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In the present study, we identified and characterized two small heat shock protein genes from Apis cerana cerana, named AccHsp24.2 and AccHsp23.0. An alignment analysis showed that AccHsp24.2 and AccHsp23.0 share high similarity with other members of the α-crystallin/sHSP family, all of which contain the conserved α-crystallin domain. The recombinant AccHsp24.2 and AccHsp23.0 proteins were shown to have molecular chaperone activity by the malate dehydrogenase thermal aggregation assay. Three heat shock elements were detected in the 5′-flanking region of AccHsp24.2 and eleven in AccHsp23.0, and two Drosophila Broad-Complex genes for ecdysone steroid response sites were found in each of the genes. The presence of these elements suggests that the expression of these genes might be regulated by heat shock and ecdysone, which was confirmed by quantitative RT-PCR (RT-qPCR). The results revealed that the expression of the two genes could be induced by cold shock (4°C) and heat shock (37°C and 43°C) in an analogous manner, and AccHsp24.2 was more susceptible than AccHsp23.0. In addition, the expression of the two genes was induced by high concentrations of ecdysone in vitro and in vivo. The accumulation of AccHsp24.2 and AccHsp23.0 mRNA was also detected in different developmental stages and tissues. In spite of the differential expression at the same stage, these genes shared similar developmental patterns, suggesting that they are regulated by similar mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: 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 1998bp long and contains an open reading frame (ORF) of 1485bp. The deduced AccTrxR1 protein has 494 aa, a calculated molecular mass (MW) of 54.24kDa, and an isoelectric point of pH 7.35. The full-length genomic DNA of AccTrxR1 is 3500bp 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°C). These results indicate that AccTrxR1 may play a key role in protection against oxidant stress. [Copyright &y& Elsevier]

Abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPX) plays a crucial role in maintaining the integrity of membrane by reducing hydroperoxides of phospholipids. Here, we report the identification and characterization of two genes, designated AccGtpx-1 and AccGtpx-2, encoding PHGPX proteins from the Chinese honeybees, Apis cerana cerana. Alignment analysis showed that AccGtpx-1 and AccGtpx-2 shared high similarity with other known PHGPXs, which show similar structure to thioredoxin. These single copy genes showed complex exon–intron structures. The mRNA of AccGtpx-1 was detected in larvae, pupae and adults and that AccGtpx-2 was only found in adult worker bees. Furthermore, the expression of AccGtpx-1 could be induced by H2O2, ultraviolet (UV) light, heat shock (37°C), HgCl2, imidacloprid, cyhalothrin, pyriproxyfen and methomyl. In contrast, AccGtpx-2 expression could only be induced by UV. These results indicated for the first time that the AccGtpx-1 and AccGtpx-2 genes encoding A. cerana cerana PHGPXs are regulated differently in response to environmental stressors. [Copyright &y& Elsevier]

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]

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]

Mitogen-activated protein kinase kinase kinases (MAPKKKs) function at the top level of MAPK cascades and play important roles in plant development and stress responses. Although MAPKKKs comprise the largest family in the MAPK cascades, very few Raf-like MAPKKKs have been functionally identified, especially in the economically important crop cotton. In this study, a Raf-like MAPKKK gene, GhRaf19 , was characterized for the first time in cotton. Our data show that the expression of GhRaf19 was inhibited by PEG and NaCl and induced by cold (4 °C) and H 2 O 2 . Furthermore, when GhRaf19 was silenced in cotton using virus-induced gene silencing (VIGS), tolerance to drought and salt stress were enhanced, the accumulation of reactive oxygen species (ROS) was reduced, and ROS-related gene expression was increased. Consistent with these results, in N. benthamiana , overexpressing- GhRaf19 reduced tolerance to drought and salt. However, GhRaf19 -silenced plants showed lowered resistance to cold in cotton, and this effect was correlated with the accumulation of ROS. In contrast, overexpressing GhRaf19 in N. benthamiana increased resistance to cold by inducing higher levels of expression and activity of ROS-related antioxidant genes/enzymes. These results indicate that GhRaf19 negatively regulates tolerance to drought and salt and positively regulates resistance to cold stress by modulating cellular ROS in cotton. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) cascades mediate various responses in plants. As the top component, MAP3Ks deserve more attention; however, little is known about the role of MAP3Ks, especially in cotton, a worldwide economic crop. In this study, a gene encoding a putative Raf-like MAP3K, GhMAP3K40, was isolated. GhMAP3K40 expression was induced by stress and multiple signal molecules. The plants overexpressing GhMAP3K40 had an enhanced tolerance to drought and salt stress at the germination stage. However, at the seedling stage, the transgenic plants suffered more severe damage after drought, exposure to pathogens and oxidative stress. The defence-related genes and the antioxidant system were activated in transgenic palnts, suggesting that GhMAP3K40 positively regulate the defence response. The transgenic plants were less able to prevent pathogenic invasion, which was due to defects in the cell structure of the leaves. The root system of the control plants were stronger compared with the transgenic plants. These results indicated a negative role of GhMAP3K40 in growth and development and GhMAP3K40 possibly caused the defects by down-regulating the lignin biosynthesis. Overall, these results suggest that GhMAP3K40 may positively regulate defence response but cause reduced tolerance to biotic and abiotic stress by negatively regulating growth and development. [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]

