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BACKGROUND: Environmental stress can induce oxidative stress in Apis cerana cerana, leading to cellular oxidative damage, reduced vitality, and even death. Currently, owing to an incomplete understanding of the molecular mechanisms by which A. cerana cerana resists oxidative damage, there is no available method to mitigate the risk of this type of damage. Cyclin plays an important role in cell stress resistance. The aim of this study was to explore the in vivo protection of cyclin H against oxidative damage induced by abiotic stress in A. cerana cerana and clarify the mechanism of action. We isolated and identified the AccCyclin H gene in A. cerana cerana and analysed its responses to different exogenous stresses. RESULTS: The results showed that different oxidative stressors can induce or inhibit the expression of AccCyclin H. After RNA‐interference‐mediated AccCyclin H silencing, the activity of antioxidant‐related genes and related enzymes was inhibited, and trehalose metabolism was reduced. AccCyclin H gene silencing reduced A. cerana cerana high‐temperature tolerance. Exogenous trehalose supplementation enhanced the total antioxidant capacity of A. cerana cerana, reduced the accumulation of oxidants, and improved the viability of A. cerana cerana under high‐temperature stress. CONCLUSION: Our findings suggest that trehalose can alleviate adverse stress and that AccCyclin H may participate in oxidative stress reactions by regulating trehalose metabolism. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The widespread use of glyphosate has many adverse effects on Apis cerana cerana. Due to the incomplete understanding of the molecular mechanisms of glyphosate toxicity, there are no available methods for mitigating the threat of glyphosate to Apis cerana cerana. Small heat shock proteins (sHSPs) play an important role in resisting oxidative stress, but their mechanism of action in Apis cerana cerana remains unclear. RESULTS: In this experiment, we cloned and identified AccsHSP21.7. Studies have shown that AccsHSP21.7 contains binding motifs for various transcription factors related to oxidative stress. Abiotic stresses induced the expression of AccsHSP21.7. Bacteriostatic testing of a recombinant AccsHSP21.7 protein proved that Escherichia coli overexpressing AccsHSP21.7 showed increased resistance to oxidative stress. Knocking down the AccsHSP21.7 gene caused significant damage to midgut cells, which seriously disrupted the antioxidant system in Apis cerana cerana and greatly increased mortality under glyphosate stress. CONCLUSION: This study investigated the relationship between antioxidant regulation and the AccsHSP21.7 gene at the molecular level, and the results have guiding significance for the improvement of stress resistance in Apis cerana cerana. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Cuticular proteins (CPs) are known to play important roles in insect development and defence responses. The loss of CP genes can lead to changes in insect morphology and sensitivity to the external environment. In this study, we identified the AccCPR2 gene, which belongs to the CPR family (including the R&R consensus motif) of CPs, and explored its function in the response of Apis cerana cerana to adverse external stresses. Our results demonstrated that AccCPR2 was highly expressed in the late pupal stage and epidermis, and the expression of AccCPR2 may be induced or inhibited under different stressors. RNA interference experiments showed that knockdown of AccCPR2 reduced the activity of antioxidant enzymes, led to the accumulation of oxidative damage and suppressed the expression of several antioxidant genes. In addition, knockdown of AccCPR2 also reduced the pesticide resistance of A. cerana cerana. The overexpression of AccCPR2 in a prokaryotic system further confirmed its role in resistance to various stresses. In summary, AccCPR2 may play pivotal roles in the normal development and environmental stress response of A. cerana cerana. This study also enriched the theoretical knowledge of the resistance biology of bees. [ABSTRACT FROM AUTHOR]

