Rice is a staple food for over half of the global population, and the postharvest quality deterioration of paddy rice is closely linked to lipid degradation. Low-temperature storage represents an effective strategy for maintaining rice quality and achieving green storage. However, the intrinsic mechanisms by which prolonged low-temperature storage coordinately regulates rice lipid metabolism at the level of metabolite dynamics and gene expression networks to maintain its stability have not been fully elucidated.

This study aimed to integrate multi-omics technologies to systematically elucidate biochemical and molecular mechanisms underlying lipid stability in japonica rice during long-term low-temperature storage.

Fresh Nanjing 46 paddy rice was stored at 25 and 15 ℃ for 360 days, with sampling every 30 days. An integrated approach combining physiological and biochemical analyses, lipidomics, and transcriptomics was employed to systematically investigate stabilization mechanisms.

Low-temperature storage effectively maintained rice lipid stability through a multi-layered regulatory network. Regarding membrane lipid metabolism, low-temperature storage downregulated PLDα1, thereby delaying the hydrolysis of phospholipids, including phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine, and helping to maintain cellular membrane integrity. Additionally, reduced expression of OsCDase limited sphingolipid degradation, further enhancing plasma membrane stability. In terms of lipid hydrolysis, lipase activity was suppressed under low-temperature conditions, inhibiting triglyceride hydrolysis. In oxidative metabolic pathways, the downregulation of OsFAD2 and ACX1 genes inhibited polyunsaturated fatty acid synthesis and β-oxidation, thus alleviating oxidative stress. Reduced lipoxygenase (LOX，Lipoxygenase) activity at low temperatures further mitigated the oxidation of unsaturated fatty acids, thereby minimizing off-flavor formation.

During rice storage, lipid hydrolysis served as a critical precursor to oxidation, with both processes jointly determining quality deterioration. Low-temperature storage simultaneously inhibited lipid hydrolysis and oxidation pathways, consequently maintaining lipid compositional stability at the metabolomic level and delaying quality decline at the phenotypic level.

Glutathione S-transferases (GSTs) are key detoxification enzymes in insects and play important roles in the development of insect tolerance to chemical insecticides. Ethyl formate (EF), characterized by high efficacy, low toxicity, and low residue, is regarded as a promising green fumigant for stored-grain pest control.

To elucidate the molecular mechanisms underlying pest tolerance to EF, this study focuses on the important stored-grain pest Cryolestes ferrugineus, aiming to analyze the relationship between GST genes (CfGSTe1 and CfGSTd) and EF tolerance.

The present study conducted bioassays to determine the susceptibility of C. ferrugineus with three different levels of phosphine resistance to EF. Through synergistic assays with diethyl maleate (DEM), the potential enhancement of EF fumigation efficacy was evaluated, and the effects of EF treatment on GST activity were analyzed. According to the previous transcriptome data of C. ferrugineus, two key GST genes (CfGSTe1 and CfGSTd1) were identified and subjected to amino acid sequence and phylogenetic analyses. The temporal and spatial expression patterns of these two genes, as well as their transcriptional responses to EF fumigation stress, were further analyzed by using real-time quantitative PCR (RT-qPCR). Finally, the effects of CfGSTe1 and CfGSTd1 on EF tolerance were analyzed by individually silencing these genes using RNA interference (RNAi) technology.

Bioassay results showed that C. ferrugineus with varying levels of phosphine resistance exhibited no significant differences in sensitivity to EF, confirming the absence of cross-resistance between the two fumigants. The synergist DEM significantly enhanced the fumigant toxicity of EF, and the GST activity in insects was markedly increased under EF stress, suggesting that GSTs play an important role in the detoxification metabolism of EF. Sequence and phylogenetic analyses indicated that CfGSTe1 and CfGSTd1 encode 216 and 215 amino acids, respectively, both containing conserved GST catalytic sites and belonging to the Epsilon and Delta families. The RT-qPCR results indicated that both genes were highly expressed at the adult stage, primarily in the midgut, fat body, and Malpighian tubules, and could be significantly induced by EF exposure. After effectively silencing CfGSTe1 and CfGSTd1 via RNAi, the tolerance of C. ferrugineus to EF was significantly reduced, as evidenced by markedly increased adult mortality following fumigation.

The CfGSTe1 and CfGSTd1 may play important roles in the detoxification metabolism of EF in C. ferrugineus, suggesting a close association with insect tolerance to this fumigant.

Rice grain undergoes various physiological and biochemical changes during long-term storage, impacting both stability and quality. Those factors contribute to rice grain quality deterioration during its storage stage, such as lipid oxidation, starch degradation, protein modifications, membrane homeostasis imbalance, and oxidative stress collectively. However, the molecular mechanisms underlying these changes remain elusive.

