Abstract: 【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.

Key words: Megalurothrips usitatus; secondary symbionts; Rickettsia, Wolbachia, Arsenophonus, fluorescence in situ hybridization, environmental factors