【Aim】 Using CRISPR/Cas9 gene editing technology to perform single-target knockout of Spodoptera frugiperda doublesex (Sfdsx), this study aims to explore the effect of Sfdsx on the wing development of male adults of S. frugiperda. 【Methods】CRISPR/Cas9 gene editing technology was used to knock out the female and male common region of Sfdsx in embryos of S. frugiperda and construct Sfdsx mutant. After pupation, the differences in the morphological characteristics of pupa and adult wings of the Sfdsx mutant were observed.【Results】 The Sfdsx mutant of S. frugiperda exhibited significant male-biased sex ratio (female∶male=0∶14), and the genital pores on the 8th-9th abdominal segments of its male pupae were severely twisted. The wing traits of the male adult mutant tended to be neutral in sex, in which，the renal patches in the center of the forewing were deformed, the black spots at the end of the wing disappeared, and the wing scales were arranged in disorder. The hind wings were deformed in wingspan, the arrangement of wing scales changed, and small black spots developed.【Conclusion】CRISPR/Cas9-mediated knockout of the common region of Sfdsx led to wing deformation of male adults of S. frugiperda. The results of our study provide an ideal gene target and theoretical basis to modulate the development of S. frugiperda through sterile insect technology (SIT).

【Aim】To assess the conditions of gene RNA interference mediated by the nanocarrier-delivery system in the pea aphid (Acyrthosiphon pisum). 【Methods】The cuticle development-related gene chitin synthase 2 (CHS2) gene, the metabolism-related enzyme vacuolar H⁺ATPase AC39 (VhaAC39) gene, the water-specific aquaporin (Wsa) gene and the key enzyme of N6 methyladenosine (m6A) modification methyltransferase-like protein 14 (METTL14) gene of A. pisum were selected. Different concentrations (10-5 000 ng/μL, 5-250 ng/individual) of dsRNAs of the above four genes were injected into the 4th instar nymphs of A. pisum through the nanocarrier-delivery system, and the expression levels of the above four genes at 48 h after injection were determined by qRT-PCR, to screen the optimal concentration of dsRNA. qRT-PCR was used to detect the expression levels of the above four genes at 12, 24, 36, 48, 60 and 72 h after injecting the dsRNAs of the above four genes at the optimal concentration into the 4th instar nymphs of A. pisum through the nanocarrier-delivery system, to screen the optimal time for RNA interference mediated by the nanocarrier-delivery system.【Results】The expression level of CHS2 was significantly decreased by 47.5% at 48 h after injecting 50 ng/μL (25 ng/individual) of dsCHS2 into the 4th instar nymph of A. pisum as compared to that of the control group injected with dsEGFP. The expression levels of METTL14 decreased significantly after injecting 50-500 ng/μL (25-100 ng/individual) of dsMETTL14 into the 4th instar nymphs of A. pisum as compared to that of the control group injected with dsEGFP, exhibiting a dose-dependent effect. The expression level of VhaAC39 decreased significantly by 53.4% at 48 h after injecting 50 ng/μL (25 ng/individual) of dsVhaAC39 into the 4th instar nymph of A. pisum as compared to that of the control group injected with dsEGFP, while the expression level of Wsa showed no significant change at 48 h after dsWsa injection. The expression levels of CHS2, METTL14 and VhaAC39 all decreased most significantly at 48 h after injection of dsRNAs of target genes, while the expression level of Wsa decreased most significantly at 60 h after injection of dsWsa, as compared to those in the control group injected with dsEGFP.【Conclusion】 The optimal concentration of dsRNA in RNA interference mediated by nanocarrier-delivery system is 50 ng/μL (25 ng/individual), and higher RNA interference efficiency appears at 48-60 h after treatment. This study provides new clues for aphid RNA interference with nanocarrier-delivery system.

