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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., (© 2022 Royal Entomological Society.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Odorant-binding proteins (OBPs) play an important role in odour perception and transport in insects. However, little is known about whether OBPs perform other functions in insects, particularly in Apis cerana cerana. Within this study, an OBP gene (AccOBP10) was isolated and identified from A. c. cerana. Both homology and phylogenetic relationship analyses indicated that the amino acid sequence of AccOBP10 had a high degree of sequence identity with other members of the gene family. Analysis of quantitative real-time PCR (qRT-PCR) showed that AccOBP10 mRNA was expressed at higher levels in the venom gland than in other tissues. The mRNA transcript expression of AccOBP10 was upregulated by low temperature (4°C), hydrogen peroxide (H2O2), pyridaben, methomyl and imidacloprid but downregulated by heat (42°C), ultraviolet light, vitamin C, mercuric chloride, cadmium chloride, paraquat and phoxim. Expression of AccOBP10 under abiotic stress was analysed by western blotting, and the results were consistent with those of qRT-PCR. And as a further study of AccOBP10 function, we demonstrated that knockdown of AccOBP10 by RNA interference could slightly increase the expression levels of some stress-related genes. Collectively, these results suggest that AccOBP10 is mainly involved in the response to stress conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

N-Terminal asparagine amidohydrolase is a component of the ubiquitin-dependent N-end rule pathway of protein degradation that has been implicated in a variety of physiological functions, including the sensing of heme, oxygen, nitric oxide, selective elimination of misfolded proteins and the repair of DNA. We identified the Apis cerana cerana N-terminal asparagine amidohydrolase (AccNtan1) gene from A. cerana cerana and investigated its role in oxidation resistance. Multiple sequence alignments and phylogenetic analysis revealed that N-terminal asparagine amidohydrolase is highly conserved in insect species. Quantitative real-time polymerase chain reaction analysis indicated that the expression levels of AccNtan1 were significantly lower in the wing, honey sac and abdomen than in other tissues and were significantly higher in early stages of development, including the larva, prepupa and pink-eyed pupa stages, than in later stages. We further observed that AccNtan1 expression was induced by several environmental stressors, including aberrant temperature, H2O2, UV, heavy metals and pesticides. Moreover, a bacteriostatic assay suggested that overexpression of AccNtan1 enhances the resistance of bacteria to oxidative stress. In addition, knockdown of AccNtan1 using RNA interference significantly affected the expression levels of most antioxidant genes and the activity levels of several antioxidant enzymes. Thus, we hypothesize that AccNtan1 plays important roles in environmental stress responses and antioxidative processes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

MAP kinase phosphatase 3 (MKP3), a member of the dual-specificity protein phosphatase (DUSP) superfamily, has been widely studied for its role in development, cancer, and environmental stress in many organisms. However, the functions of MKP3 in various insects have not been well studied, including honeybees. In this study, we isolated an MKP3 gene from Apis cerana cerana and explored the role of this gene in the resistance to oxidation. We found that AccMKP3 is highly conserved in different species and shares the closest evolutionary relationship with AmMKP3. We determined the expression patterns of AccMKP3 under various stresses. qRT-PCR results showed that AccMKP3 was highly expressed during the pupal stages and in adult muscles. We further found that AccMKP3 was induced in all the stress treatments. Moreover, we discovered that the enzymatic activities of peroxidase, superoxide dismutase, and catalase increased and that the expression levels of several antioxidant genes were affected after AccMKP3 was knocked down. Collectively, these results suggest that AccMKP3 may be associated with antioxidant processes involved in response to various environmental stresses.

