The Effects of Temperature and Humidity around the Beehives on the Distribution of Nosema ceranae, and also Geographical and Seasonal Activity of the Infection in the Eastern Black Sea Region of Turkey.

20 localities were randomly selected in Eastern Black Sea Region of Turkey and samples were collected from around the beehives from April to September. Total of 4,640 dead adult worker bees were examined during the study. Total infection rate in worker bees was 21.23%. Nosema ceranae was identified in all localities with molecular techniques. Temperature and humidity values were measured from around the beehives during field studies. The infection rate ofN. ceranae increased proportionally with increasing temperature and humidity factors. Humidity was more effective than temperature on the infection rate ofN. ceranae. The seasonal activity ofN. ceranae was studied. The highest infection rates were observed in June and July. N. ceranae infection rate was higher in localities that were in low-altitude than in localities that were in high-altitude.

Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

Propolis collected by stingless bees from various types of plants has been used as an antimicrobial agent in several previous studies. We assessed the effect of propolis produced by a stingless bee, Trigona apicalis, on Apis florea experimentally infected with Nosema ceranae, a parasite of honeybees. For parasite inoculation each Nosema free-bee was fed 2μL of 50% （w/v） sucrose solution containing N. ceranae spores at 40,000 spores/bee and 0 as a negative control （CO）. Treated bees were provided with 0%, 10%, 20% and 50% propolis （w/v） in water, defined as 0P, 10P, 20P and 50P, respectively. We assessed the effects of propolis 14 days post inoculation. All propolis-treated bees had significantly higher survival than untreated bees. However, survival of Nosema-inoculated bees was lower than that of control bees. Bees treated with the highest propolis concentration （50P） had the highest survival ratio. No control bees became infected over the course of the study. However, N. ceranae infection rates of bees treated with 0P, 10P, 20P and 50P were 75 ± 1.4%, 72 ± 5.6%, 69± 4.2% and 47± 1.4%, respectively. In addition, propolis-treated bees had hypopharyngeal gland protein content that was significantly higher than 0P and CO bees. Overall, propolis treatment significantly reduced N. ceranae infection rate and bee mortality and was associated with increased hypopharyngeal gland protein concentration.

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.

意大利蜜蜂(Apis mellifera ligustica)与中华蜜蜂(Apis cerana ceraca)的生态位比较

为了明确中华蜜蜂和意大利蜜蜂在皖南山区生态适应性,研究两种蜜蜂的食物生态位、时间生态位和空间生态位及其差异,结果是中蜂与意蜂食物(蜜源植物)资源生态位宽度分别是0.923、0.765,中蜂对蜜源植物采集喜好性差异小,而意蜂差异大,中蜂对意蜂生态位重迭为0.160,中蜂对意蜂生态位相似性为0.755;油菜花期,中蜂与意蜂时间资源生态位宽度分别是0.879、0.801,枇杷花期,分别是0.760、0.677,中蜂与意蜂的空间资源生态位宽度分别是0.797、0.670。中蜂3种生态位宽度均大于意蜂,中蜂三维生态位值是意蜂的1.61倍和1.57倍。表明中蜂在皖南山区生态适应性比意蜂强。

兔耳兰食源性欺骗传粉的研究

兰科植物具有精巧、多样化的花部结构以及高度多样的吸引传粉者方式。作者对广西雅长兰科植物自治区级保护区内的一个兔耳兰(Cymbidium lancifolium)居群进行了连续2年的观察和研究。观察发现兔耳兰唯一的传粉者为膜翅目蜜蜂科的中华蜜蜂(Apis cerana cerana)。中华蜜蜂一般直接落在唇瓣外弯的中裂片上,然后调整身体的方向,进入花中,当发现花中无蜜液等回报时,借助于后足的蹬力退出花朵。在退出的过程中,花粉块连同药帽会通过粘盘粘附在中华蜜蜂的胸部。中华蜜蜂在花内的停留时间为8-71s,平均18.3s(N=11)。根据观察我们推测兔耳兰可能是通过其唇瓣上无规则的紫栗色小斑点(假蜜导)来吸引中华蜜蜂为其传粉,属于食源性欺骗方式。在传粉过程中兔耳兰的药帽与花粉团和粘盘一起粘在中华蜜蜂背部。药帽的存在能够阻止下一朵被拜访的花实现雌性功能。兔耳兰药帽高度(0.154±0.032cm)(N=10)加上传粉昆虫胸高(2005年为0.37±0.03cm(N=10),2006年0.35±0.04cm(N=7))大于传粉通道入口的高度(0.29±0.04cm)(N=21),支持兔耳兰可能通过药帽来减少同株异花授粉现象的推测。2005和2006年该兔耳兰居群的自然繁殖成功率分别为21.13%和21.28%。繁育系统实验证明兔耳兰是高度自交亲和物种,自交和异交的繁殖成功率没有显著性差异,表明该种在结实过程中未显示近交衰退。兔耳兰不存在无融合生殖和自花授粉的现象,其结实依赖传粉者。TTC法检测结果显示兔耳兰种子活力达85.78%(N=11),可见种子活力不是制约兔耳兰种子萌发的主要原因。因此传粉者的密度和访问频率可能是影响兔耳兰结实的重要因素,并最终影响兔耳兰种群的维持和扩张。

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.

