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.

Low temperatures can induce adaptive responses in ectothermic animals, the grain pest Cryptolestes ferrugineus has exhibited a remarkable adaptability to cold environment. Mitochondrial protein-coding genes are crucial for maintaining respiration metabolism and ATP synthesis in organisms, and they play various functions in insects. However, the role of these genes in cold adaptation is poorly understood.

The purpose of this study is to elucidate the roles of mitochondrial protein-coding genes in the formation of cold tolerance of C. ferrugineus.

The cold tolerance of C. ferrugineus ST and CK populations was determined at a lethal low temperature (-20 ℃). The respiration rate and ATP content of C. ferrugineus ST and CK populations were measured using a CO₂ detector and an ATP content assay kit. The relative expression levels of 13 mitochondrial protein-coding genes between the ST and CK populations were assessed by using RT-qPCR. RNA interference (RNAi) technology was employed to knock down the key mitochondrial protein-coding genes ND6 and ATP6 in C. ferrugineus, and then the expression levels of the remaining 12 mitochondrial protein-coding genes, respiration rate, ATP content, and changes in cold tolerance were explored after effective silencing of ND6 and ATP6.

The cold tolerance of C. ferrugineus CK population was higher than that of the ST population, while the respiration rate and ATP content of the CK population were only 58.68% and 62.54% of those in the ST population, respectively. Additionally, the expression levels of 12 mitochondrial protein-coding genes (except ND3) in the CK population were significantly lower than those in the ST population. These results suggested a negative correlation between cold tolerance and physiological indicators (respiration rate, ATP content, and the expression levels of mitochondrial protein-coding genes). When the key mitochondrial protein-coding genes ND6 and ATP6 were effectively silenced via dsRNA feeding, the respiration rate and ATP content were significantly reduced, while cold tolerance in C. ferrugineus was significantly enhanced.

The mitochondrial protein-coding genes ND6 and ATP6 are involved in the formation of cold tolerance by regulating energy metabolism in C. ferrugineus.