Inositol phosphate synthase (IPS) is a rate-limiting enzyme in myo-inositol biosynthesis, which can regulate stress responses in plants and animals. However, there are few studies on the function of IPS in insects, especially in Apis cerana cerana. In this study, the inositol-3-phosphate synthase 1-B gene (AccIPS1-B) was isolated from Apis cerana cerana, and its connection to antioxidant defence was investigated. The open reading frame of AccIPS1-B was 1542 bp, encoding a 513 amino acid polypeptide. Quantitative real-time PCR analysis revealed that the expression level of AccIPS1-B was highest in pupae of Apis cerana cerana, and it was expressed at higher levels in the thorax than in other tissues tested. Moreover, the expression of AccIPS1-B was significantly upregulated by abiotic stresses. The recombinant AccIPS1-B also displayed significant tolerance to cumene hydroperoxide and HgCl 2 . In addition, knockdown of AccIPS1-B significantly suppressed the expression of most of the antioxidant genes and decreased the antioxidant enzymatic activities of SOD, POD, and GST. Taken together, these findings indicate that AccIPS1-B may be involved in the response to antioxidant defence and development in Apis cerana cerana.

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., (Copyright © 2019. Published by Elsevier Inc.)

Clip-domain serine proteases (Clip-SPs) mediate innate immunity and embryonic development in insects. However, the function of Clip-SPs in Apis cerana cerana is little known. Here, a Clip-SP gene, AccSp1, was identified. AccSp1 was mainly detected in third and sixth day instar larvae, dark-eyed pupae, and adults (1and 30 days post-emergence). In addition, AccSp1 was expressed at its highest level in the venom gland and epidermis than tentacle, abdomen, muscle, honey sac, head, leg, chest, hemolymph, rectum, and midgut. AccSp1 was induced by 4, 24, and 44 °C; H 2 O 2 ; CdCl 2; HgCl 2 ; and pesticides (paraquat, pyridaben, and methomyl) and was inhibited by UV light and cyhalothrin treatments. When adults that had been pretreated with dsRNA 6 h prior (knocking AccSp1 down) were challenged with Bacillus bombysepticus for 18 h, the survival rate of bees greatly decreased, the activity of PO (phenoloxidase) was reduced, revealing that AccSp1 may play a critical role in assisting bees to survive the microbial infection and participate in regulating PO activity. The antioxidant enzymatic activities of catalase, peroxidase, and superoxide dismutase; the contents of hydrogen peroxide and malondialdehyde; and the ratio of NADP + /NADPH were all lower in samples containing dsRNA-AccSp1 interference than in control groups, but the content of carbonyl was not significantly different. These findings suggest the knockdown of AccSp1 may influence melanization so that the antioxidant enzyme activities and the harmful metabolites decreased. These results collectively suggest that AccSp1 plays critical roles in abiotic stresses responses and resistance to pathogens.

Various environmental stresses, such as heat shock, heavy metals, ultraviolet (UV) radiation and different pesticides, induce a cellular oxidative stress response. The cellular oxidative stress response is usually regulated by heat shock proteins (Hsps) acting as molecular chaperones. Stress-induced phosphoprotein 1 (STIP1), one of the most widely studied co-chaperones, functions as an adaptor that directs Hsp90 to Hsp70-client protein complexes. However, the biological functions of STIP1 remain poorly understood in honeybee (Apis cerana cerana). In this study, AccSTIP1 was identified in Apis cerana cerana. AccSTIP1 transcription was found to be induced by heat (42 °C), HgCl 2 , H 2 O 2 and different pesticides (emamectin benzoate, thiamethoxam, hexythiazox and paraquat) and inhibited by CdCl 2 , UV and kresoxim-methyl. Moreover, western blot analysis indicated that the expression profiles of AccSTIP1 were consistent with its transcriptional expression levels. The disc diffusion assay showed that chemically competent transetta (DE3) bacteria expressing a recombinant AccSTIP1 protein displayed the smaller death zones than did control bacteria after exposure to paraquat and HgCl 2 . The DNA nicking assay suggested that recombinant purified AccSTIP1 protected supercoiled pUC19 plasmid DNA from damage caused by a thiol-dependent mixed-function oxidation (MFO) system. After knocking down AccSTIP1 gene expression via RNA interference (RNAi), the transcript levels of antioxidation-related genes were obviously lower in dsAccSTIP1 honeybees compared with those in the uninjected honeybees. Collectively, these results demonstrated that AccSTIP1 plays an important role in counteracting oxidative stress. This study lays a foundation for revealing the mechanism of AccSTIP1 in the Apis cerana cerana antioxidant system.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is known that melatonin plays an indispensable role in the defense against some environment-induced stresses. The melatonin receptor (MTNR) is also closely linked to the environmental stress response in mammals. However, little is known about the function of the MTNR in insects, including honeybees. In this study, we identified a MTNR from Apis cerana cerana named AccMTNR1A, which contained a typical seven-transmembrane domain common to this family of receptors. A subcellular localization analysis showed that AccMTNR1A was localized in the cytomembrane. Additionally, we found that cold stress apparently boosted AccMTNR1A transcription, indicating that AccMTNR1A possibly connects to the cold stress response. The knockdown of AccMTNR1A attenuated the expression level of some genes associated with the cold stress response, suggesting that AccMTNR1A likely plays an analogous role with these genes during low temperature stress response. Moreover, silencing of AccMTNR1A also suppressed the transcription of some antioxidant genes, prompting the possibility that the response of AccMTNR1A to cold stress response may be related to antioxidant signaling pathways. Collectively, the findings presented here provide evidence that AccMTNR1A may play essential roles in protecting Apis cerana cerana from cold stress.