This study aimed to analyze differentially expressed genes (DEGs) in stored rice grian, construct a co-expression network, identify core genes using WGCNA and explore regulatory mechanisms associated with rice storage stability.

Transcriptomic analysis was conducted on Japonica rice (Nanjing 46) grain stored for different durations (0, 3, 6, 9 and 12 months) to obtain gene expression profiles. WGCNA was employed to identify highly variable genes during storage, construct a weighted gene co-expression network, and identify storage-time-specific modules. Core genes screening was based on network connectivity, followed by functional enrichment analysis and physiological and biochemical assays to explore their potential roles in storage quality regulation.

A total of 9 050 DEGs were identified, with 8 654 showing variations across storage stages, and 396 were expressed consistently across all time points. WGCNA identified 17 gene co-expression modules, of which four showed strong associations with storage duration. Connectivity analysis further highlighted key genes with regulatory potential：OsOLE4 and OsCDAP3, involved in lipid metabolism; OsLEA32, OsAGP24 and OsRHD3 associated with maintaining cellular stability; OsERF064 linked to the ethylene signaling pathway and OsEMF2a, an epigenetic regulator. Additionally, five candidate genes lacking functional annotation were identified for further study.

This study systematically analyzed the molecular regulatory network of rice grain storage using transcriptomics and WGCNA, revealing that rice grain adapts to storage environments through multi-level gene regulatory mechanisms. Core genes within specific modules played pivotal roles in antioxidant activity, nutrient metabolism, membrane stability, and cellular function maintenance. These findings provided a biological basis for delaying rice quality deterioration and offered potential genetic resources for improving rice grain storage stability.

Heat shock proteins (Hsps) are essential molecular chaperones in organisms and play the crucial roles in resisting adverse environmental stresses. Cryptolestes ferrugineus is a cosmopolitan pest of stored grains with strong environmental adaptability. This study aims to elucidate the crucial roles of heat shock protein genes CfHsp70-1 and CfHsp70-2 in the development of high-temperature tolerance in this pest.

C. ferrugineus was acclimated to sub-lethal temperatures (37 and 42 ℃) for 2 h to examine the tolerance changes to lethal high temperature (50 ℃). Two key heat shock protein genes (CfHsp70-1 and CfHsp70-2) were identified based on the transcriptome data of C. ferrugineus, and the amino acid sequences and phylogenetic analysis of Hsp70 proteins were further conducted. The quantitative real-time PCR method was employed to analyze the expression patterns of CfHsp70-1 and CfHsp70-2 in response to sub-lethal heat stress. The RNA interference (RNAi) technology was used to silence CfHsp70-1 and CfHsp70-2, and then the changes in high-temperature tolerance of C. ferrugineus under different conditions were analyzed.

The median lethal time (LT₅₀) of different C. ferrugineus populations was significantly increased under lethal heat temperature conditions (50 ℃) after acclimation of insects to sub-lethal temperatures (37 and 42 ℃) for 2 h, indicating a substantial enhancement of the high-temperature tolerance. The further sequence and phylogenetic analysis revealed that the amino acid sequences of CfHsp70-1 and CfHsp70-2 contained three conserved Hsp70 family signature motifs, and they clustered together with Hsp70 proteins of other Coleoptera insects. The results of quantitative real-time PCR analysis suggested that the expression levels of heat shock protein genes CfHsp70-1 and CfHsp70-2 were significantly up-regulated after acclimation to 37 and 42 ℃ for 2 h in C. ferrugineus. The gene functional analysis revealed that the high-temperature tolerance of C. ferrugineus was significantly reduced after the effective silencing of CfHsp70-1 and CfHsp70-2 via RNAi, that is, the mortality of the test insects at 50 ℃ increased significantly.

The heat shock protein genes CfHsp70-1 and CfHsp70-2 are involved in enhancing the high-temperature tolerance after heat acclimation of C. ferrugineus.

Cuticle proteins (CPs) are the main components of insect cuticle, and numerous studies have confirmed that the CP genes were involved in insecticide resistance. The objective of this study is to clarify the roles of TcCP14.6 (cuticle protein CP14.6) and TcLCPA3A (larval cuticle protein A3A) in phosphine resistance of Tribolium castaneum.

The FAO (Food and Agriculture Organization of the United Nations)-recommended bioassay method was used to determine the phosphine resistance levels of five different T. castaneum populations. The TcCP14.6 and TcLCPA3A sequences were downloaded from the T. castaneum genome data, and their encoded amino acid sequences, signal peptides and conserved domains were predicted via the online services. The total RNAs were extracted from different tissues (the cuticles of head, thorax and abdomen, wing, leg, gut, Malpighian tubules and fat body), different phosphine resistance levels, as well as phosphine induction of T. castaneum, respectively. With TcRPS and TcRPL as internal reference genes, the RT-qPCR was used to analyze the expression patterns of TcCP14.6 and TcLCPA3A in different tissues, different phosphine resistance levels, and in response to phosphine induction. Lastly, the RNA interference (RNAi) technology and bioassay method were used to explore the relationship between the two CP genes and phosphine resistance in T. castaneum.

