Cloning and Expression of Gonadotrophin Releasing Hormone Receptor in Apis cerana cerana

[Objective] This study was to clone the GnRH （gonadotropin-releasing hormone）, and to investigate its expression in Apis cerana cerana. [Method] The cDNA sequence of GnRHR gene was amplified from Apis cerana cerana by using RT-PCR techniques. It was conducted with bioinformatics analysis and the in situ hybridization histochemistry of its expression products was studied. [Result] The sequence analy- sis showed that the full cDNA sequence was 1 050 bp with the open reading frame of 1 050 bp, and it encoded 349 amino acid residues. The deduced amino sequence included 7 transmembrane regions, and the predicted molecular mass and isoelectric point were 40.6 kD and 9.54, respectively. The cluster analysis showed that the GnRHR from ＇,4. cerana cerana had close relationship to the GnRHR II from other insects. In situ hybridization showed that Bee-GnRHR staining was specifically localized to the brain, intestine, fat body and testis. [Conclusion] The results indicated that the GnRHR provided molecular bond for the reproduction and metabolism for insects, and suggested a functional role for bee-GnRHR signaling in the coupling of reproduction activities and environment conditions.

The Influence of Bt-Transgenic Maize Pollen on the Bacterial Diversity in the Midgut of Chinese Honeybees, Apis cerana cerana

Using culture-independent technique polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） and conventional culture techniques, ecological risk of transgenic maize pollen on gut bacteria of the Chinese honeybee, Apis cerana cerana, was assessed. Honeybees were fed with Bt-transgenic maize pollen, non-transgenic near isoline pollen, linear crylAh gene （800 ng mL^-1） and supercoiled plasmid DNA （800 ng mL^-1） under laboratory conditions. The DGGE profile showed that the number of DGGE bands varied from 10.7 to 14.7 per sample, and the Shannon＇s index ranged from 0.85 to 1.00. The similarity calculated by PAST was mostly above 92%, indicating no obvious changes among treatments or within replicates. 14 bacterial strains affiliated with Alphaproteobacteria, Gammaproteobacteria, Firmicutes, and Actinobacteria were isolated and characterized on media under aerobic and anaerobic conditions. These results demonstrated that transgenic crylAh maize pollen did not induce significant changes of the honeybee gut bacterial community composition under laboratory conditions.

Cloning and Sequence Analysis of cDNA Encoding MRJP3 of Apis cerana cerana

By screening the worker （Apis cerana cerana） heads cDNA library using a fragment of the mrjp3 gene of Apis cerana as probe, 120 positive clones were obtained. The clone containing A. cerana cerana MRJP3 （AccMRJP3） cDNA was selected. Based on the sequencing of the inserts of the positive clone, a sequence of AccMRJP3 cDNA which is 1 887 bp long including a poly （A） tail was obtained. The AccMRJP3 cDNA encompassed an open-reading frame （ORF） with 1 779 bp encoding 593 amino acids. The un-translated regions （UTR） of the 5′ end and 3′end are 46 bp and 160 bp in length, respectively. Similar to AmMRJP3 and AdMRJP3, the putative AccMRJP3 also has a repetitive region. The comparison of the repetitive region of AccMRJP3, AmMRJP3 and AdMRJP3 shows some differences between them.

Role of c-Jun NH_(2)-terminal kinase-mediated mitogen-activated protein kinase pathway in response to pesticides in Apis cerana cerana

The mitogen-activated protein kinase(MAPK)cascade pathway plays an important role in regulating stress responses.The function of the c-Jun NH_(2)-terminal kinase(JNK),a component of the MAPK cascade pathway,in Apis cerana cerana(Acc)remains unclear.Here,JNK was isolated and identified from Acc.Bioinformatics analyses revealed there is a typical serine/threonine protein kinase catalytic domain in the AccJNK protein.An expression profile analysis showed that AccJNK was significantly induced by pesticide treatments.To further explore the functional mechanisms of AccJNK,a yeast 2-hybrid screen was performed,activator protein-1(AP-1)was screened as the interaction partner of AccJNK,and the interaction relationship was further verified by pull-down assay.Quantitative real-time polymerase chain reaction showed the expression pattern of AccAP-I was similar to that of AccJNK.After a knockdown of AccJNK or AccAP-I by RNA interference,the survival rate of Acc after pesticide treatments increased.Additionally,the expression levels of antioxidant-related genes and the activities of antioxidant enzymes increased,suggesting that the knockdown of AccJNK or AccAP-I increased the antioxidant capacity of bees.Our study revealed that the JNK-mediated MAPK pathway responds to pesticide stress by altering the antioxidant capacity of Acc.