Cyclin-dependent kinase 5 (CDK5) is an unusual CDK whose function has been implicated in protecting the central nervous system (CNS) from oxidative damage. However, there have been few studies of CDK5 in insects. In this study, we identified the AccCDK5 gene from Apis cerana cerana and investigated its role in oxidation resistance. We found that AccCDK5 is highly conserved across species and contains conserved features of the CDK5 family. The results of qPCR analysis indicated that AccCDK5 is highly expressed during the larval and pupal stages and in the adult head and muscle. We further observed that AccCDK5 is induced by several environmental oxidative stresses. Moreover, the overexpression of the AccCDK5 protein in E. coli enhances the resistance of the bacteria to oxidative stress. The activation of CDK5 requires binding to its activator. Therefore, we also identified and cloned cyclin-dependent kinase 5 regulatory subunit 1, which we named AccCDK5r1, from Apis cerana cerana. AccCDK5r1 contains a conserved cell localization targeting domain as well as binding and activation sites for CDK5. Yeast two-hybrid analysis demonstrated the interaction between AccCDK5 and AccCDK5r1. The expression patterns of the two genes were similar after stress treatment. Collectively, these results suggest that AccCDK5 plays a pivotal role in the response to oxidative stresses and that AccCDK5r1 is a potential activator of AccCDK5.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

RNA-binding motif proteins (RBMs) belong to RNA-binding proteins that display extraordinary posttranscriptional gene regulation roles in various cellular processes, including development, growth, and stress responses. Nevertheless, only a few examples of the roles of RBMs are known in insects, particularly in Apis cerana cerana. In the present study, we characterized the novel RNA-binding motif protein 11 from Apis cerana cerana, which was named AccRBM11 and whose promoter sequence included abundant potential transcription factor binding sites that are connected to responses to adverse stress and early development. Quantitative PCR results suggested that AccRBM11 was expressed at highest levels in 1-day postemergence worker bees. AccRBM11 mRNA and protein levels were higher in the poison gland and the epidermis than in other tissues. Moreover, levels of AccRBM11 transcription were upregulated upon all the simulation of abiotic stresses. Furthermore, Western blot analysis indicated that AccRBM11 protein expression levels could be induced under some abiotic stressors, a result that did not completely in agree with the qRT-PCR results. It is also noteworthy that the expression of some genes that connected with development or stress responses were remarkably suppressed when AccRBM11 was silenced, which suggested that AccRBM11 might play a similar role in development or stress reactions with the above genes. Taken together, the data presented here provide evidence that AccRBM11 is potentially involved in the regulation of development and some abiotic stress responses. We expect that this study will promote future research on the function of RNA-binding proteins.