As molecular chaperones, DnaJs play critical roles in maintaining cytoplasmic structure and resisting various stresses. However, the functions of DnaJs in insects are poorly understood. In this study, we identified a DnaJC3 from Apis cerana cerana (AccDnaJC3) and investigated its roles in adverse conditions. Real-time quantitative PCR analysis showed that AccDnaJC3 was highly expressed in muscle and epidermis. In addition, AccDnaJC3 was induced by a variety of stresses, such as 4 °C, 24 °C, 44 °C, H 2 O 2 , HgCl 2 , VC, UV, cyhalothrin, abamectin and emamectin benzoate treatments, whereas it was inhibited by CdCl 2 and paraquat treatments. Disc diffusion experiments indicated that overexpression of recombinant AccDnaJC3 enhanced Escherichia coli tolerance to some stress conditions. In contrast to the control group, when AccDnaJC3 was knocked down with RNAi technology, several other antioxidant genes were downregulated, suggesting that AccDnaJC3 may play important roles in stress response. Furthermore, we found that the enzyme activities of superoxide dismutase, peroxidase and catalase were lower in AccDnaJC3 -knockdown bees than in control bees. Taken together, these results suggest that AccDnaJC3 may be involved in various stress responses in Apis cerana cerana. Unlabelled Image • A DnaJC3 gene from Apis cerana cerana was isolated and characterized. • The expression of AccDnaJC3 was induced by various environmental stresses. • AccDnaJC3 enhances the tolerance of Escherichia coli to stress conditions. • RNA interference experiments suggested that AccDnaJC3 is important for resistance against stresses. [ABSTRACT FROM AUTHOR]

Y-box binding protein 1 (YB-1) is a member of the cold shock domain protein superfamily and is involved in development, environmental stresses and DNA oxidative damage in many organisms. However, the precise functions of YB-1 are still not well understood in various insects, including bees. In the current study, we identified a YB-1 gene in Apis cerana cerana ( AccYB-1 ). The predicted cis-acting elements in the promoter sequence of AccYB-1 indicated its possible roles in development and stress responses. AccYB-1 expression was higher in one-day-old larvae and dark-eyed pupae than in other development stages. Tissue-specific expression analysis showed that the mRNA level of AccYB-1 was higher in the thorax and midgut than in other tissues. The results from real-time PCR showed that AccYB-1 was induced by many environmental stresses. Silencing AccYB-1 downregulated the transcriptional level of some growth- and development-related genes and antioxidant genes and decreased the enzyme activities of several antioxidant-related enzymes, further indicating a possible function of AccYB-1 in growth, development and stress responses. Taken together, our findings suggest that AccYB-1 may play an indispensable role in growth and development and environmental stress responses in Apis cerana cerana . To our knowledge, this is the first paper to explore the role of YB-1 in bees. [ABSTRACT FROM AUTHOR]

Previous studies examining the functions of cyclin-dependent kinases (CDKs) have mainly focused on the regulation of the cell cycle. Recent studies have found that cyclin-dependent kinase 7 (CDK7) and cyclin-dependent kinase 9 (CDK9) play important roles in cell stress, metabolism of toxic substances and maintaining the stability of the internal environment. Here, we found that under stress conditions, the transcription and protein expression of AccCDK7 and AccCDK9 were induced to varying degrees. Meanwhile, the silencing of AccCDK7 and AccCDK9 also affected the expression of antioxidant genes and the activity of antioxidant enzymes, and reduced the survival rate of bees under high temperature stress. Furthermore, the exogenous overexpression of AccCDK7 and AccCDK9 improved the viability of yeast under stress conditions. Therefore, AccCDK7 and AccCDK9 may play roles in A.cerana cerana resistance to oxidative stress caused by external stimuli, potentially revealing a new mechanism of the honeybee response to oxidative stress. • Isolation of AccCDK7 and AccCDK9 from Apis cerana cerana. • The expression of AccCDK7 and AccCDK9 is induced by oxidative stress. • AccCDK7 and AccCDK9 can enhance the stress resistance of E. coli and yeast. • RNA interference experiments suggested that AccCDK7 and AccCDK9 is important for resistance against stresses. