Crystal structure of peptidoglycan recognition protein SA in Apis mellifera (Hymenoptera: Apidae)

Peptidoglycan recognition protein SA (PGRP‐SA) is a key pattern recognition receptor in the insect innate immune system. PGRP‐SA can bind to bacterial PGN and activate the Toll pathway, which triggers the expression and release of antimicrobial peptides to prevent bacterial infection. Here, we report the first structure of Apis mellifera PGRP‐SA from Hymenoptera at 1.86 Å resolution. The overall architecture of Am‐PGRP‐SA was similar to the Drosophila PGRP‐SA; however, the residues involved in PGN binding groove were not conserved, and the binding pocket was narrower. This structure gives insight into PGN binding characteristics in honeybees.     

意大利蜜蜂胚后发育过程中中肠上皮组织细胞的更替

中肠是昆虫消化、 吸收营养物质的主要部位。本研究通过形态解剖、 BrdU免疫组织化学和原位末端转移酶标记(TUNEL)细胞凋亡检测等技术, 对意大利蜜蜂Apis mellifera ligustica中肠胚后发育过程中细胞的增殖和凋亡模式进行了比较研究。结果表明：意大利蜜蜂幼虫发育早期, 中肠的增加主要来自于上皮细胞的分裂以及再生细胞的增殖。在变态发育期间, 中肠上皮经历了广泛的重组, 由再生细胞重新形成的蛹上皮替代了幼虫上皮。再生细胞在蜜蜂中肠的整个发育阶段始终存在, 为中肠的生长和更替提供了主要的细胞来源。本研究为昆虫组织细胞自噬和凋亡机制的研究提供一定的证据。     

意大利蜜蜂不同日龄工蜂脑部三种miRNA的表达水平

【目的】意大利蜜蜂 Apis mellifera ligustica（简称“意蜂”）是社会性昆虫,蜂群中的工蜂表现出年龄依赖性的行为转变,其行为转变的机制一直是研究的热点。本研究分别检测了3种miRNA（ame-let-7, ame-miR-13b和ame-miR-279）在不同日龄意蜂工蜂脑部的表达情况,以期为探究意蜂工蜂年龄依赖性行为变化机制提供重要线索。【方法】通过荧光定量PCR分别检测了不同发育时期（4, 8, 12, 17, 22, 26和30日龄）意蜂工蜂脑部3种miRNA的表达情况,并检验miRNA的表达差异情况。【结果】ame-let-7的表达量随工蜂日龄的增加逐渐降低,在17日龄后其表达量趋于稳定;ame-miR-13b的表达量则随着工蜂日龄的增加逐渐增加,但在26和30日龄的工蜂中稳定表达;ame-miR-279的表达量则呈类似正态分布状态,其中在12日龄工蜂中表达量最高,且在17日龄后稳定表达。这3种miRNA表达均具有明显的时间特异性。【结论】 ame-let-7, ame-miR-13b和ame-miR-279的表达与工蜂年龄依赖性行为变化具有相关性和规律性,对于深入探究miRNA的作用位点具有重要参考价值。     

成年蜜蜂脑神经细胞的培养和电生理特征

为了研究杀虫剂等对蜜蜂毒性作用的神经机制,需在体外建立成年蜜蜂脑神经细胞的分离培养和电生理记录技术并研究其正常电生理特征,而对成年蜜蜂脑神经细胞的分离培养和电生理特性的研究报道甚少。我们采用酶解和机械吹打相结合的方法获得了数量较多且活力较好的成年意大利蜜蜂Apis mellifera脑神经细胞,并用全细胞膜片钳技术研究了成年意大利蜜蜂脑神经细胞对电流和电压刺激的反应,获得了成年意蜂脑神经细胞的基本电生理特征以及钠电流和钾电流的特性。全细胞电流钳的记录结果表明,在体外培养条件下,细胞无自发放电发生,注射电流后仅引起细胞单次放电,引起细胞放电的阈电流平均为60.8±63 pA; 细胞动作电位产生的阈电位平均为−27.4±2.3 mV。用全细胞电压钳记录了神经细胞的钠电流和钾电流。钠电流的分离是在电压刺激下通过阻断钾通道和钙通道实现。细胞的内向钠电流在指令电压为−40～−30 mV左右激活,−10 mV达峰值,钠通道的稳态失活电压V_1/2为−58.4 mV; 外向钾电流成份至少包括较小的快速失活钾电流和和较大的缓慢失活钾电流（占总钾电流的80%）,其半激活膜电位V_1/2为3.86 mV,无明显的稳态失活。结果提示缓慢失活钾电流的特征可能是细胞单次放电的机制之一。     