To tolerate many acute and chronic oxidative stress-producing agents that exist in the environment, organisms have evolved many classes of signal transduction pathways, including the transforming growth factor β (TGFβ) signal pathway. Decapentapletic gene (Dpp) belongs to the TGFβ superfamily, and studies on Dpp have mainly focused on its role in the regulation of development. No study has investigated the response of Dpp to oxidative pressure in any organism, including Apis cerana cerana (A. cerana cerana). In this study, we identified a Dpp gene from A. cerana cerana named AccDpp. The 5΄ flanking region of AccDpp had many transcription factor binding sites that relevant to development and stress response. AccDpp was expressed at all stages of A. cerana cerana, with its highest expression in 15-day worker bees. The mRNA level of AccDpp was higher in the poison gland and midgut than other tissues. Furthermore, the transcription of AccDpp could be repressed by 4°C and UV, but induced by other treatments, according to our qRT-PCR analysis. It is worth noting that the expression level of AccDpp protein was increased after a certain time when A. cerana cerana was subjected to all simulative oxidative stresses, a finding that was not completely consistent with the result from qRT-PCR. It is interesting that recombinant AccDpp restrained the growth of Escherichia coli, a function that might account for the role of the antimicrobial peptides of AccDpp. In conclusion, these results provide evidence that AccDpp might be implicated in the regulation of development and the response of oxidative pressure. The findings may lay a theoretical foundation for further genetic studies of Dpp.

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H(2)O(2), and HgCl(2). The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H(2)O(2) in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.

The Omega-class of GSTs (GSTOs) is a class of cytosolic GSTs that have specific structural and functional characteristics that differ from those of other GST groups. In this study, we demonstrated the involvement of the GSTO1 gene from A. cerana cerana in the oxidative stress response and further investigated the effects of three cysteine residues of GSTO1 protein on this response. Real-time quantitative PCR (qPCR) showed that AccGSTO1 was highly expressed in larvae and foragers, primarily in the midgut, epidermis, and flight muscles. The AccGSTO1 mRNA was significantly induced by cold and heat at 1 h and 3 h. The TBA (2-Thiobarbituric acid) method indicated that cold or heat resulted in MDA accumulation, but silencing of AccGSTO1 by RNAi in honeybees increased the concentration of MDA. RNAi also increased the temperature sensitivity of honeybees and markedly reduced their survival. Disc diffusion assay indicated that overexpression of AccGSTO1 in E. coli caused the resistance to long-term oxidative stress. Furthermore, AccGSTO1 was active in an in vitro DNA protection assay. Mutations in Cys-28, Cys-70, and Cys-124 affected the catalytic activity and antioxidant activity of AccGSTO1. The predicted three-dimensional structure of AccGSTO1 was also influenced by the replacement of these cysteine residues. These findings suggest that AccGSTO1 plays a protective role in the response to oxidative stress.

Cuticular proteins (CPs) are key components of insect cuticle, a structure that plays a pivotal role in insect development and defense. In this study, we cloned the full-length cDNA of a CP gene from Apis cerana cerana (AccCPR24). An amino acid sequence alignment indicated that AccCPR24 contains the conserved Rebers and Riddiford consensus sequence and shares high similarity with the genes from other hymenopteran insects. We then isolated the genomic DNA and found that the first intron, which is present in other CP genes, is absent in AccCPR24. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that AccCPR24 is highly expressed in the late pupal stage and midgut. Expression was inhibited by an exogenous ecdysteroid in vitro but was enhanced by this hormone in vivo; environmental stressors, such as heavy metals and pesticides, also influenced gene expression. In addition, a disc diffusion assay showed that AccCPR24 enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results that AccCPR24 acts in honeybee development and in protecting these insects from abiotic stresses., (© 2013 Wiley Periodicals, Inc.)