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CarEs) play vital roles in metabolising different physiologically important endogenous compounds and in detoxifying various harmful exogenous compounds in insects. Multiple studies of CarEs have focused on pesticide metabolism in insects, while few studies have aimed to identify CarE functions in oxidative resistance, particularly in Apis cerana cerana . In this study, we isolated a carboxylesterase gene, esterase FE4, from Apis cerana cerana and designated it towards an exploration of its roles as an antioxidant and in detoxification. We investigated AcceFE4 expression patterns in response to various stressors. A quantitative real-time PCR analysis revealed that AcceFE4 was up-regulated by H 2 O 2 , imidacloprid, and paraquat, and was down-regulated by 4 °C, UV radiation, CdCl 2 , and HgCl 2 . Additionally, the protein expression of this gene was down-regulated at 4 °C and up-regulated by H 2 O 2 . Disc diffusion assays showed that the AcceFE4 recombinant protein-expressing bacteria had a smaller killing zone than the control group with the paraquat, HgCl 2 and cumyl hydroperoxide treatments. Moreover, when the gene was knocked down by RNA interference, we observed that multiple oxidant genes (i.e., AccSOD , AccGST , AccTrx , AccMsrA, and others) were down-regulated in the knockdown samples. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity levels were reduced in the knockdown samples relative to the control group. Finally, we measured the enzyme activity of carboxylesterase and found that the enzyme activity was also reduced in the silent samples. Together, these data suggest that AcceFE4 may be involved in the oxidative resistance response during adverse stress. [ABSTRACT FROM AUTHOR]

In eukaryotes, cytochrome c oxidase (COX) is a multimeric protein complex that is the last enzyme in the respiratory electron transport chain of mitochondria. Syntheses of cytochrome c oxidase (SCO) proteins are copper-donor chaperones involved in metalation of the CuA redox center of COX. However, its other precise actions are not yet understood. Here, we report the characterization of AccSCO2 from Apis cerana cerana ( Acc ). Our data showed that AccSCO2 expression was induced by cold (4 °C), CdCl 2 , HgCl 2 , ultraviolet (UV) light, and H 2 O 2 and was inhibited by different pesticide treatments. In addition, a disc diffusion assay of recombinant AccSCO2, AccSCO2-R1, and AccSCO2-R2 proteins showed that they played a functional role in protecting cells from oxidative stress involved in copper-dependent manner. Further, following knockdown of AccSCO2 in A. cerana cerana using RNA interference (RNAi), the expression levels of most antioxidant genes ( AccGSTD , AccGSTO1 , AccGSTS4 , AccSOD1 , AccSOD2 , etc.) were significantly decreased in the AccSCO2 -silenced bees compared with the control bees. Moreover, the antioxidant enzymatic activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were all lower in the silenced bees than in the control bees. Finally, the in vivo activity of COX was measured after AccSCO2 knockdown, which revealed a strong reduction in COX activity in the silenced bees. Thus, we hypothesize that AccSCO2 plays important roles in cellular stress responses and anti-oxidative processes, which help to regulate the production of mitochondrial reactive oxygen species and/or the impairment of mitochondrial activity under oxidative stress. [ABSTRACT FROM AUTHOR]

Vitellogenin (Vg) is a yolk precursor protein in most oviparous females. However, Vg has not been studied in the Apis cerana cerana. In this work, the Vg gene of the A. cerana cerana has been cloned and sequenced. The gene codes for a protein consisting of 1770 amino acids in seven exons with a predicted molecular mass and isoelectric point of 200 kDa and 6.46, respectively. Additionally, an 807-bp 5′-flanking region was isolated, and potential transcription factor binding sites associated with development and stress response were identified. Quantitative real-time PCR analysis showed that AccVg is highly expressed in pupae during different developmental stages. In addition, the expression of AccVg could be induced by cold (4 and 16 °C), CdCl, and pesticide treatment. Real-time quantitative PCR (RT-PCR) and western blot analyses indicated that AccVg transcription was induced by various abiotic stresses. Western blot was used to measure the expression levels of AccVg protein. Taken together, these results suggest that AccVg most likely plays essential roles in antioxidant defence and that it may be of critical importance to honey bee survival. [ABSTRACT FROM AUTHOR]