Honeybees show an important pollination ability and play vital roles in improving crop yields and increasing plant genetic diversity, thereby generating tremendous economic benefits for humans. However, honeybee survival is affected by a number of biological and abiotic stresses, including the effects of fungi, bacteria, viruses, parasites, and especially agrochemicals. Glyphosate, a broad-spectrum herbicide that is primarily used for weed control in agriculture, has been reported to have lethal and sublethal effects on honeybees. Here, we summarize recent advances in research on the effects of glyphosate on honeybees, including effects on their behaviors, growth and development, metabolic processes, and immune defense, providing a detailed reference for studying the mechanism of action of pesticides. Furthermore, we provide possible directions for future research on glyphosate toxicity to honeybees. [ABSTRACT FROM AUTHOR]

Abstract: Thioredoxin peroxidases (Tpxs), members of the antioxidant protein family, play critical roles in resisting oxidative stress. In this work, a novel 1-Cys thioredoxin peroxidase gene was isolated from Apis cerana cerana and was named AccTpx5. The open reading frame (ORF) of AccTpx5 is 663bp in length and encodes a 220-amino acid protein with a predicted molecular mass and isoelectric point of 24,921kDa and 5.45, respectively. Promoter sequence analysis of AccTpx5 revealed the presence of putative transcription factor binding sites related to early development and stress responses. Additionally, real-time quantitative PCR (Q-PCR) analysis indicated that AccTpx5 was primarily present in some developmental stages, with the highest expression levels in the first-instar larvae. The expression level of AccTpx5 was up-regulated under various abiotic stresses, including 4°C, 42°C, HgCl2, H2O2, phoxim and acaricide treatments. Conversely, it was down-regulated by UV and pyriproxyfen treatments. Moreover, H2O2 concentration dramatically increased under a variety of stressful conditions. Finally, the purified recombinant AccTpx5 protein protected the supercoiled form of plasmid DNA from damage in the thiol-dependent mixed-function oxidation (MFO) system. These results suggest that AccTpx5 most likely plays an essential role in antioxidant defence. [Copyright &y& Elsevier]

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]

Highlights: [•] Cloned a mitochondrial manganese superoxide dismutase gene, AccSOD2, from Apis cerana cerana. [•] Examined the expression profiles of AccSOD2 under a variety of oxidative stresses. [•] Evaluated the antioxidant activity of the recombinant AccSOD2 protein under oxidative stresses. [•] Measured the in vivo activities of AccSOD2 in response to oxidative conditions. [ABSTRACT FROM AUTHOR]

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 407bp containing a 318bp open reading frame (ORF) that encodes a predicted protein of 105 amino acids, 11.974kDa 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 729bp in length and encodes a predicted protein of 242 amino acids, 27.084kDa and an isoelectric point of 8.70. Furthermore, the 980bp 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, H2O2, cyhalothrin, acaricide and phoxime treatments. In contrast, AccTpx-3 transcription was downregulated by other abiotic stresses, including 16°C, 25°C, ultraviolet light and HgCl2. 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 &y& Elsevier]