The bioassay analysis showed that Jiangsu (JS, RR=1.7) and Yunnan (YN, RR=3.0) belonged to susceptible populations, Hunan (HN, RR=20.2) belonged to moderately resistant population, Sichuan (SC, RR=395.4) and Guangdong (GD, RR=862.7) belonged to highly resistant populations. The sequence analysis demonstrated that both TcCP14.6 and TcLCPA3A proteins consisted of signal peptides and chitin binding domains. The RT-qPCR analysis suggested that TcCP14.6 and TcLCPA3A all had a higher expression in the peripheral tissues (the cuticles of head, thorax and abdomen, wings and legs) of T. castaneum, and with a lower expression in the internal tissues, such as fat body, gut and Malpighian tube. Besides, with the increase of phosphine resistance levels in T. castaneum, the expression level of TcCP14.6 was up-regulated, while the expression level of TcLCPA3A was down-regulated. After phosphine induction for 6 h in T. castaneum, the expression levels of TcCP14.6 and TcLCPA3A were up-regulated and down-regulated, respectively. The injection of dsRNA could significantly inhibit the expression of TcCP14.6 and TcLCPA3A in phosphine resistance (GD) and susceptible (YN) populations of T. castaneum. When treated with phosphine (LC₃₀), the mortality significantly increased after the TcCP14.6 was silenced. Instead, the mortality significantly decreased after the TcLCPA3A was silenced.

The two CP genes TcCP14.6 and TcLCPA3A are involved in phosphine resistance of T. castaneum.

As an important structural component of insect cuticle, the cuticle protein (CP) plays an important role in the formation of cuticle penetration resistance to pesticides. The phosphine resistance of Cryptolestes ferrugineus is increasingly prominent, and the current study was conducted to reveal the roles of CP genes in the formation of phosphine resistance in C. ferrugineus.

According to the phosphine bioassay method that recommended by the Food and Agriculture Organization of the United Nations (FAO), the difference in phosphine sensitivity from five geographical populations (Zhangjiagang, Xiangyin, Huaian, Huaihua and Taicang populations) of C. ferrugineus was analyzed. The four CP genes were identified from the previous transcriptome data of C. ferrugineus, and then the phylogenetic tree of CPs was constructed and the corresponding amino acid sequence of C. ferrugineus CPs was further analyzed. Afterwards, the RT-qPCR was used to analyze the spatio-temporal (different developmental stages and different tissues of adults) expression patterns of four CP genes, and their expression levels under different phosphine resistance levels, as well as the expression patterns of four CP genes in response to phosphine stress were explored. Subsequently, a specific CP gene (CfRR2-1) was selected to be knocked down by using RNAi (RNA interference) technology, and the change of phosphine sensitivity of C. ferrugineus was determined.

The results of phosphine sensitivity bioassay analysis showed that there were significant differences in phosphine resistance levels of different geographical populations, and the range of insecticide resistance ratio (RR) was 7.2-1 906.8. The further sequence analysis suggested that the four CPs all contained chitin binding domain, which belonged to the RR2 subfamily of CPR family, and they were named as CfRR2-1, CfRR2-2, CfRR2-3 and CfRR2-4, respectively. The gene expression patterns demonstrated that four CP genes were specifically highly expressed in the pupal stage of C. ferrugineus, and the high expression levels of four CP genes were detected in the peripheral tissues of C. ferrugineus as well. Besides, the CP genes were highly expressed in the phosphine resistant population (Taicang population, RR=1 906.8), and their expression levels could be significantly induced by phosphine in C. ferrugineus. Lastly, a CP gene CfRR2-1 was selected for the further functional study. After the gene expression level of CfRR2-1 was significantly knocked down in phosphine resistance (TC) population of C. ferrugineus via the injection of dsRNA, the sensitivity of C. ferrugineus to phosphine was significantly increased.

The over-expression of CP gene is involved in the formation of phosphine resistance.

Mitochondria is an important organelle in the organism, which is the primary site for cellular oxygen consumption and the production of the energy substance adenosine triphosphate (ATP), playing a significant role in the organism’s resistance to adversity. The rusty grain beetle (Cryptolestes ferrugineus) is a type of global stored grain pest, possessing extremely strong environmental adaptability.