Insect cytochrome P450 monooxygenases (P450s or CYPs) perform important functions in the metabolic detoxification of both endogenous and exogenous substrates. However, the mechanism of action of the P450 genes in bees is unclear. In this study, we investigated the effects of AccCYP6k1 on the metabolism and detoxification of Apis cerana cerana. Spatiotemporal expression profiling revealed that the expression of AccCYP6k1 was the highest in foragers (A15) and was mainly expressed in the leg, midgut and head. RT–qPCR results showed that AccCYP6k1 exhibited different expression patterns following exposure to xenobiotics. In addition, silencing AccCYP6k1 increased the pesticides sensitivity and affected the detoxification system and antioxidant process of A. cerana cerana. In brief, the induced expression of AccCYP6k1 is related to the resistance of A. cerana cerana , while knockdown AccCYP6k1 affect the pesticides resistance and metabolic detoxification system of A. cerana cerana. These findings not only support the theoretical basis of metabolic detoxification in bees but also provide a better understanding of P450-mediated resistance to pesticides in insects. [Display omitted] • The level of inducible expression of AccCYP6k1 is related to pesticide resistance. • Silencing AccCYP6k1 increased the sensitivity of Apis cerana cerana to abamectin and imidacloprid. • Silencing AccCYP6k1 had adverse effects on the detoxification system of Apis cerana cerana. [ABSTRACT FROM AUTHOR]

Tyrosine aminotransferase (TATN) is the first enzyme involved in the metabolic degradation of tyrosine, and it plays an important role in tyrosine detoxification and helps the body resist oxidative damage. However, the function of TATN in Apis cerana cerana (A. c. cerana) remains unclear. To explore the role of TATN in the response to pesticide and heavy metal stress in A. c. cerana , AccTATN was isolated and identified. AccTATN was highly expressed in the integument and the adult stage. Exposure to multiple pesticides and heavy metal stress upregulated AccTATN expression. RNA interference experiments showed that silencing AccTATN reduced the resistance of A. c. cerana to glyphosate and avermectins stress. The expression of antioxidant-related genes and the activity of antioxidant enzymes were reduced after AccTATN was silenced, leading to the accumulation of oxidative damage. Overexpression of the recombinant AccTATN protein in a prokaryotic system also confirmed its role in heavy metal stress and improved antioxidant capacity. Our study showed that AccTATN may promote resistance to pesticide and heavy metal stress by regulating the antioxidant capacity of A. c. cerana. This study provides a valuable theoretical basis for A. c. cerana conservation. [Display omitted] • AccTATN gene was isolated and characterized from (A. c. cerana) • Pesticide and heavy metal stress can affect the expression of AccTATN. • AccTATN overexpression enhanced the tolerance of E. coli cells to oxidative stress. • AccTATN knockdown disrupted the original redox balance and caused oxidative stress. • AccTATN promotes A. c. cerana to resist pesticide and heavy metal stress. [ABSTRACT FROM AUTHOR]

Activating transcription factor 2 (ATF2), a basic leucine zipper (bZIP) transcription factor, plays a crucial role in immune and DNA damage response in mammals. However, the function of ATF2 in insects remains unknown. Here, we isolated the ATF2 gene from Apis cerana cerana (AccATF2) and found that AccATF2 was a main regulator of the honeybee response to oxidative stress. Our results showed that AccATF2 was highly expressed in the head, thorax and integument. AccATF2 was expressed throughout the development period of honeybees, and the highest AccATF2 transcript level was noted in brown-eyed pupae, indicating its indispensable roles in honeybee survival. Antioxidant function analysis showed that AccATF2 expression was markedly induced in response to oxidative stress caused by various environmental stresses. AccATF2 overexpression substantially enhanced the tolerance