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins that are involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin 2 gene was isolated from Apis cerana cerana, AccTrx2. The full-length cDNA sequence of AccTrx2 was composed of 407 bp containing a 318 bp open reading frame (ORF) that encodes a predicted protein of 105 amino acids, 11.974 kDa and an isoelectric point of 4.45. Expression profile of AccTrx2 as determined by a quantitative real-time PCR (qRT-PCR) analysis was higher in brain than in other tissues, with its highest transcript occurring on the 15day post-emergence adult and upregulated by such abiotic stresses as 4 °C, 16 °C, 25 °C, H2O2, cyhalothrin, acaricide, paraquat, phoxime and mercury (HgCl2) treatments. However, AccTrx2 was slightly repressed when exposed to 42 °C treatment. Characterisation of the recombinant protein showed that the purified AccTrx2 had insulin disulfide reductase activity and could protect DNA from ROS damage. These results indicate that AccTrx2 functions as an antioxidant that plays an important role in response to oxidative stress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Thioredoxin peroxidase (Tpxs) plays an important role in maintaining redox homeostasis and in protecting organisms from the accumulation of toxic reactive oxygen species (ROS). Here, we isolated a mitochondrial thioredoxin peroxidase gene from Apis cerana cerana, AccTpx-3. The open reading frame (ORF) of AccTpx-3 is 729 bp in length and encodes a predicted protein of 242 amino acids, 27.084 kDa and an isoelectric point of 8.70. Furthermore, the 980 bp 5' flanking region was cloned, and the transcription factor binding sites were predicted. A quantitative RT-PCR (Q-PCR) analysis indicated that AccTpx-3 was expressed higher in muscle than other tissues, with its highest expression occurring on the fourth day of the larval stage, followed by the fifteenth day of the adult stage. Moreover, the expression of the AccTpx-3 transcript was upregulated by such abiotic stresses as 4°C, 42°C, H(2)O(2), cyhalothrin, acaricide and phoxime treatments. In contrast, AccTpx-3 transcription was downregulated by other abiotic stresses, including 16°C, 25°C, ultraviolet light and HgCl(2). Recombinant AccTpx-3 protein acted as a potent antioxidant that resisted paraquat-induced oxidative stress and protected DNA from oxidative damage. Taken together, these results suggest that the AccTpx-3 protein is an antioxidant enzyme that may protect organisms from oxidative stress., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fatty acid-binding proteins (FABPs) play pivotal roles in cellular signaling, gene transcription, and lipid metabolism in vertebrates and invertebrates. In this study, a putative FABP gene, referred to as AccFABP, was isolated from the Asian honeybee, Apis cerana cerana Fabricius (Hymenoptera: Apidae). The full-length cDNA consisted of 725 bp, and encoded a protein of 204 amino acids. Homology and phylogenetic analysis indicated that AccFABP was a member of the FABP multifamily. The genomic structure of this gene, which was common among FABP multifamily members, spanned 1,900 bp, and included four exons and three introns. Gene expression analysis revealed that AccFABP was highly expressed in the dark-pigmented phase of pupal development, with peak expression observed in the fat bodies of the dark-pigmented phase pupae. The AccFABP transcripts in the fat body were upregulated by exposure to dietary fatty acids such as conjugated linoleic acid, docosahexaenoic acid, and arachidonic acid. Transcription factor binding sites for Caudal-Related Homeobox and functional CCAAT/enhancer binding site, which were respectively associated with tissue expression and lipid metabolism, were detected in the 5' promoter sequence. The evidence provided in the present study suggests that AccFABP may regulate insect growth and development, and lipid metabolism.

Calcyclin binding protein (CacyBP), a homolog of Sgt1, was shown to interact with some S100 proteins, Skp1, tubulin, actin and ERK1/2 kinases. Studies have also shown that CacyBP is a neuronal protein in mammals. Limited information is available regarding the properties and functions of CacyBP in insects. Here, we cloned and characterized a novel CacyBP gene, named AccCacyBP, from honeybee (Apis cerana cerana). Bioinformatic analysis indicated that AccCacyBP was highly conserved and closely related to the CacyBP of other insects. Promoter analysis revealed a number of putative tissue, development and stress-related transcription factor-binding sites. RT-qPCR demonstrated that AccCacyBP was expressed at all of the stages of development, especially in the brains of honeybees. Moreover, immunohistochemistry analysis showed the presence of AccCacyBP in the brain. The transcript levels of AccCacyBP in the brains of honeybees were developmentally induced and upregulated by exposure to oxidative stresses, including UV-light, acetamiprid and HgCl(2). This study demonstrates that the CacyBP gene in honeybees may be a neuronal protein involved in the developmental regulation and the stress-response of the brain of honeybees.