意大利蜜蜂刚出房与性成熟雄蜂的蛋白质组差异分析

【目的】对意大利蜜蜂Apis mellifera ligustica刚出房雄蜂与性成熟雄蜂的蛋白质组进行比较,探讨雄蜂在性成熟过程中蛋白质表达变化,为进一步研究雄蜂发育生物学获得差异表达蛋白质方面的依据。【方法】采用双向电泳法建立意大利蜜蜂雄蜂发育过程中刚出房时与性成熟期的蛋白质表达谱,通过质谱分析与数据库检索,鉴定部分差异蛋白。【结果】在意大利蜜蜂刚出房雄蜂和性成熟雄蜂中分别检测到2 490和2 317个蛋白点,其中差异表达蛋白点有157个。在刚出房雄蜂中高度表达的蛋白点有102个;在性成熟雄蜂中高度表达的蛋白点有55个。对部分差异蛋白进行质谱分析,共鉴定了18个蛋白点,其中在刚出房雄蜂中上调表达的蛋白有肌钙蛋白、SEC13蛋白、DJ蛋白等,在性成熟雄蜂中上调表达的蛋白有副肌球蛋白、精氨酸激酶、肌动蛋白解聚因子等。【结论】意大利蜜蜂雄蜂在性成熟发育过程中,其体内大量蛋白表达发生了变化,其差异表达的蛋白质可能与骨骼、飞行肌以及精子发育等机能有关。     

意蜂工蜂酸性磷酸酶的纯化及其酶学特性

从意蜂Apis mellifera工蜂体内分离提纯酸性磷酸酶（ACPase, EC3.1.3.2）,并对其性质进行了研究。将工蜂酸性磷酸酶的初提物经分段盐析、DEAE-Sepharose FF离子交换层析及Sephadex G-200 凝胶过滤等纯化步骤,得到经聚丙烯酰胺凝胶电泳为单一蛋白区带的酶液。提纯倍数为77.24,酶液比活力为16.22 U/mg（对硝基苯磷酸二钠作底物）。利用凝胶过滤法测定酶的相对分子质量为135 kD,SDS-PAGE测定酶的亚基相对分子质量为63 .1 kD。酶的等电点为4.46和4.79。非还原/还原（NR/R）单向、双向SDS-PAGE显示酶分子含有链内二硫键。对二级结构圆二色谱分析显示,酶分子中α-螺旋占13.84%,β-折叠占25.68%,无规则卷曲占56.34%。氨基酸组成分析结果表明, 酸性磷酸酶约含有507个氨基酸残基,富含门冬氨酸残基。     