to oxidative stress of Escherichia coli cells compared with control cells. AccATF2 knockdown significantly increased the production of malondialdehyde (MDA), the transcription of antioxidant genes and the activity of antioxidant enzymes in honeybees, suggesting that AccATF2 knockdown resulted in oxidative damage to honeybees. Moreover, AccATF2 knockdown decreased honeybee resistance to oxidative stress caused by high temperature. Overall, AccATF2 plays an important role in maintaining redox homeostasis and protecting honeybees from oxidative stress caused by various environmental stimuli. Our discoveries add to a growing understanding of how honeybees cope with various adverse environmental conditions to ensure their survival. [Display omitted] • AccATF2 gene was isolated and characterized from Apis cerana cerana. • The expression of AccATF2 was induced by oxidative stress caused by adverse environment. • AccATF2 overexpression enhanced the tolerance of E. coli cells to oxidative stress. • AccATF2 knockdown disrupted the original redox balance and caused oxidative stress. [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) play an important role in protecting against stress-induced cell damage and fundamental physiological processes. In this study, we identified an intronless sHsp gene from Apis cerana cerana ( AccsHsp22.6 ). The open reading frame of AccsHsp22.6 was 585 bp and encoded a 194 amino acid protein. Furthermore, a 2064 bp 5'-flanking region was isolated, and potential transcription factor binding sites associated with development and stress response were identified. Quantitative PCR and western blot analyses demonstrated that AccsHsp22.6 was detected at higher levels in the midgut than in other tissues tested, and it is highly expressed during the shift to different development stages. Moreover, AccsHsp22.6 was significantly up-regulated by abiotic and biotic stresses, such as 4 °C, 16 °C, 42 °C, cyhalothrin, pyridaben, H 2 O 2 , UV, CdCl 2 , 20-hydroxyecdysone and Ascosphaera apis treatments. However, AccsHsp22.6 was slightly repressed by other stresses, including 25 °C, phoxim, paraquat and HgCl 2 treatments. The recombinant AccsHSP22.6 also exhibited significant temperature tolerance, antioxidation and molecular chaperone activity. In addition, we found that knockdown of AccsHsp22.6 by RNA interference remarkably reduced temperature tolerance in A . cerana cerana . Taken together, these results suggest that AccsHsp22.6 plays an essential role in the development stages and defence against cellular stress. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase (CDK) protein family plays an important role in regulating life functions, such as the cell cycle and metabolism. This study reports the first cloning and functional analysis of A. cerana cerana CDK1 (AccCDK1). The distribution profile of AccCDK1 in different developmental periods and different tissues was determined. The experimental results showed that the distribution of AccCDK1 was tissue-specific. AccCDK1 distribution at the transcriptional and translational levels was affected by stress conditions induced by H 2 O 2 , UV, HgCl 2 , CdCl 2 , extreme temperatures (4 °C, 44 °C) and pesticides (avermectin, lambda-cyhalothrin, haloxyfop- R -methyl, and glyphosate), which resulted in changes in the expression levels. These results suggest that AccCDK1 may have an important part to play in honey bee resistance to stress. The expression of a recombinant AccCDK1 protein in vitro enhanced the antistress capacities of E. coli and yeast, which suggests that AccCDK1 is related to the stress response. When AccCDK1 was silenced, the expression of some antioxidant genes was downregulated, and the enzymatic potencies of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were reduced, which suggests that AccCDK1 takes part in the body's resistance to oxidative stress upon external stimulation by influencing relevant antioxidants. Notably, the survival rate of A. cerana cerana under high-temperature-induced stress decreased after AccCDK1 silencing, which verifies our results. In conclusion, we found that AccCDK1 played an indispensable function in resisting oxidative stress and maintaining normal cellular functions. [Display omitted] • Oxidative stress can affect the expression of cyclin-dependent kinase1. • AccCDK1 silencing affects cellular antioxidant genes and enzymes. • AccCDK1 promotes the survival of eukaryotic and prokaryotic cells under stress. • AccCDK1 promotes bees to resist high temperature stress. [ABSTRACT FROM AUTHOR]

