Ethyl formate (EF), a naturally occurring fumigant, has attracted widespread attention owing to its low toxicity in mammals. Here, Direct Immersion Solid-Phase Microextraction (DI-SPME) was employed for sample preparation in mass spectrometry-based untargeted metabolomics to evaluate the effects on Tribolium castaneum (Herbst) strains with different levels of PH₃ resistance (sensitive, TC-S; moderately resistant, TC-M; strongly resistant, TC-SR) when exposed to a sub-lethal concentration (LC₃₀) of EF. The bioassay indicated that T. castaneum strains with varying PH₃ resistance levels did not confer cross-resistance to EF. A metabolomic analysis revealed that exposure to sublethal doses of EF significantly altered 23 metabolites in T. castaneum, including 2 that are unique to the species which remained unaffected by external conditions, while 11 compounds showed a strong response. A pathway topology analysis indicated that EF caused changes to several metabolic pathways, mainly involving fatty acids and their related metabolic pathways. This study showed that EF can induce highly similar metabolic responses in insects across varying levels of PH₃ resistance, suggesting that the mechanisms driving the toxicity of EF and PH₃ are distinct. These insights significantly extend our knowledge of the toxic mechanisms of EF and provide direct evidence for the efficacy of EF treatment for managing PH₃ resistance in insects.

Bactrocera dorsalis is a major pest that causes serious damage to many fruits. Although phytosanitary treatment methods have been developed for Bactrocera control, there is a lack of information related to the gene expression pattern of B. dorsalis subjected to phytosanitary treatment conditions. Prior to quantitative reverse transcription polymerase chain reaction analysis of the most stable reference genes in B. dorsalis (Diptera: Tephritidae), B. dorsalis third-instar larvae were exposed to various phytosanitary treatments; seven candidate reference genes (18S, G6PDH, GAPDH, RPL-13, RPL-32, RPS-3, and α-Tub) were amplified and their expression stabilities were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Different reference genes were found under different stress conditions. G6PDH was the most stable gene after heat treatment. After cold treatment, α-Tub exhibited the highest expression stability. G6PDH expression stability was the highest after fumigation with methyl bromide. RPL-32 showed the highest expression stability after irradiation treatment. Collectively, RefFinder analysis results revealed G6PDH and RPL-32 as the most suitable genes for analyzing phytosanitary treatment in B. dorsalis. This study provides an experimental basis for further gene expression analyses in B. dorsalis subjected to various phytosanitary treatments, which can aid in the development of novel phytosanitary treatments against insect pests.

The metabolites produced by the larvae of Bactrocera dorsalis (Diptera: Tephritidae) exposed to different doses of irradiation were analyzed using solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS), and a metabonomic analysis method of irradiated insects based on GC-MS was established. The analysis revealed 67 peaks, of which 23 peaks were identified. The metabolites produced by larvae treated with different irradiation doses were compared by multivariate statistical analysis, and eight differential metabolites were selected. Irradiation seriously influenced the fatty acid metabolic pathway in larvae. Using the R platform combined with the method of multivariate statistical analysis, changes to metabolite production under four irradiation doses given to B. dorsalis larvae were described. Differential metabolites of B. dorsalis larvae carried chemical signatures that indicated irradiation dose, and this method is expected to provide a reference for the detection of irradiated insects.

The management of phosphine (PH3) resistance in stored grain pests is an essential component of implementing timely and effective pest control strategies. The prevailing standard method for PH3 resistance testing involves the exposure of adult insects to a specific concentration over a fixed period. Although it is widely adopted, this method necessitates an extensive period for assay preparation and diagnosis. To address this issue, this study employed Direct Immersion Solid-Phase Microextraction (DI-SPME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) to compare and analyze the metabolic profiles of PH3-sensitive (TC-S), PH3 weak-resistant (TC-W), and PH3 strong-resistant (TC-SR) Tribolium castaneum (Herbst) adults. A total of 36 metabolites were identified from 3 different PH3-resistant strains of T. castaneum; 29 metabolites were found to present significant differences (p < 0.05) across these groups, with hydrocarbon and aromatic compounds being particularly prevalent. Seven metabolites showed no significant variations among the strains, consisting of four hydrocarbon compounds, two iodo-hydrocarbon compounds, and one alcohol compound. Further multivariate statistical analysis revealed a total of three, two, and nine differentially regulated metabolites between the TC-S versus TC-W, TC-S versus TC-SR, and TC-W versus TC-SR groups, respectively. Primarily, these metabolites comprised hydrocarbons and iodo-hydrocarbons, with the majority being associated with insect cuticle metabolism. This study demonstrates that DI-SPME technology is an effective method for studying differentially expressed metabolites in T. castaneum with different levels of PH3 resistance. This approach may help to provide a better understanding of the development of insect PH3 resistance and act as a valuable reference for the establishment of rapid diagnostic techniques for insect PH3 resistance.