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。

Molecular identification of Nosema species in provinces of Fars,Chaharmahal and Bakhtiari and Isfahan(Southwestern Iran)

Objective: To molecularly identify Nosema species in provinces of Isfahan, Fars,Chaharmahal and Bakhtiari.Methods: One hundred and eighty adult honey bees suspected with nosemosis from provinces of Fars(different counties), Isfahan, and Chaharmahal and Bakhtiari were tested. In order to determine the species of Nosema, previously developed PCR and primers based on 16 S r RNA gene were used. PCR products were puri fi ed and sent to the Korean company of Macrogen for sequencing.Results: Only Nosema ceranae was determined in all samples based on their molecular profile. Sequences of the 16 S r RNA gene were sent to Gen Bank/NCBI(samples accession numbers KP318660–KP318663).Conclusions: This species currently exists in European honeybee apiaries of Apis mellifera in the studied provinces.

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%。

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.

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。

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.

常春油麻藤有气味花蜜及其生态功能

一些被子植物能够分泌有气味的花蜜,但这一自然现象很少被关注。作为嗅觉信号线索,有气味的花蜜可能是将访花者和气味信号结合在一起的特征,它与传粉者及盗蜜者的关系值得探索。本研究以常春油麻藤(Mucuna sempervirens)为对象,研究了其开花动态及泊氏长吻松鼠(Dremomy spernyi)和赤腹松鼠(Callo sciuruserythraeus)的访花行为,采用顶空固相微萃取和气相色谱-质谱法收集并分析了花蜜的挥发物成分,探讨了花蜜对中华蜜蜂(Apiscerana cerana)的吸引作用及对酸臭蚁(Tapinoma sp.)的毒杀作用。结果表明:常春油麻藤花蜜释放的挥发物以脂肪族化合物为主(87.2%),其中酮类占56.1%,无含硫挥发性成分,这和该属其他蝙蝠传粉种类花蜜释放含硫化合物的结果不一致。此外,常春油麻藤的花蜜对酸臭蚁有慢性毒杀作用,而对中华蜜蜂则有吸引作用。未发现蝙蝠访花,但观察到泊氏长吻松鼠和赤腹松鼠可能为常春油麻藤传粉。因此,常春油麻藤可能不属于蝙蝠传粉的种类。希望本研究能为该属亚洲类群的传粉机制提供数据,并为其他植物类群花蜜成分及功能研究提供新的视角。

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.

Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen, Paenibacillus larvae

American foulbrood (AFB) disease is caused by Paenibacillus larvae. Currently, this pathogen is widespread in the European honey bee— Apis mellifera. However, little is known about infectivity and pathogenicity of P. lan'ae in the Asiatic cavity-nesting honey bees, Apis cerana. Moreover, comparative knowledge of P. larvae infectivity and pathogenicity between both honey bee species is scarce. In this study, we examined susceptibility, larval mortality, survival rate and expression of genes encoding antimicrobial peptides (AMPs) including defensin, apidaecin, abaecin, and hymenoptaecin in A. mellifera and A. cerana when infected with P. larvae. Our results showed similar effects of P. larvae on the survival rate and patterns of AMP gene expression in both honey bee species when bee larvae are infected with spores at the median lethal concentration (LC5 0 ) for A. mellifera. All AMPs of infected bee larvae showed significant upregulation compared with noninfected bee larvae in both honey bee species. However, larvae of A. cerana were more susceptible than A. mellifera when the same larval ages and spore concentration of P. larvae were used. It also appears that A. cerana showed higher levels of AMP expression than A. mellifera. This research provides the first evidence of survival rate, LC50 and immune response profiles of Asian honey bees, A. cerana, when infected by P. larvae in comparison with the European honey bee, A. mellifera.

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.

Genetic study on the behavior of honeybees influenced by polyandry system

Within a honeybee population, due to polyandry, there are super-sister and half-sister relations, thus many sub-families exist. For the Chinese honeybee (Apis cerana cerana F.) a phylogenetic dendrogram has been constructed in which 4 sub-families are clustered based on DNA fingerprint patterns. And it has been observed that each kind of workers is distributed to several different sub-families.