Abstract: Glutathione-S-transferases (GSTs) play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). However, no information is available for GSTs in the Chinese honey bee (Apis cerana cerana). In this study, we isolated and characterized a zeta class GST gene (AccGSTZ1) from the Chinese honey bee. This gene is present in a single copy and harbors five exons. The deduced amino acid sequence of AccGSTZ1 shared high sequence identity with homologous proteins and contained the highly conserved features of this gene family. The temporal and spatial expression profiles of AccGSTZ1 showed that AccGSTZ1 was highly expressed in fourth instar larvae during development, and the mRNA level of AccGSTZ1 was higher in the epidermis than that in other tissues. The expression pattern under oxidative stress revealed that AccGSTZ1 transcription was significantly upregulated by external factors, such as temperature challenges and H2O2 treatment. The characterization of the purified protein revealed that AccGSTZ1 had low glutathione-conjugating activity, but the recombinant AccGSTZ1 protein displayed high antioxidant activity under oxidative stress. These data suggest that AccGSTZ1 is an oxidative stress-inducible antioxidant enzyme that plays an important role in the protection against oxidative stress and may be of critical importance for the survival of the honey bees. [Copyright &y& Elsevier]

Abstract: Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainly involved in detoxification of endogenous and xenobiotic compounds and oxidative stress resistance in insects. In this study, we identified a sigma class GST from Apis cerana cerana (AccGSTs4). The open reading frame of cDNA was 612bp and encoded a 203 amino acid polypeptide, which exhibited the structural motif and domain organization characteristic of GST. Homology and evolutionary analysis indicated that the induced amino acid sequence of AccGSTs4 belonged to an insect sigma class group. Expression analysis indicated that AccGSTs4 was presented in all stages of development with high level in 4th instar larvae. Immunolocalization further revealed the distribution of AccGSTs4 in 4th instar larvae. RT-qPCR showed that the transcripts of AccGSTs4 from the larvae were upregulated under dietary HgCl2. The GST activity under stress was higher than the controls fed on HgCl2-free diet. Disc diffusion assay provided evidence of recAccGSTs4 resistance to long-term exposure of HgCl2 stress. Additionally, analysis of 5′-flanking region further clarified the probable expression patterns of AccGSTs4. Taken together, our findings indicate that the larvae AccGSTs4 may play a role in mercury stress response, and it will help to protect honeybees from heavy metals. [Copyright &y& Elsevier]

Abstract: The RNA-dependent RNA polymerases (RDRs) play a key role in RNA silencing, heterochromatin formation and natural gene regulation. Here, a novel RDR gene was isolated from Nicotiana glutinosa, designated as NgRDR1. The full-length cDNA of NgRDR1 encodes a 1117-amino acid protein which harbors the five conserved regions in plant RDRs, including the most remarkable motif DbDGD (b is a bulky residue). Amino acid sequence alignment revealed that NgRDR1 exhibited a high degree of identity with other higher plant RDR genes. Five exons were detected in the genomic DNA sequence, and the fourth exon is 151bp, the location and the length of which are conserved among different plant species. From the phylogenetic tree constructed with different kinds of plant RDRs, it is determined that NgRDR1 falls into group I, and is closely associated with the dicotyledons RDRs. The analysis of the 5′-flanking region of NgRDR1 revealed a group of putative cis-acting elements. The results of expression analysis showed that the transcripts of NgRDR1 can be induced by biotic stresses, such as exogenous signaling molecules including salicylic acid (SA), SA analogues, hydrogen peroxide (H2O2), and methyl jasmonate (MeJA). Furthermore, NgRDR1 expression can be up-regulated by potato virus Y (PVY), tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV), but not by potato virus X (PVX). Besides, different kinds of fungi can also induce NgRDR1 expression. These results indicate that NgRDR1 may play an important role in response to biotic and abiotic stresses. [Copyright &y& Elsevier]

• A micro/nano bulge and crack structures were fabricated to improve the sensitivity and measurement range of the sensor. • A simple tear and stick method was proposed to obtain micro/nano structures. • The measurement range of the flexible sensor can be up to 80% and maximum sensitivity is 20. • The flexible sensor can perform real-time monitoring of human joint motion. [Display omitted] Flexible strain sensors have been garnering substantial attention since being proven to be an effective means to acquire critical biomarkers for monitoring the states of the movement of organisms. This work introduces a flexible metal thin film strain sensor with micro/nano structure which has large measurement range and high sensitivity strain measurement. The large measurement range can be realized by the bugles that avoiding expanding of cracks and the high sensitivity is attributed to the disconnection-reconnection process by nanoscale cracks. In experiments, a flexible strain sensor with 30 nm Au nano-film was researched, and the results show that the maximum strain measurement range of the Au nano-film flexible sensor with micro/nano bulge structure can reach to 80%, and the gauge factor (GF) can reach to 20. In addition, the good repeatability and stability were proved by more than 1000 times cycles. Finally, this flexible sensor was attached to human's fingers for real-time monitoring of the state of human joints and was demonstrated to have good comprehensive performance and usability. These features create opportunities for applying in many fields like wearable medical devices, e-skin, flexible robots and so on. [ABSTRACT FROM AUTHOR]