X-ray irradiation and modified atmospheres (MAs) provide eco-friendly, chemical-free methods for pest management. Although a low-oxygen atmospheric treatment improves the performance of some irradiated insects, its influence on the irradiation of quarantine insects and its impacts on pest control efficacy have yet to be investigated. Based on bioassay results, this study employed direct immersion solid-phase microextraction (DI-SPME) combined with gas chromatography-mass spectrometry (GC-MS) to determine metabolic profiles of late third-instar B. dorsalis larvae under normoxia (CON, Air), hypoxia (95% N₂ + 5% O₂, HY), super-hypoxia (99.5% N₂ + 0.5% O₂, Sup-HY), irradiation-alone (116 Gy, IR-alone), hypoxia + irradiation (HY + IR) and super-hypoxia + irradiation (Sup-HY + IR). Our findings reveal that, compared to the IR-alone group, the IR treatment under HY and Sup-HY (HY + IR and Sup-HY + IR) increases the larval pupation of B. dorsalis, and weakens the delaying effect of IR on the larval developmental stage. However, these 3 groups further hinder adult emergence under the phytosanitary IR dose of 116 Gy. Moreover, all IR-treated groups, including IR-alone, HY + IR, and Sup-HY + IR, lead to insect death as a coarctate larvae or pupae. Pathway analysis identified changed metabolic pathways across treatment groups. Specifically, changes in lipid metabolism-related pathways were observed: 3 in HY vs. CON, 2 in Sup-HY vs. CON, and 5 each in IR-alone vs. CON, HY + IR vs. CON, and Sup-HY + IR vs. CON. The treatments of IR-alone, HY + IR, and Sup-HY + IR induce comparable modifications in metabolic pathways. However, in the HY + IR, and Sup-HY + IR groups, the third-instar larvae of B. dorsalis demonstrate significantly fewer changes. Our research suggests that a low-oxygen environment (HY and Sup-HY) might enhance the radiation tolerance in B. dorsalis larvae by stabilizing lipid metabolism pathways at biologically feasible levels. Additionally, our findings indicate that the current phytosanitary IR dose contributes to the effective management of B. dorsalis, without being influenced by radioprotective effects. These results hold significant importance for understanding the biological effects of radiation on B. dorsalis and for developing IR-specific regulatory guidelines under MA environments.

Mealybugs are high-priority quarantine pests in fresh-produce trade due to cryptic habits, broad host ranges, and market-access risks. Phytosanitary irradiation (PI) provides a non-residual, process-controlled option that is increasingly integrated with modified-atmosphere (MA/MAP) logistics. Because molecular oxygen enhances indirect radiation damage (oxygen enhancement ratio, OER), oxygen limitation may modulate PI outcomes in mealybugs. The Jack Beardsley mealybug (Pseudococcus jackbeardsleyi) has an IPPC-adopted PI treatment of 166 Gy (ISPM 28, PT 45). We exposed adult females to 166 Gy under air and 1% O₂ and generated whole-transcriptome profiles across treatments. Differentially expressed genes and co-differentially expressed genes (co-DEGs) were integrated with protein–protein interaction (PPI) and regulatory networks, and ten hubs were validated by reverse transcription quantitative PCR (RT-qPCR). Hypoxia attenuated irradiation-induced transcriptional disruption. Expression programs shifted toward transport, redox buffering, and immune readiness, while morphogen signaling (Wnt, Hedgehog, BMP) was coherently suppressed; hubs including wg, hh, dpp, and ptc showed stronger down-regulation under hypoxia + irradiation than under irradiation alone. Despite these molecular differences, confirmatory bioassays at 166 Gy under both atmospheres (air and 1% O₂) achieved complete control. These results clarify how oxygen limitation modulates PI responses in a quarantine mealybug while confirming the operational efficacy of the prescribed 166 Gy dose. Practically, they support the current international standard and highlight the value of documenting oxygen atmospheres and managing dose margins when PI is applied within MA/MAP supply chains.