TF binds to alpha helices S5, P1, and S6, but not to the linker helices S4-S5 within either PyR1 or PyR2. In agreement with the model, functional examination of 15 AaNa1-1 mutants demonstrated that substitutions of DMT/PMT-sensing residues in helices S5, P1, and S6, but not in the linker- helices S4-S5, altered channel sensitivity to TF. These results revealed the unique action of TF on channel gating and suggest a distinct subtype of type I pyrethroids with a previously uncharacterized pattern of interactions with residues at the dual pyrethroid receptor sites.

Parasitoids often exhibit high flexibility in their development depending on stages of their host at the parasitism, yet little is known about the mechanism underlying such flexibility. In the study, we evaluated the larval development time of the parasitoid Exorista sorbillans (Diptera: Tachinidae) on the lepidopteran model insect Bombyx mori (Lepidoptera: Bombycidae). We found that the development duration of E. sorbillans larvae parasitizing on the late-developmental silkworms was significantly shorter than that of the larvae parasitizing on the early-developmental hosts. Intriguingly, the 2nd-3rd instar molting of parasitoid always occurred when the ecdysteroid titers in the host were increased to higher levels. Furthermore, inhibiting the release of ecdysteroids to parasitic abdomen by thorax-abdomen ligation of the host only repressed the 2nd-instar growth and molting of E. sorbillans larvae, but had no effect on their pupation. Meanwhile, the ecdysone synthesis and 20-hydroxyecdysone (20 E) signaling in larval parasitoids were impeded after ligation treatment. Moreover, exogenous 20 E application could largely rescue the defect in 2nd instar growth and molting through stimulating ecdysone synthesis and signaling in E. sorbillans. Our results indicate that the parasitoid requires the host ecdysteroids to stimulate 20 E signaling and the subsequent 2nd-instar growth and molting. These findings will improve our understanding of the host utilization strategies of parasitoids, and contribute to the development of in vitro rearing procedures of tachinid parasitoids for biological control.

Plants produce complex chemical defenses against herbivores, resulting in the emergence of detoxification strategies in phytophagous insects. While enzymatic detoxification and target site mutagenesis are well-documented, the quantitative contribution of excretion remains less studied. We focus on the cabbage looper (Trichoplusia ni), a generalist herbivore, to elucidate the detoxification of a steroidal alkaloid, solanidine, produced in potato (Solanum tuberosum). Through larval feeding experiments and chemical analysis of metabolites using high-resolution mass spectrometry, we identify solanidine 3-O-β-glucopyranoside and solanidine 3-phosphate as major metabolization products of solanidine. Glycosylation and phosphorylation reactions have not previously been observed in cabbage looper. Modified solanidine derivatives exhibit reduced lipophilicity, preventing passive transport as predicted by physicochemical analyses, and only solanidine was detected in body tissue. In addition, the metabolism of solanidine in a T. ni mutant strain with midgut cadherin protein knocked out was also investigated to examine the potential role of the cadherin, an important receptor for Bt toxins, in steroidal alkaloid detoxification. T. ni cadherin-knockout strain showed lower solanidine conversion (33.9% ± 2.2) and uptake (27.41 ± 0.49 nmol/g) compared to the wild-type strain (51.3% ± 4.1, 33.66 ± 2.48 nmol/g) but similar excretion kinetics. Although solanidine negatively impacted the feeding performance of both strains the cadherin-knockout does not affect the feeding performance. Our study expands the metabolization enzyme repertoire in cabbage loopers, emphasizing the complexity of detoxification mechanisms in generalist herbivores.

The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.

Winged parthenogenetic aphids are mainly responsible for migration and dispersal. Aphid alarm pheromone (E)-β-Farnesene (EBF) has dual effects on repelling and stimulating wing differentiation in aphids. Previous studies have shown that the odorant coreceptor SmisOrco is involved in the perception of EBF by S. miscanthi; however, its EBF-specific odorant receptor (OR) and the difference between winged and wingless aphids remain unclear. In this study, the Xenopus oocyte expression system and RNAi technology were used to detect the transmission of EBF signals, and it was found that the olfactory receptor SmisOR5 is an EBF-specific OR in S. miscanthi and is specifically highly expressed in the antennae of winged aphids. Furthermore, when OR5 was silenced with dsRNA, the repellent effect of EBF was weakened, and aphids showed more active aimless movements. Therefore, as a specific OR for EBF, the high expression level of SmisOR5 in winged aphids suggests a molecular basis for its high sensitivity to EBF. This study advances our understanding of the molecular mechanisms of aphid EBF perception and provides novel ideas for effective management and prevention of the migration of winged aphids.

Uridine diphosphate-glycosyltransferases (UGTs) catalyze sugar conjugation of endogenous and exogenous molecules in insects. In this study, 45 putative UGT genes in 11 families were identified from the genome of S. litura. Exposure to Bt toxins in 5th-instar larvae of the WT strain led to a significant upregulation of midgut UGT40 expression, particularly of SlUGT40D20, SlUGT40D22, and SlUGT40F25. This upregulation was not observed following exposure to chemical pesticides. Knockout of the UGT genes SlUGT40D20 and SlUGT40D22 in S. litura (mutant strains SlUGT40D20-KO and SlUGT40D22-KO) via CRISPR/Cas9-mediated mutagenesis increased susceptibility of S. litura to Bacillus thuringiensis (Bt) insecticidal proteins. However, in comparison with the wild-type (WT) strain, the mutants did not change susceptibility to chemical pesticides. Observations of 5th-instar larval midgut by electron microscopy revealed severe damage to the midgut epithelium caused by Cry1Ac toxin at 10 μg/g in the SlUGT40D20-KO strain compared to the WT. SDS-PAGE and LC MS/MS analyses identified a specific protein band corresponding to putative proteoglycans in the peritrophic matrix of the WT strain, which was absent in the SlUGT40D20-KO strain. Our study suggests an inverse correlation between expression of some UGTs and the susceptibility of S. litura larvae to some Bt toxins.