SEQUENCE ANALYSIS OF A NOVEL INSECT PHOSPHOGLYCERATEMUTASE GENE FROM THE CHINESE HONEYBEE, APIS CERANA

A clone inserted with 1 104 bp fragment containing a 765bp Open Reading Frame(ORF), encoding a putative 2,3-bisphosphoglycerate(2,3BPG) dependent Phosphoglycerate mutase(dPGAM) that catalyzes the transfer of a phosphate group from the C3 carbon atom to the C2 carbon atom of phosphoglycerate, was screened by mass sequencing from the cDNA library of the venom glands of Apis cerana. The deduced amino acid sequence shared high similarities (39%-88%)with the dPGAM of 7 other organisms, but the similarities with the iPGAM of 4 other organisms were low (10%-12%). Moreover, the alignment of Ac-PGAM with the dPGAMs from 7 other organisms showed that all the active site amino acid residues were conserved. This result shows that Ac-PGAM is a typical dPGAM. Thus, this is the second PGAM gene reported in Insecta. Furthermore, phylogenetic analysis showed that the evolutionary tree of PGAMs reflects the systematic relationship of species.

Purification and characterization of α-glucosidase in Apis cerana indica

Apis cerana indica foragers were used for the isolation of a full-length α- glucosidase cDNA, and for purification of the active nascent protein by low salt extraction of bee homogenates, ammonium sulphate precipitation and diethylaminoethyl-cellulose and Superdex 200 c hromatographies. The molecular mass of the purified protein was estimated by polyacrylamide gel electrophoresis resolution, and the pH, temperature, incubation, and substrate optima for enzymic activity were determined. Conformation of the purified enzyme as α-glucosidase was performed by BLAST software homology comparisons between matrix assisted laser desorption ionization time of flight mass spectroscopy analysed partial tryptic peptide digests of the purified protein with the predicted amino acid sequences deduced from the α-glucosidase cDNA sequence.

Expression of a bee venom phospholipase A_2 from Apis cerana cerana in the baculovirus-insect cell

Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2) gene from the Chinese honeybee(Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector.Tn-5B-4(Tn) cells were transfected with the recombinant bacmid DNA for expression.Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa.Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins.The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2(BvPLA2 of the European honeybee,Apis mellifera) polyclonal serum.The reaction resulted in a double glycosylation band,which agrees with the band generated by the native AmPLA2 in Western blot analysis.The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg).In summary,the recombinant AccPLA2 protein,a native BvPLA2-like structure with corresponding biological activities,can be glycosylated in Tn cells.These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.

Insulin receptor participates in the peripheral olfactory processes of honey bees (Apis cerana cerana)

Insulin receptors(InR)are an integral component of the insulin/insulin-like growth factor signaling pathway,which plays a vital role in insect development,lifespan,reproduction,and olfactory sensitivity.However,whether InR participate in the periph-eral olfactory system of insects remains unclear.Recently,we found that 2-heptanone(2-HT)affects AcerlnR expression,the gene for an InR protein,in Apis cerana cerana.We then examined the spatiotemporal expression profile of the gene in A.cerana cerana.The mRNA of AcerlnR was primarily expressed in the antennae,wings,and legs of forager bees,which are probable chemosensory tissues.The results of fluorescence competitive binding assays,combined with site-directed mutagenesis,demonstrated that AcerOBP6 and AcerOBP14 exhibit strong binding affinities to 2-HT.Furthermore,after foragers were fed with double-stranded AcerlnR,the expression levels of AcerOBP6 and AcerOBP14 decreased significantly,as did the electroantennogram responsiveness to 2-HT and some other odorants.In conclusion,our findings provide a foundation for understanding the in-volvement of AcerlnR in the odor perception of A.cerana cerana.Moreover,they offer novel insights into the olfactory recognition mechanism in insects.

