Characterization of a mitochondrial manganese superoxide dismutase gene from Apis cerana cerana and its role in oxidative stress.

Mitochondrial manganese superoxide dismutase (mMnSOD) plays a vital role in the defense against reactive oxygen species (ROS) in eukaryotic mitochondria. In this study, we isolated and identified a mMnSOD gene from Apis cerana cerana, which we named AccSOD2. Several putative transcription factor-binding sites were identified within the 5'-flanking region of AccSOD2, which suggests that AccSOD2 may be involved in organismal development and/or environmental stress responses. Quantitative real-time PCR analysis showed that AccSOD2 is highly expressed in larva and pupae during different developmental stages. In addition, the expression of AccSOD2 could be induced by cold (4 °C), heat (42 °C), H2O2, ultraviolet light (UV), HgCl2, and pesticide treatment. Using a disc diffusion assay, we provide evidence that recombinant AccSOD2 protein can play a functional role in protecting cells from oxidative stress. Finally, the in vivo activities of AccSOD2 were measured under a variety of stressful conditions. Taken together, our results indicate that AccSOD2 plays an important role in cellular stress responses and anti-oxidative processes and that it may be of critical importance to honeybee survival.

Cloning, expression patterns, and preliminary characterization of AccCPR24, a novel RR-1 type cuticle protein gene from Apis cerana cerana.

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.

Characterization of the response to ecdysteroid of a novel cuticle protein R&R gene in the honey bee, Apis cerana cerana.

Genes encoding cuticle proteins are helpful subjects to study the molecular mechanisms of insect molting and metamorphosis. In this study, we isolated and characterized a novel cuticle protein R&R gene, referred to as AccCPR1, from Apis cerana cerana. The open reading frame of AccCPR1 has a length of 573 nt and encodes a protein of 190 amino acids that contains a chitin binding region and is a typical cuticle R&R-2 protein. Five putative E74 binding sites and four BR-C binding sites were predicted in the 5'-flanking region, which suggests a potential function in molting and metamorphosis. RT-qPCR showed that AccCPR1 transcript occurred as the ecdysteroid titer decreased after reaching a peak, which suggests AccCPR1 expression requires a "pulse" regimen of ecdysteroids. This hypothesis was tested using different experimental strategies. When larvae were reared with different concentrations of 20E in their diet, the ecdysteroid peak repressed AccCPR1 expression. Exposure of the thoracic integument of the pupae in vitro to different concentration of 20E repressed AccCPR1 expression, which recovered after the removal of 20E. These results suggest that AccCPR1 is a typical cuticle R&R-2 protein that plays an important role in development, and an ecdysteroid pulse is critical for high AccCPR1 gene expression.

Identification and characterization of a novel calcyclin binding protein (CacyBP) gene from Apis cerana cerana.

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.

Characterization of a sigma class glutathione S-transferase gene in the larvae of the honeybee (Apis cerana cerana) on exposure to mercury.

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 612 bp 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 HgCl(2). The GST activity under stress was higher than the controls fed on HgCl(2)-free diet. Disc diffusion assay provided evidence of recAccGSTs4 resistance to long-term exposure of HgCl(2) 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.