Abstract: Glutaredoxins (Grxs) and thioredoxins (Trxs) play important roles in maintaining intracellular thiol-redox homeostasis by scavenging reactive oxygen species. However, few Grxs and Trxs have been functionally characterized in Apis cerana cerana. In this study, we identified three genes, AccGrx1, AccGrx2, and AccTrx1, and investigated their connection to antioxidant defense. AccGrx1 and AccGrx2 were mainly detected in dark-eyed pupae, whereas AccTrx1 was highly concentrated in 15-day postemergence adults. The expression levels of AccGrx1 and AccTrx1 were the highest in fat body and epidermis, respectively. However, the expression level of AccGrx2 was the highest in muscle, followed by the epidermis. AccGrx1, AccGrx2, and AccTrx1 were induced by 4, 16, and 42°C; H2O2; and pesticide (acaricide, paraquat, cyhalothrin, and phoxime) treatments and repressed by UV light. AccGrx1 and AccGrx2 were upregulated by HgCl2 treatment, whereas AccTrx1 was downregulated. We investigated the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 in A. cerana cerana and surprisingly found that knockdown of the these four genes enhanced the enzymatic activities of CAT and POD; the metabolite contents of hydrogen peroxide, carbonyls, and ascorbate; and the ratios of GSH/GSSG and NADP+/NADPH. In addition, we also analyzed the transcripts of other antioxidant genes and found that some were upregulated and others were downregulated, revealing that the upregulated genes may be involved in compensating for the knockdown of AccGrx1, AccGrx2, AccTpx-3, and AccTrx1. Taken together, these results suggest that AccGrx1, AccGrx2, AccTpx-3, and AccTrx1 may play critical roles in antioxidant defense. [Copyright &y& Elsevier]

• AccT5H-1 gene from Apis cerana cerana was isolated and characterized. • AccT5H-1 knockdown caused oxidative stressors of Apis cerana cerana. • The mRNA level of AccT5H-1 was induced by various environmental stressors. • Adding 10 µg/mL of melatonin to the diet could improve the cold resistance of bee. Tryptophan-5-hydroxylase-1 (T5H-1) is the rate-limiting enzyme in the biosynthesis of serotonin, which is involved in the biosynthesis of melatonin (Mel). Mel, a biological hormone, plays crucial roles in stressors tolerance, such as cold, hot, Ultraviolet (UV) and pesticide tolerance. However, the direct correlation between T5H-1 and Mel and the underlying mechanism in organisms remains elusive. Mel-mediated cold tolerance was studied extensively in plants and somewhat in insects, including bees. The present study isolated the Mel synthesis gene T5H-1 from Apis cerana cerana for the first time. qRT-PCR analysis indicated that AccT5H-1 played vital roles during some adverse conditions, including 4 °C, 8 °C, 10 °C, 45 °C, UV, cyhalothrin, abamectin, paraquat and bifenthrin exposure. Knockdown of AccT5H-1 using RNA interference (RNAi) technology upregulated most antioxidant genes. Additionally, an enzyme activity assay revealed higher contents of Malondialdehyde (MDA) and Hydrogen peroxide (H 2 O 2), lower content of Vitamin C (VC), and higher activities of Glutathione S-transferase (GST), Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD) in the AccT5H-1 silenced group than the control group. These results suggest that AccT5H-1 is involved in the response to different oxidative stressors in A. cerana cerana. The survival rate of A. cerana cerana exposed to low temperature treatment revealed that the optimal concentration of Mel in the diet was 10 µg/mL. We also found that the antioxidant enzyme (GST, SOD, POD and CAT) concentrations at 10 µg/mL Mel increased to different degrees, and the content of oxidizing substances (MDA and H 2 O 2) decreased, the content of VC increased, and the content of substances that promote cold resistance (glycerol and glycogen) increased. Mel increased the resistance of A. cerana cerana exposed to low temperatures. The expression of AccT5H-1 decreased after the feeding of exogenous Mel to bees. These results provide a reference for other insect studies on Mel and T5H-1. [ABSTRACT FROM AUTHOR]