Defining honeybee subspecies in an evolutionary context warrants strategized conservation

Despite the urgent need for conservation consideration,strategic action plans for the preservation of the Asian honeybee,Apis cerana Fabricius,1793,remain lacking.Both the convergent and divergent adaptations of this widespread insect have led to confusing phenotypical traits and inconsistent infraspecific taxonomy.Unclear subspecies boundaries pose a significant challenge to honeybee conservation efforts,as it is difficult to effectively prioritize conservation targets without a clear understanding of subspecies identities.Here,we investigated genome variations in 362 worker bees representing almost all populations of mainland A.cerana to understand how evolution has shaped its population structure.Whole-genome single nucleotide polymorphisms(SNPs)based on nuclear sequences revealed eight putative subspecies,with all seven peripheral subspecies exhibiting mutually exclusive monophyly and distinct genetic divergence from the widespread central subspecies.Our results demonstrated that most classic morphological traits,including body size,were related to the climatic variables of the local habitats and did not reflect the true evolutionary history of the organism.Thus,such morphological traits were not suitable for subspecific delineation.Conversely,wing vein characters showed relative independence to the environment and supported the subspecies boundaries inferred from nuclear genomes.Mitochondrial phylogeny further indicated that the present subspecies structure was a result of multiple waves of population divergence from a common ancestor.Based on our findings,we propose that criteria for subspecies delineation should be based on evolutionary independence,trait distinction,and geographic isolation.We formally defined and described eight subspecies of mainland A.cerana.Elucidation of the evolutionary history and subspecies boundaries enables a customized conservation strategy for both widespread and endemic honeybee conservation units,guiding colony introduction and breeding.

中华蜜蜂养殖业在我国的发展方向和解决方案

分析中华蜜蜂养殖规模小和多点性特点,提出解决我国中华蜜蜂养殖业的方案。建议采用活节叠加式仿生蜂箱,使养蜂技术标准化和简单化、门槛降低,可扩大养殖的规模;使用网络技术、大数据分析技术实现养殖的精准管理;最终在国家政策的引领下,实现中华蜜蜂产业集团化。

多花黄精花粉萌发与传粉生物学特性

以同质园移栽的多花黄精Polygonatum cyrtonema Hua为研究对象,对其开花物候、访花昆虫种类、访花行为及传粉过程进行观察,并采用单因素和正交设计研究不同培养基(蔗糖、H_(3)BO_(3)、CaCl_(2))对多花黄精离体花粉萌发的影响,比较I_(2)-KI染色法、MTT染色法与TTC染色法测定花粉活力的差异,检测其花粉活力与柱头可授性,并统计其自然结实率。结果表明:(1)多花黄精花期为4月末到6月初,盛花期在5月中旬,单株、花序和单花水平的花期依次为8~13、3~7、1~3 d,开花振幅为多峰曲线式样,开花同步性较高,花期同步指数为0.81±0.12;(2)离体花粉萌发的最适条件是150.00 g/L蔗糖+200.00 mg/L H_(3)BO_(3)+250.00 mg/L CaCl_(2);(3)TTC染色法是快速且适宜的测定多花黄精离体花粉活力的方法;(4)花粉在开花前1~2 d开始有活力,在开花后2~3 d花粉活力最高,此后随着花药散粉活力逐渐下降,花枯萎后花粉活力丧失;(5)柱头在开花前1~2 d至开花后4~5 d均有可授性,其中开花后2~3 d柱头可授性最高;(6)多花黄精的传粉昆虫为中华蜜蜂Apis cerana、黑带食蚜蝇Episyrphus balteatus和熊蜂Bombus latreille,中华蜜蜂是其主要的传粉昆虫;(7)多花黄精自然结实率为46.35%。

重庆彭水县中华蜜蜂形态指标测定及比较研究

采集重庆彭水县中华蜜蜂(Apis cerana cerana)主要栖息区内18个乡镇的蜜蜂样本,对工蜂吻长、前翅长等6项主要形态指标进行测定,并将彭水县中华蜜蜂形态指标数据与湖北、江西等17地已报道的中华蜜蜂形态指标进行比较。结果显示,彭水县中华蜜蜂的翅长、翅宽、T3+T4背板长平均值处于中上水平,暗示其采集能力和花蜜储存能力有一定优势。试验结果可为当地中华蜜蜂遗传资源调查与保护提供形态学数据参考。