• 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]

Understanding the causes of the decline in bee population has attracted intensive attention worldwide. The indiscriminate use of agrochemicals is a persistent problem due to their physiological and behavioural damage to bees. Glyphosate and its commercial formulation stand out due to their wide use in agricultural areas and non-crop areas, such as parks, railroads, roadsides, industrial sites, and recreational and residential areas, but the mode of action of glyphosate on bees at the molecular level remains largely unelucidated. Here, we found that the numbers of differentially expressed genes and metabolites under glyphosate commercial formulation (GCF) stress were significantly higher in Apis cerana cerana than in Apis mellifera ligustica. Despite these differences, the number of differentially expressed transcripts increased following an increase in the GCF treatment time in both A. cerana cerana and A. mellifera ligustica. GCF exerted adverse impacts on the immune system, digestive system, nervous system, amino acid metabolism, carbohydrate metabolism, growth and development of both bee species by influencing their key genes and metabolites to some extent. The expression of many genes involved in immunity, agrochemical detoxification and resistance, such as antimicrobial peptides, cuticle proteins and cytochrome P450 families, was upregulated by GCF in both bee species. Collectively, our results indicate that both A. cerana cerana and A. mellifera ligustica strive to mitigate the pernicious effects caused by GCF by regulating detoxification and immune systems. Moreover, A. cerana cerana might be better able to withstand the toxic effects of GCF with lower fitness costs than A. mellifera ligustica. Our work will contribute to elucidating the deleterious physiological and behavioural impacts of GCF on bees. Unlabelled Image • Bees were fed with glyphosate commercial formulation (GCF). • GCF had adverse impacts on the digestive system and development of bees. • Bees regulate detoxification and immune system to mitigate pernicious effects. • A. cerana cerana might be better able to withstand GCF than A. mellifera ligustica. [ABSTRACT FROM AUTHOR]

There is clear evidence of severe honeybee declines in recent years, and parallel declines of plant community and crop productivity that rely on them. Different stresses, including heat stress, are among the primary drivers of this decline. However, the mechanisms by which honeybees respond to heat stress are elusive. Though heat shock proteins (Hsps) play important roles in heat stress response, the function of DnaJs (a subfamily of Hsps) is unclear. Here, we aimed to determine the underlying regulatory mechanism of honeybees to heat stress mediated by DnaJs. We found that several DnaJ genes, including DnaJA1 , DnaJB12 and DnaJC8 , are key for honeybee heat tolerance. DnaJA1 and DnaJB12 are cytoplasmic proteins, and DnaJC8 is a nuclear protein. The expression of DnaJA1 , DnaJB12 and DnaJC8 was induced at different levels under short-term and long-term heat stress. Phenotypic analysis indicated that DnaJA1 , DnaJB12 and DnaJC8 knockdown attenuated honeybee heat resistance. In addition, DnaJA1 participated in the heat stress response by upregulating many heat-inducible genes at the transcriptome-wide level, especially LOC108002668 and LOC107995148. Importantly, the upregulation of LOC108002668 and LOC107995148 was significantly repressed under heat stress when DnaJA1 was knocked down. We also found that knockdown of DnaJA1 , DnaJB12 and DnaJC8 decreased antioxidant defense ability and increased the degree of oxidative damage in the honeybee. Taken together, our results indicate that DnaJ genes play important roles under heat stress in the honeybee. Overexpression of DnaJ genes may protect honeybees from heat stress-induced injuries and increase their survival rate. Unlabelled Image • DnaJ genes play important roles in heat stress response in honeybee. • Upregulation of DnaJ genes, such as DnaJA1 , DnaJB12 and DnaJC8 , is essential for honeybee survival under heat stress. • DnaJ family proteins improved honeybee heat tolerance by regulating the antioxidant defense system. [ABSTRACT FROM AUTHOR]