Background Phosphine (PH3) is widely used for controlling pests in stored products. Its repeated application has caused strong resistance in Rhyzopertha dominica. However, most studies on phosphine resistance in R. dominica to date have mainly identified biochemical changes at the molecular level. Therefore, this study aims to elucidate the resistance mechanisms toward PH3 in R. dominica using metabolomic techniques in cooperation with proteomic and physiological analyses. Results Phylogenetic analysis confirmed that tested strains belonged to R. dominica. The PH3-resistant strain (PH3-R strain) exhibited 15% lower body weight compared with that of the PH3-susceptible strain (PH3-S strain). The PH3-R strain demonstrated a 97.5-fold higher LC50 than the PH3-S strain. The PH3-R strain exhibited a P49S point mutation in the dihydrolipoamide dehydrogenase (DLD) gene and showed reduced activities in cytochrome c oxidase, glutathione S-transferase, and carboxylesterase. Whole-proteome analysis using Orbitrap LC-MS/MS revealed upregulation of 22 proteins and downregulation of 7 proteins in the PH3-R strain. In particular, DLD, a key enzyme involved in energy metabolism, showed significantly reduced abundance in the PH3-R strain. Metabolomic analysis using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed that 6-phosphogluconic acid (× 19-fold), ribulose (× 2.76-fold), and ribose (× 3.53-fold) in the pentose phosphate pathway were highly overproduced in the PH3-R strain, enhancing NADPH generation. In addition, the upregulation of kynurenine (× 2.86-fold) and protocatechuic acid (× 2.51-fold) in the PH3-R strain indicated activation of NAD(P)+ production and sialic acid biosynthesis. Conclusions PH3 resistance in R. dominica was associated with reduced body weight, DLD P49S mutation and reduced DLD abundance, reduced COX, GST, and CE activities, and increased abundance of pentose phosphate pathway-related metabolites. These findings suggest that PH3 resistance involves coordinated biochemical, proteomic, and metabolomic changes linked to energy and redox metabolism. Graphical Abstract

Whole proteome and metabolome analyses of phosphine resistance in the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae)

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2026年7月22日

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Jinuk Yang,
Donghyeon Kim,
Junyeong Park,
Minseo Kang,
Ji-Woo Yu,
Min-Ho Song,
Jung-Hoon Lee,
Ji-Ho Lee,
Sung-Eun Lee

摘要

Background

Phosphine (PH₃) is widely used for controlling pests in stored products. Its repeated application has caused strong resistance in Rhyzopertha dominica. However, most studies on phosphine resistance in R. dominica to date have mainly identified biochemical changes at the molecular level. Therefore, this study aims to elucidate the resistance mechanisms toward PH₃ in R. dominica using metabolomic techniques in cooperation with proteomic and physiological analyses.

Results

Phylogenetic analysis confirmed that tested strains belonged to R. dominica. The PH₃-resistant strain (PH₃-R strain) exhibited 15% lower body weight compared with that of the PH₃-susceptible strain (PH₃-S strain). The PH₃-R strain demonstrated a 97.5-fold higher LC₅₀ than the PH₃-S strain. The PH₃-R strain exhibited a P49S point mutation in the dihydrolipoamide dehydrogenase (DLD) gene and showed reduced activities in cytochrome c oxidase, glutathione S-transferase, and carboxylesterase. Whole-proteome analysis using Orbitrap LC-MS/MS revealed upregulation of 22 proteins and downregulation of 7 proteins in the PH₃-R strain. In particular, DLD, a key enzyme involved in energy metabolism, showed significantly reduced abundance in the PH₃-R strain. Metabolomic analysis using gas chromatography-tandem mass spectrometry (GC-MS/MS) revealed that 6-phosphogluconic acid (× 19-fold), ribulose (× 2.76-fold), and ribose (× 3.53-fold) in the pentose phosphate pathway were highly overproduced in the PH₃-R strain, enhancing NADPH generation. In addition, the upregulation of kynurenine (× 2.86-fold) and protocatechuic acid (× 2.51-fold) in the PH₃-R strain indicated activation of NAD(P)⁺ production and sialic acid biosynthesis.

Conclusions

PH₃ resistance in R. dominica was associated with reduced body weight, DLD P49S mutation and reduced DLD abundance, reduced COX, GST, and CE activities, and increased abundance of pentose phosphate pathway-related metabolites. These findings suggest that PH₃ resistance involves coordinated biochemical, proteomic, and metabolomic changes linked to energy and redox metabolism.

Graphical Abstract