西方蜜蜂和兰州熊蜂在设施桃园的访花偏好性比较

【目的】为了比较西方蜜蜂 Apis mellifera 和兰州熊蜂 Bombus lantschouensis 在设施桃园内对不同时期桃花的访花偏好性、以及这种偏好性与花粉活力和采集花粉花蜜之间的关系。【方法】记录2种蜂在温室桃园内访问早期花、中期花和晚期花的比例,测定桃花不同时期的花粉活力以及2种蜂携带的花粉活力,观察2种蜂采集花蜜和采集花粉的成功率,统计2种蜂访花过程中桃花所处的枝条数及植株数。【结果】桃花不同时期的花粉活力差异显著,早期花花药未开裂,花粉未释放,中期花花粉活力（58.3%）显著高于晚期花花粉活力（34.2%）（P<0.01）;西方蜜蜂更加偏好访问中期花,对中期花的访问率高达75.3%,显著高于兰州熊蜂对中期花的访问率（49.2%）（P<0.01）;西方蜜蜂携带的花粉活力（92.1%）显著高于兰州熊蜂携带的花粉活力（72.9%）,但是西方蜜蜂采集花粉和采集花蜜的成功率均低于兰州熊蜂（P<0.01）;在访问一定数量的桃花过程中,兰州熊蜂在设施桃园内访问的枝条数和植株数较多,分布范围较广（P<0.01）。【结论】和兰州熊蜂相比,西方蜜蜂对活力花粉的辨别能力更强,更加偏好访问花粉活力较高的花朵,这种偏好性导致其采集花粉花蜜的成功率降低。     

Mitochondrial DNA variability in the Canary Islands honeybees (Apis mellifera L.)

The mitochondrial DNA (mtDNA) of individuals from 79 colonies of Apis mellifera from five Canary Islands was studied using the Dra I test based on the restriction of PCR products of the tRNAleu–COII intergenic region. Five haplotypes of the African (A) lineage and one of the west European (C) lineage were found. The haplotypes A14 and A15 are described for the first time. These haplotypes have a new P sequence named P₁. The wide distribution and high frequency of haplotype A15 suggest that it is characteristic of the Canarian Archipelago. Sources of haplotype variability of honeybee mtDNA in the Canary Islands (waves of colonization from Africa, queen importations, habitat diversification) are discussed.     

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected approximately 1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive eubacteria), Lactobacillus (Gram-positive eubacteria), and Gluconacetobacter (Gram-negative alpha-proteobacteria); two sequences each clustered with Simonsiella (beta-proteobacteria) and Serratia (gamma-proteobacteria); and three sequences each clustered with Bartonella (alpha-proteobacteria). Although the sequences relating to these six bacterial genera initially were obtained from either A. m. capensis or A. m. scutellata or both, newly designed honeybee-specific 16S rRNA primers subsequently amplified all sequences from all individual workers of both subspecies. Attempts to amplify these sequences from eggs have failed. However, the wsp primers designed to amplify Wolbachia DNA from arthropods, including these bees, consistently produced a 0.6-kb DNA band from individual eggs, indicating that amplifiable bacterial DNA was present. Hence, the 10 bacteria could have been acquired orally from workers or from other substrates. This screening of 16S rRNA sequences from A. m. capensis and A. m. scutellata found sequences related to Lactobacillus and Bifidobacterium which previously had been identified from other honeybee subspecies, as well as sequences related to Bartonella, Gluconacetobacter, Simonsiella/Neisseria, and Serratia, which have not been identified previously from honeybees.     

噻虫胺及其混配制剂对意大利蜜蜂和玉米螟赤眼蜂的急性毒性

【目的】明确新烟碱类杀虫剂噻虫胺及其2种混剂对意大利蜜蜂 Apis mellifera ligustica 和玉米螟赤眼蜂 Trichogramma ostriniae 的毒性。【方法】采用摄入法、接触法和药膜法分别测定3种制剂对意大利蜜蜂成年工蜂和玉米螟赤眼蜂成蜂的急性毒性。【结果】急性毒性测定结果表明,30%噻虫胺悬浮剂、30%吡蚜酮·噻虫胺悬浮剂和20%醚菊酯·噻虫胺悬浮剂对意大利蜜蜂成年工蜂的急性摄入毒性均为剧毒,LC₅₀ 值(48 h)分别为0.0200（0.0143~0.0272）, 0.084（0.0658~0.1157）和0.1594（0.1200~0.2056） mg a.i./L;3种制剂对意大利蜜蜂成年工蜂急性接触毒性均为高毒,LD₅₀ 值(48 h)分别为0.0155（0.0114~0.0197）, 0.0426（0.0335~0.0539）和0.1122（0.0796~0.1385）μg a.i./蜂。药膜法测定3种制剂对玉米螟赤眼蜂成蜂的LD₅₀ 值(24 h)分别为0.0232（0.0180~0.0295）, 0.1050（0.0940~0.1170）和0.0059（0.0054~0.0065） mg a.i./L;安全性评价结果表明,3种制剂对玉米螟赤眼蜂成蜂均存在极高风险性,安全系数分别为5.95×10^-4, 2.69×10^-3和9.50×10^-5。【结论】噻虫胺及其混剂对意大利蜜蜂和玉米螟赤眼蜂均存在较高的毒性风险,在害虫综合治理中应谨慎使用。     