Lipid storage droplet 1 (LSD-1), a PAT family protein located around lipid droplets in insects, is intimately linked to lipid droplets formation and lipid metabolism. Conjugated linoleic acid (CLA) and rosiglitazone (Rosi) have previously been shown to modulate the expression of several PAT family proteins through peroxisome proliferator-activated receptor-γ (PPARγ). In the present study, we isolated and characterized a novel LSD-1 gene, referred to AccLSD-1, from Chinese honeybee (Apis cerana cerana). Sequence analysis indicated that the central region of LSD-1 protein had significant sequence similarity and a typical LSD-1 gene was composed of 8 exons and 7 introns. Interestingly, the first intron of AccLSD-1 including several PPARγ-response elements (PPREs) was located in 5' UTR. Analysis of 5'-flanking region of AccLSD-1 revealed a number of putative cis-acting elements, including three PPREs. Quantitative real-time PCR showed that AccLSD-1 expressed ubiquitously from feeding larva to adult, and its expression level was highest at brown-eyed pupae (Pb) stage. The effect of CLA, Rosi and combination on AccLSD-1 expressions indicated 1% CLA and 0.5 mg/ml Rosi were considered as the suitable diets for rearing adult workers in laboratory, and AccLSD-1 was down-regulated by CLA whereas up-regulated by Rosi. Furthermore, the combination of CLA and Rosi remarkly rescued the suppression of AccLSD-1 expression by CLA alone. These results suggest that AccLSD-1 is associated with A. cerana cerana development, especially during pupal metamorphosis, and can be regulated by CLA or Rosi possibly via activating PPARγ.

Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), plays roles in a variety of cellular responses. However, limited information is available on the relationship between ERKs and environmental stresses. In this report, an ERK gene, AccERK2, was cloned and characterized from Apis cerana cerana. Polypeptide sequence alignment revealed that the single-copied AccERK2 shares high identity with other known ERKs and contains the typical conserved Thr-Glu-Tyr (TEY) motif in its activation loop. Genomic sequence analysis revealed that the seven exons of AccERK2 are interrupted by six introns, and the seventh intron is located in the 3' untranslated region. Semi-quantitative reverse transcription (RT-PCR) indicated that AccERK2 was expressed at higher levels in the larval and pupal stages than in the adult stage. AccERK2 was also most highly expressed in the brain. The expression of AccERK2 was induced by abiotic stresses, including heat, ion irradiation, oxidative stress, and heavy metal ions. Based on these results, it appears that AccERK2 in A. cerana cerana participates in developmental processes, the nervous system, and responses to environmental stressors., (© 2012 Wiley Periodicals, Inc.)

Previous studies have demonstrated that mammalian thioredoxin reductases 1 (TrxRs; EC 1.6.4.5) play roles in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. However, little is known about the function of TrxRs in insects, especially in Apis cerana cerana. To gain a better understanding of its role in insects, we cloned TrxR1 from A.cerana cerana (AccTrxR1) and investigated its structural features and expression. The full-length cDNA is 1998 bp long and contains an open reading frame (ORF) of 1485 bp. The deduced AccTrxR1 protein has 494 aa, a calculated molecular mass (MW) of 54.24 kDa, and an isoelectric point of pH 7.35. The full-length genomic DNA of AccTrxR1 is 3500 bp and contains eight exons and seven introns. In addition, the sequence and putative transcription factor-binding sites of the 5'-flanking region were examined. We also looked for conserved domains/motifs, generated homologous alignments, conducted phylogenetic analysis, and made secondary and tertiary structure predictions using web software. RT-PCR revealed that the expression of AccTrxR1 could be induced by UV and heat (37 degrees C). These results indicate that AccTrxR1 may play a key role in protection against oxidant stress., ((c) 2010 Elsevier Inc. All rights reserved.)