Tissue regeneration is an efficient strategy developed by animals to compensate for damaged tissues, involving various types of progenitor cells. Deciphering the signal network that modulates the activity of these progenitors during regeneration is crucial for understanding the differences in regenerative capacities across species. In this study, we evaluated the expression profile and phenotypic function of Notch signaling during limb regeneration in arthropod Chinese mitten crabs. The expression of key components of the Notch signaling pathway was upregulated at 7-day post-autotomy (7 DPA), and declined later at 18-day post-autotomy (18 DPA). To assess the role of Notch, we injected dsRNA targeting the Notch gene into the automized area and evaluated the regeneration efficiency. Our results indicated that blocking Notch signaling led to regenerative defects, manifested by delays in the wound closure and blastema emergence processes. Furthermore, the expression of Notch target genes, Hes1 and HeyL, was significantly reduced following Notch knockdown by dsRNA. Knockdown of Hes1 specifically impaired the proliferation and expression of neural progenitor cell markers, without affecting myogenic cells. In contrast, blockage of HeyL inhibited the proliferation and expression of markers in both activated neurogenic and myogenic progenitor cells, while up-regulating markers of quiescent neural progenitor cells. These findings suggest that Notch signaling plays an important role in limb regeneration of E. sinensis by activating downstream effectors Hes1 and HeyL, regulating neurogenesis and myogenesis through distinct mechanisms.

Male-specific lethal-3 (MSL3) is a component of the dosage compensation complex in Drosophila melanogaster, where its mutation leads to male-specific lethality. However, the function of MSL3 in hemipteran insects remains unclear. This study investigated the role of the MSL3 homolog in a major rice pest, the brown planthopper (Nilaparvata lugens). We cloned and characterized the gene NlMSL3 from N. lugens, which is 1467-bp long and encodes a protein of 488 amino acids. Phylogenetic analysis revealed that MSL3 is conserved across various insect orders, with high conservation in the chromo-barrel domain. Quantitative real-time polymerase chain reaction indicated differential expression levels of NlMSL3 between male and female insects during development, with the highest expression in the testes. RNA interference-mediated knockdown of NlMSL3 in N. lugens resulted in significant mortality in later instar nymphs and adults compared with the control group. In females, NlMSL3 knockdown impaired feeding behavior, leading to decreased body weight, notably reduced honeydew excretion, flat abdomens, decreased vitellogenin expression, and defective ovarian development. When dsNlMSL3-treated males were mated with control females, the number of eggs laid was similar to that laid by the females mated with control males; however, none of the eggs laid by the former hatched into nymphs. These results highlight the crucial role of NlMSL3 in the development and fecundity of N. lugens.

Sugar consumption increases the fecundity and longevity in many species of parasitic wasps (parasitoids) but whether these insects use sugars to synthesize significant amounts of fatty acids and storage fat de novo (lipogenesis) is discussed controversially. It has long been assumed that parasitic wasps lost this ability during evolution, mainly because in several species wasps with ad libitum access to sugar did not increase teneral lipid levels. Recent studies demonstrated that many species are nonetheless capable of synthesizing fatty acids de novo from glucose. It is unclear, however, whether also other sugars are used for fatty acid biosynthesis and whether an increase of sugar concentration to levels occurring in natural sugar sources translates into higher fatty acid production. Furthermore, it has been suggested that fatty acid production in parasitoids is negligible compared to species increasing teneral fat reserves such as Drosophila melanogaster. Here we show by stable isotope labeling experiments that females of Nasonia vitripennis convert D-glucose, D-fructose, sucrose, and α,α-trehalose, major sugars consumed by adult parasitoids in nature, equally well to palmitic, stearic, oleic, and linoleic acid. Lipogenesis from D-galactose occurs as well albeit to a lesser extent. Sugar concentration is crucial for lipogenic activity, and almost 80% of de novo synthesized fatty acids were incorporated into storage fat (triacylglycerides). Comparison of fatty acid biosynthesis within a 48-h feeding period with D. melanogaster revealed that N. vitripennis produced approximately half as many fatty acids per body mass unit. Both species fed equal amounts of the glucose offered. We conclude that lipogenesis is far from negligible in N. vitripennis and plays an important role for the energy balance when teneral lipid reserves deplete.

RNA interference (RNAi)-based products have the potential to significantly contribute to insect pest control. However, RNAi efficiency varies widely among different insect orders, particularly in Lepidoptera, where it is often low. One key factor affecting RNAi efficiency is the presence of double-stranded ribonuclease (dsRNase) in the digestive tract, which can degrade dsRNA prior to uptake by gut cells. In this study, four dsRNases were identified in the beet armyworm, Spdoptera exigua, of which two were highly expressed gut dsRNases, SedsRNase1 and SedsRNase2. To assess their effect on dsRNA degradation activity via the oral route, CRISPR/Cas9-based gene editing was employed to knock out these gut dsRNases. The results indicate that all mutant strains, including SeKO1 (knockout SedsRNase1), SeKO2 (knockout SedsRNase2), and SeKO1KO2 (knockout SedsRNase1 and SedsRNase2), showed significantly decreased dsRNA degradation activity, particularly in the SeKO1KO2 mutant strain, where the weakest degradation occurred in both the gut and whole body. Additionally, we noticed that the lack of gut SedsRNases led to a slight extended developmental period and reduced reproductive capacity in S. exigua. Collectively, these findings deepen our understanding of gut SedsRNases and how they can impact the biology of the beet armyworm and can support the exploration dsRNA-based approaches for pest control.