Development of Beauveria bassiana dry formulation for vectoring by honey bees Apis mellifera (Hymenoptera: Apidae) to the flowers of crops for pest control

Using bee pollinators as a means for the dissemination of microbial control agents, such as Beauveria bassiana, against insect pests of agricultural crops is a novel and interesting approach to biological control. In four laboratory trials, one in Canada and three in Jordan, factors affecting the acquisition of B. bassiana by honey bees were evaluated using hive-mounted inoculum dispensers. The numbers of conidia carried by bees emerging from the dispensers differed according to the type of carrier used. Bees that passed through corn flour acquired more inoculum than did those that walked through wheat flour, durum semolina, corn meal, potato starch, potato flakes, oat flour or barley flour. The numbers of conidia acquired by the bees increased with decreasing particle size and moisture content of the carrier, and with increasing density of B. bassiana conidia in the formulation. Time required for a bee to pass through the dispenser did not significantly affect the acquisition of conidia. This study indicated that honeybees (Apis mellifera carnica) have a great potential for vectoring B. bassiana in crop systems. It also opens more avenues for studies on bee delivery of other microbial biological control agents.     

亚致死剂量吡虫啉对意大利蜜蜂中肠细菌群落的影响（英文）

【目的】在实验室条件下,研究亚致死剂量吡虫啉对意大利蜜蜂 Apis mellifera ligustica 中肠细菌群落的影响,以期为杀虫剂的生物安全性评价体系提供理论依据。【方法】分别用3种不同浓度（0.005, 0.015 和0.045 mg/L）的吡虫啉蔗糖溶液以及含丙酮（0.03%）的糖水(空白对照)在实验室内饲喂刚出房的意大利蜜蜂,在饲喂3, 6, 9, 12和15 d时分别取样。提取中肠细菌基因组DNA,采用下一代高通量测序技术对4个处理组蜜蜂中肠细菌群落进行检测分析。【结果】细菌序列分析表明,意大利蜜蜂中肠细菌主要属于变形菌门（Proteobacteria）和厚壁菌门（Firmicutes）,同一时间内各处理组间主要细菌数量差异不显著（P>0.05）,同时细菌群落多样性指数差异也不显著（P>0.05）。【结论】在实验室条件下亚致死剂量吡虫啉对意大利蜜蜂中肠细菌群落结构影响不显著。     

意大利蜜蜂和小峰熊蜂在温室桃园的传粉行为及其影响因素

意大利蜜蜂Apis mellifera ligustica和小峰熊蜂Bombus hypocrita是我国设施农业中的两种主要授粉昆虫, 二者的传粉方式和传粉效率因不同作物而各有不同。为了选用最理想的蜂种为温室作物授粉, 提高作物的授粉效率, 我们于2008-2010年连续3年在北京的温室桃园进行了意大利蜜蜂和小峰熊蜂的传粉行为及其影响因素的研究。结果表明, 两种蜂的日活动规律不同, 和意大利蜜蜂相比, 小峰熊蜂的活动起点温度低、 日工作时间长、 单花访问时间长, 采集蜂在温室内距离蜂巢不同距离之间扩散比较均匀。而意大利蜜蜂采集蜂的数量随授粉半径的增大而明显减少。温室内环境因子对两种蜂传粉活动的影响基本一致, 温度对两种蜂的传粉活动影响最大, 其次是湿度、 单花花蜜浓度和光照强度, 而单花花蜜体积对蜂活动无明显影响。本研究认为对于温室桃授粉应优先选用授粉性能稳定的小峰熊蜂, 并且适当调节温室内环境条件, 以提高其授粉效率。     