【Aim】 To develop a nanocomplex-mediated delivery system for enhancing RNA interference (RNAi) efficiency and dsRNA stability in Bactrocera dorsalis, along with its preparation method and application strategies. 【Methods】Agarose gel shifting test was used to detect the ability of the adult B. dorsalis hemolymph to degrade dseGFP, the optimal mass ratio for the combination of star polycation (SPc) and dseGFP, and the protective effect of SPc on dseGFP. The mortality rate of adult B. dorsalis was calculated to assess the toxicity of the surfactant alkyl polyglucoside (APG), commonly used in dsRNA delivery systems, by injecting APG into the abdomen of adult B. dorsalis. After mixing in vitro synthesized dshsp68 with the nanocarrier SPc, the nanocomplex was injected into the abdomen of adult B. dorsalis using microinjection method, and the RNAi efficiency was detected using RT-qPCR. 【Results】Agarose gel electrophoresis result showed that B. dorsalis hemolymph was able to degrade dseGFP rapidly. When the mass ratio of SPc to dseGFP was greater than or equal to 1∶1, SPc/dseGFP nanocomplex was spontaneously formed, thus protecting dseGFP from being degraded by B. dorsalis hemolymph. The surfactant APG showed strong toxicity towards adult B. dorsalis. At 48 h after injection of APG, the average mortality rate of B. dorsalis was 78.33%, making it unsuitable as an adjuvant for injection methods. The RNAi efficiency of dshsp68 injected alone at 48 h was 49.53% and faded away at 72 h. In contrast, the RNAi efficiency of SPc/dshsp68 nanocomplex was higher, reaching 85.99% at 48 h and remaining at 40.87% at 72 h, offering prolonged duration of RNAi effect. 【Conclusion】SPc can bind dsRNA to form complex and protect dsRNA from being degraded by RNase in hemolymph, thereby achieving sustained and efficient RNAi effects. This study established a method to achieve high-efficiency RNAi with the nanocarrier SPc, which can be used to examine gene function of B. dorsalis.

【Aim】 This study aims to explore the distribution patterns of three secondary symbionts Rickettsia, Wolbachia and Arsenophonus in Megalurothrips usitatus, and evaluate how fungal infection, temperature, host plants, and acetamiprid resistance influence their contents, so as to provide insights into the potential roles of these secondary symbionts in M. usitatus and theoretical support for the development of innovative biocontrol strategies.【Methods】 Fluorescence in situ hybridization (FISH) was employed to detect the distribution of Rickettsia, Wolbachia and Arsenophonus in female and male adults and the 2nd instar nymphs of M. usitatus. RT-qPCR was used to detect the contents of Rickettsia, Wolbachia and Arsenophonus in adults of M. usitatus fed with Vigna unguiculata soaked with Beauveria bassiana spore suspension (1×10⁸ spores/mL)， exposed to different temperatures (5, 10, 15, 25, 30, 35 and 45 ℃), fed with host plants V. unguiculata and Canavalia gladiata, and from acetamiprid-susceptible and -resistant strains.【Results】Rickettsia and Wolbachia were widely distributed in both adults and the 2nd instar nymphs of M. usitatus, and mainly localized in the thorax and abdomen and present in small amounts in the mouthparts and tail regions. Arsenophonus was restricted to the abdomen of female adults of M. usitatus. The content of Rickettsia in female M. usitatus adults at 4 d after B. bassiana infection began to significantly increase, that of Wolbachia in adults after B. bassiana infection increased first and then decreased, and that of Arsenophonus in adults at 6 d after B. bassiana infection increased significantly as compared with those of the control. The contents of Rickettsia, Wolbachia and Arsenophonus in adults of M. usitatus under 25 ℃ were the highest and somewhat decreased under the other temperatures. The content of Rickettsia in adult M. usitatus fed on C. gladiata was significantly reduced as compared with that fed on V. unguiculata. The contents of Rickettsia, Wolbachia and Arsenophonus in adults of M. usitatus of the acetamiprid-resistant strain were significantly increased as compared with those of the acetamiprid-susceptible strain.【Conclusion】 Rickettsia and Wolbachia were widely distributed in adults and the 2nd instar nymphs of M. usitatus, while Arsenophonus was only distributed in female adults of M. usitatus. The contents of Rickettsia, Wolbachia and Arsenophonus were dynamically regulated by factors including fungal infection, temperature, host plants and insecticides. These results highlight the ecological plasticity of secondary symbionts and their potential utility in targeted pest management strategies.