The objective of this study is to analyze the respiratory rate of C. ferrugineus and the response of mitochondrial protein-coding genes to different environmental stresses, and to investigate the stress response of mitochondria in the adversity resistance of C. ferrugineus.

Mitochondrial protein-coding genes were identified based on the mitochondrial genome data of C. ferrugineus, and corresponding real-time fluorescence quantitative PCR (RT-qPCR) primers were designed. The toxicity regression equation and LC₃₀ of C. ferrugineus to fumigants (ethyl formate), botanical insecticides (rotenone), and stored grain protectants (avermectin) were determined by using bioassay methods, and these concentrations were used for subsequent drug stress treatment on the test insects. The spatial and temporal expression patterns (different developmental stages and different tissues of larvae) of mitochondrial protein-coding genes in C. ferrugineus were analyzed by using RT-qPCR technology. Finally, the changes in the respiratory rate of C. ferrugineus under various adversity stresses such as high temperatures (35 and 40 ℃), ethyl formate, rotenone, avermectin, and starvation, as well as the expression patterns of mitochondrial protein-coding genes, were studied by using a CO₂ detector and RT-qPCR technology, respectively.

Twelve mitochondrial protein-coding genes (excluding nad6) quantitative primers were designed. RT-qPCR results showed that these mitochondrial protein-coding genes had a higher expression level at the 3rd instar larval stage, and mitochondrial genes were specifically highly expressed in the Malpighian tubules of 3rd instar larvae. Moreover, under high-temperature stress, the respiratory rate of C. ferrugineus significantly increased, and the expression levels of mitochondrial protein-coding genes nad2, cytb, and cox2 increased significantly, while nad4, nad4L, cox3, and atp6 showed a significant downregulation trend. Under ethyl formate fumigation stress, the respiratory rate of C. ferrugineus significantly decreased, and all 12 mitochondrial protein-coding genes were significantly downregulated. Among them, the expression levels of nad4L and nad5 were only 3.48% and 1.91% of the control group, respectively. Under rotenone and avermectin stress, the respiratory rate of C. ferrugineus significantly decreased, and the expression levels of mitochondrial protein-coding genes, except for cox2, were significantly downregulated. Under starvation stress, the respiratory rate of C. ferrugineus significantly decreased, and as the stress duration increased, the downregulation of mitochondrial encoded gene expression levels became more pronounced.

The respiratory metabolism rate and mitochondrial protein-coding gene expressions of C. ferrugineus changed significantly under different environmental stresses, indicating that the mitochondria plays an important role in the adaption to high temperature, pesticides, and starvation stress in C. ferrugineus.

Cryptolestes ferrugineus is one of the most economically important stored-grain pests, and its phosphine resistance is particularly prominent. Mitochondria are important organelles in living organisms, which are the core site for insects to undergo respiratory metabolic reactions. Mitochondrial protein-coding genes (PCGs) are involved in regulating physiological processes of insects, such as respiratory rate, energy metabolism, and cell signal transduction.

The objective of this study is to clarify the roles of mitochondrial PCGs in phosphine resistance of C. ferrugineus.

The respiratory rates of different phosphine resistant populations of C. ferrugineus were measured by using a CO₂ detector. The Taicang and Shanghai populations of C. ferrugineus with greatest differences in phosphine resistance levels and respiratory rates were selected to analyze the expression patterns of mitochondrial PCGs by using RT-qPCR technology, and the activities of mitochondrial complexes I and V were measured as well. The expression levels of three key mitochondrial PCGs including Nad5, Nad6 and Atp6, and the activities of mitochondrial complex I and V were determined after phosphine fumigation treatments in C. ferrugineus. RNA interference (RNAi) was used to silence Nad5, Nad6 and Atp6, and then the changes of respiratory rate and phosphine sensitivity of C. ferrugineus were analyzed.

There was a negative correlation between the respiratory rate and phosphine resistance levels of C. ferrugineus, that is, the respiratory rates significantly decreased with the increase of phosphine resistance levels. The RT-qPCR results showed that the expression levels of mitochondrial PCGs in the highly phosphine resistant population (Taicang population, RR=1 906.8) of C. ferrugineus were significantly lower than those in the relatively sensitive population (Shanghai population, RR=1.4), and the enzyme activities of mitochondrial complexes I and V were consistent with the expression patterns of mitochondrial PCGs. The expression levels of three key mitochondrial PCGs, Nad5, Nad6 and Atp6, and the activities of mitochondrial complexes I and V were significantly inhibited after phosphine fumigation treatments in C. ferrugineus. The key mitochondrial PCGs, Nad5, Nad6 and Atp6 were silenced by injecting dsRNA, which resulted in a significant decrease in respiratory rate and phosphine sensitivity of C. ferrugineus.

The mitochondrial PCGs are involved in phosphine resistance of C. ferrugineus.