Review on effects of some insecticides on honey bee health.

Insecticides, one of the main agrochemicals, are useful for controlling pests; however, the indiscriminate use of insecticides has led to negative effects on nontarget insects, especially honey bees, which are essential for pollination services. Different classes of insecticides, such as neonicotinoids, pyrethroids, chlorantraniliprole, spinosad, flupyradifurone and sulfoxaflor, not only negatively affect honey bee growth and development but also decrease their foraging activity and pollination services by influencing their olfactory sensation, memory, navigation back to the nest, flight ability, and dance circuits. Honey bees resist the harmful effects of insecticides by coordinating the expression of genes related to immunity, metabolism, and detoxification pathways. To our knowledge, more research has been conducted on the effects of neonicotinoids on honey bee health than those of other insecticides. In this review, we summarize the current knowledge regarding the effects of some insecticides, especially neonicotinoids, on honey bee health. Possible strategies to increase the positive impacts of insecticides on agriculture and reduce their negative effects on honey bees are also discussed.

Identification of an MGST2 gene and analysis of its function in antioxidant processes in Apis cerana cerana.

MGST2 is a member of the MAPEG superfamily, which participates in LTC4 synthesis and plays important roles in the regulation of the oxidative stress pathway and some diseases. Here, we isolated a previously uncharacterized gene in Apis cerana cerana named AccMGST2 by reverse transcription-polymerase chain reaction. The biological characteristics of AccMGST2 were analyzed by bioinformatics. The amino acid sequence similarity between AccMGST2 and AmMGST2 of Apis mellifera reached 96.08%. The expression characteristics of AccMGST2 were explored in several tissues. The quantitative real-time polymerase chain reaction results showed that the AccMGST2 gene was highly expressed in the head and muscle and that AccMGST2 expression responded to oxidative stress caused by different abiotic stresses. AccMGST2 was silenced using RNA interference, which decreased the expression levels of some MAPK and antioxidant genes. Therefore, we conclude that AccMGST2 is involved in the regulation of oxidative stress in A. cerana cerana.

Molecular and functional characaterization of the novel odorant-binding protein gene AccOBP10 from Apis cerana cerana.

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.

Molecular Mechanism of the UDP-Glucuronosyltransferase 2B20-like Gene (AccUGT2B20-like) in Pesticide Resistance of Apis cerana cerana.

UDP-glucuronosyltransferases (UGTs), being multifunctional detoxification enzymes, play a major role in the process of resistance to various pesticides in insects. However, the mechanism underlying the molecular regulation of pesticide resistance remains unclear, especially in Apis cerana cerana. In this study, all of the UGTs in Apis cerana cerana (AccUGT) have been identified through the multiple alignment and phylogenetic analysis. Expression of AccUGT genes under different pesticides, and antioxidant genes after silencing of AccUGT2B20-like, were detected by qRT-PCR. The resistance of overexpressed AccUGT2B20-like to oxidative stress was investigated by an Escherichia coli overexpression system. Also, antioxidant-related enzyme activity was detected after silencing of the AccUGT2B20-like gene. Expression pattern analysis showed that almost all UGT genes were upregulated under different pesticide treatments. This result indicated that AccUGTs participate in the detoxification process of pesticides. AccUGT2B20-like was the major gene because it was more highly induced than the others. Overexpression of AccUGT2B20-like in E. coli could effectively improve oxidative stress resistance. Specifically, silencing the AccUGT2B20-like gene increased oxidative stress by repressing the expression of oxidation-related genes, decreasing antioxidant-related enzyme activity, and increasing malondialdehyde concentration. Taken together, our results indicate that AccUGTs are involved in pesticide resistance, among which, AccUGT2B20-like contributes to the detoxification of pesticides by eliminating oxidative stress in Apis cerana cerana. This study explains the molecular basis for the resistance of bees to pesticides and provides an important safeguard for maintaining ecological balance.

Analyses of the function of DnaJ family proteins reveal an underlying regulatory mechanism of heat tolerance in honeybee.

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.

Functional and transcriptomic analyses of the NF-Y family provide insights into the defense mechanisms of honeybees under adverse circumstances.

As predominant pollinators, honeybees are important for crop production and terrestrial ecosystems. Recently, various environmental stresses have led to large declines in honeybee populations in many regions. The ability of honeybees to respond to these stresses is critical for their survival. However, the details of the stress defense mechanisms of honeybees have remained elusive. Here, we found that the Nuclear Factor Y (NF-Y) family (containing NF-YA, NF-YB, and NF-YC) is a novel stress mediator family that regulates honeybee environmental stress resistance. NF-YA localized in the nucleus, NF-YB accumulated in the cytoplasm, and NF-YC presented in both the nucleus and cytoplasm. NF-YC interacted with NF-YA and NF-YB in vitro and in vivo, and the nuclear import of NF-YB relied on its interaction with NF-YC. We further found that the expression of NF-Y was induced under multiple stress conditions. In addition, NF-Y regulated many stress responses and antioxidant genes at the transcriptome-wide level, and knockdown of NF-Y repressed the expression of stress-inducible genes, particularly LOC108003540 and LOC107994062, under adverse circumstances. Silencing NF-Y lowered honeybee stress resistance by reducing total antioxidant capacity and enhancing oxidative impairment. Collectively, these results indicate that NF-Y plays important roles in stress responses. Our study sheds light on the underlying defense mechanisms of honeybees under environmental stress.

Molecular mechanism by which Apis cerana cerana MKK6 (AccMKK6)-mediated MAPK cascades regulate the oxidative stress response.

Mitogen-activated protein kinase kinases (MKKs) are important components of the MAPK signaling pathways, which play a key role in responding to stress and inflammatory stimuli. Here, a new MKK gene, AccMKK6, was identified and functionally analyzed in Apis cerana cerana Real-time quantitative PCR (qPCR) and Western blot analysis demonstrated that the AccMKK6 expression level was up-regulated by several environmental stresses. Moreover, the knockdown of AccMKK6 by RNA interference technology altered the expression levels of some antioxidant genes. In addition, the knockdown of AccMKK6 resulted in increased malonyldialdehyde (MDA) concentration and decreased antioxidant-related enzymes activity in honeybees. To explore the MAPK signaling pathways involved in AccMKK6, we identified the transcription factor kayak in A. cerana cerana We analyzed the interactions of AccMKK6, Accp38b, and Acckayak using the yeast two-hybrid system. AccMKK6 and Acckayak showed similar expression profiles after several stress treatments. In addition, the expression level of Acckayak was significantly increased when AccMKK6 was silenced. Therefore, we speculate that AccMKK6 may be involved in the MAPK cascades, which play a crucial role in counteracting oxidative stress caused by external stimuli.

Isolation of carboxylesterase (esterase FE4) from Apis cerana cerana and its role in oxidative resistance during adverse environmental stress.

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 H2O2, imidacloprid, and paraquat, and was down-regulated by 4 °C, UV radiation, CdCl2, and HgCl2. Additionally, the protein expression of this gene was down-regulated at 4 °C and up-regulated by H2O2. Disc diffusion assays showed that the AcceFE4 recombinant protein-expressing bacteria had a smaller killing zone than the control group with the paraquat, HgCl2 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.