【Aim】Diaphorina citri and Xanthomonas citri subsp. citri (Xcc) are two major pests and diseases that seriously hinder the sustainable development of citrus industry in our country, and their current control methods are limited. Silver nanoparticles (AgNPs) and nano-silicon dioxide (SiO₂ NPs) exhibit insecticidal and antibacterial activities, holding potential application prospects for the control of agricultural pests and diseases. This study aims to test the toxicity of Ag-SiO₂ nanocomposite to D. citri and the antibacterial activity against Xcc, offering a novel measure for effective control of citrus pests and diseases.【Methods】In a controlled laboratory setting, we tested the toxicity of Ag-SiO₂ nanocomposite at the concentrations of 1, 2, 4, 8 and 16 mg/mL to the early (1st-2nd instar) nymphs, late (3rd-5th instar) nymphs, and adults of D. citri by immersion method, and calculated the corrected mortality rates of D. citri and the median lethal concentration (LC₅₀) values of Ag-SiO₂ nanocomposite against different developmental stages of D. citri at 24, 48 and 72 h after treatment. Meanwhile we investigated the alterations in morphology and behavior of D. citri at 24 and 48 h after treatment with Ag-SiO₂ nanocomposite using optical microscopy and scanning electron microscopy, and tested the antibacterial activities of Ag-SiO₂ nanocomposite at the concentrations of 100, 200, 400, 800 and 1 600 μg/mL targeting Xcc by inhibition zone method, measured the diameter of the inhibition zone using the cross method, and calculated the relative antibacterial rate and the medium effective concentration (EC₅₀) value at 48 h after treatment. 【Results】At 72 h after treatment, the corrected mortality rates of the early and late nymphs, and adults of D. citri caused by Ag-SiO₂ nanocomposite at the concentration of 8 mg/mL reached 97.50%, 85.00% and 57.14%, respectively, and the LC₅₀ values of Ag-SiO₂ nanocomposite against the early and late nymphs, and adults of D. citri were 1.3949, 1.9650 and 5.4648 mg/mL, respectively. At 48 h after treatment, the relative antibacterial rate of Ag-SiO₂ nanocomposite at the concentration of 1 600 μg/mL against Xcc reached 53%, and the EC₅₀ value of Ag-SiO₂ nanocomposite against Xcc in 48 h was 1.1853 mg/mL.【Conclusion】Ag-SiO₂ nanocomposite exhibited obvious toxicity to D. citri and also inhibited the activity of Xcc. Ag-SiO₂ nanocomposite exhibited promising potential as both efficient insecticide against D. citri and efficient fungicide for Xcc in the control of citrus pests and diseases, providing a theoretical basis for the pesticide development and field application in the integrated management of citrus pests and diseases.

【Aim】 The English grain aphid, Sitobion avenae, is a severe agricultural pest in China. This study aims to elucidate the interfering effects of miR-14 antagomir on the population of S. avenae, providing a novel technical reference for its prevention and control. 【Methods】Employing a nanomaterial/adjuvant delivery system as the carrier, 0.2 μL of 300 nmol/L miR-14 antagomir was administered to the 1st instar nymphs of S. avenae newborn within 24 h with topical application. Concurrently, nano-material/adjuvant negative control (NNC) and water negative control (WNC) groups were established. The parameters including the proportion of winged morphs, nymphal duration, adult longevity, total developmental duration, mean longevity, reproductive period, number of nymphs produced per female, pre-adult survival rate, total pre-nymphal period and adult pre-nymphal period were calculated. The effects of miR-14 antagomir on the growth, development and fecundity of S. avenae were analyzed using the age-stage, two-sex life table. 【Results】miR-14 antagomir had a significant inhibitory effect on the population of S. avenae. Treatment with miR-14 antagomir resulted in a significant decrease in the pre-adult survival rate and the proportion of winged morphs of S. avenae compared to the controls NNC and WNC. In the miR-14 antagomir treatment, the duration of the 1st instar nymphs of S. avenae was significantly prolonged by 0.17 and 0.29 d, respectively, and that of the 2nd instar nymphs was significantly prolonged by 0.24 and 0.25 d, respectively, as compared to that in the controls NNC and WNC. The mean longevity of S. avenae treated with miR-14 antagomir was significantly shortened by 4.05 and 4.72 d, respectively, as compared to that of the controls NNC and WNC. The adult pre-nymphal period of S. avenae treated with miR-14 antagomir was extended by 0.27 and 0.22 d, respectively, and the total pre-nymphal period of S. avenae treated with miR-14 antagomir was extended by 0.53 d in comparison to those in both the controls NNC and WNC. In addition, the intrinsic rate of increase (r), net reproductive rate (R₀), and finite rate of increase (λ) of S. avenae treated with miR-14 antagomir were significantly decreased as compared to those of the controls NNC and WNC. After treatment with miR-14 antagomir, the population size of S. avenae was approximately 0.33 million after 60 d, significantly lower than that of the controls NNC and WNC (3.24 million and 4.42 million, respectively). 【Conclusion】 miR-14 antagomir prolongs the duration of low instar nymphs, reduces the proportion of winged morphs and inhibits the population growth of S. avenae. These findings provide valuable insights for developing miRNA-based nucleic acid pesticides against S. avenae.

【Aim】The oriental fruit fly (Bactrocera dorsalis), a globally significant pest of fruits and vegetables, has been effectively managed using the male annihilation technique (MAT) based on male attractants, where methyl eugenol (ME) serves as a key lure with potent attraction to males. However, current techniques predominantly target males, leaving the behavioral responses of females to ME and their olfactory perception unclear. This gap has become a bottleneck in the comprehensive optimization of ME-based attractants for females. Therefore, this study aims to systematically reveal the behavioral responses of female adults of B. dorsalis to ME and their olfactory perception capabilities, explore the potential biological roles of ME in B. dorsalis females, and provide new insights for optimizing female-targeted pest control strategies using ME.【Methods】To clarify the responses of female adults of B. dorsalis to ME and their perception capabilities, active 12-day-old female and male adults of B. dorsalis were selected for the subsequent assays. First, olfactory trap experiments and a four-arm olfactometer were used to test the taxis responses to 1 000 and 100 μg of ME and 2-allyl-4,5-dimethoxy phenol (DMP) with similar male-attracting functions, and to evaluate the feeding behavior elicited by 1 000 μg of ME and DMP. Second, the electroantennogram (EAG) responses of B. dorsalis adults to ME and DMP at the doses of 0.1, 1, 10 and 100 μg were recorded in both sexes. Finally, single sensillum recording (SSR) was used to record the SSR responses of basiconic sensilla on the antennae of female and male adults of B. dorsalis to ME and DMP.【Results】Behavioral test results demonstrated that female adults of B. dorsalis displayed neither obvious attraction nor feeding preferences for ME or DMP, in contrast to male adults exhibiting strong behavioral responses. However, EAG experiments showed that female adults of B. dorsalis, similar to male adults, exhibited significant olfactory responses to ME and DMP. Further SSR experiments revealed that female and males adults of B. dorsalis possessed similar olfactory sensilla, responsible for detecting ME and DMP.【Conclusion】The findings suggest that female adults of B. dorsalis have comparable olfactory perception mechanisms to male adults for ME detection. However, the effects of ME on female adult behaviors differ from its strong attraction function in male adults, indicating that ME may have other potential biological roles in female adults. These results provide novel experimental evidence for understanding the perception and behavioral responses of B. dorsalis to ME, while offering theoretical insights for optimizing female-targeted behavioral manipulation technologies.