西方蜜蜂发育基因AmWnt1的克隆鉴定及时空表达分析

本研究旨在克隆鉴定西方蜜蜂Apis mellifera发育相关基因AmWnt1,分析其在不同发育时期和刚出房工蜂不同组织的表达特征,为进一步研究Wnt1基因功能提供理论参考。根据NCBI中AmWnt1基因序列信息,利用Primer 6.0设计引物,RT-PCR扩增AmWnt1基因完整的CDS序列,进行生物信息学预测,用推导的氨基酸序列构建系统进化树;利用荧光定量PCR检测该基因在卵（1日龄、2日龄和3日龄）、幼虫（1日龄、3日龄和5日龄）、预蛹（1日龄和3日龄）、蛹（0日龄、2日龄、4日龄、6日龄和8日龄）、刚出房工蜂、哺育蜂和采集蜂以及刚出房工蜂8个组织中相对表达量。克隆获得西方蜜蜂的Wnt1基因CDS序列,命名为AmWnt1,上传NCBI,获得GenBank登录号MT993937。全长1 239 bp,编码412个氨基酸,预测等电点为9.48,相对分子质量为46.40313 kDa。序列比对和系统进化树结果表明：AmWnt1蛋白与其它膜翅目昆虫聚为一类,其中和东方蜜蜂Apis cerana亲缘关系最近,序列相似度为99.50%。时空表达谱结果表明：AmWnt1基因在各个时期中均有表达,且在胚胎后期表达量最高,预蛹期和蛹前期表达量相对较高,其它时期表达量相对较低;AmWnt1基因在刚出房工蜂头、胸、触角表达量高于其它组织。AmWnt1可能参与西方蜜蜂胚胎晚期的神经系统发育和化蛹过程中的四肢发育等关键历程,为进一步研究AmWnt1功能提供了理论参考。     

微小RNA ame-miR-31a和ame-miR-13b在意大利蜜蜂哺育蜂和采集蜂脑部的表达差异

【目的】研究微小RNA ame-miR-31a和ame-miR-13b在意大利蜜蜂Apis mellifera ligustica哺育蜂和采集蜂脑部的表达情况,探索其在蜜蜂哺育蜂和采集蜂行为转变中的调节作用。【方法】利用qPCR分别检测ame-miR-31a和ame-miR-13b在意大利蜜蜂工蜂正常蜂群和同龄蜂群中哺育蜂和采集蜂脑部的表达量。【结果】不论在意大利蜜蜂工蜂正常蜂群中还是在同龄蜂群中,ame-miR-31a在哺育蜂脑部的表达量总是显著高于其在采集蜂脑部的表达量;在正常蜂群中,ame-miR-13b在采集蜂脑部的表达量显著高于其在哺育蜂脑部中的表达量;在同龄蜂群中,ame-miR-13b在年老哺育蜂脑部的表达量极显著高于其在同龄采集蜂脑部的表达量,在年轻哺育蜂脑部与同龄年轻采集蜂脑部的表达量差异不显著。【结论】ame-miR-13b在意大利蜜蜂哺育蜂和采集蜂脑部的表达没有明显规律,而ame-miR-31a总是在哺育蜂脑部高表达,提示ame-miR-31a在蜜蜂哺育蜂和采集蜂行为转变中可能发挥重要的调控作用。     

意大利工蜂不同发育时期抗氧化酶基因mRNA表达量的变化

超氧化物歧化酶（SOD）和过氧化氢酶（CAT）在清除工蜂体内自由基的过程中起着关键的作用, 其活性和基因表达量的高低与蜜蜂的寿命、 抗病力有直接的联系, 进而影响着蜂群的生产性能。本研究采用实时荧光定量PCR法研究意大利蜜蜂Apis mellifera ligustica不同发育时期铜锌超氧化物歧化酶（copper zinc superoxide dismutase, CuZnSOD）、 锰超氧化物歧化酶（manganese superoxide dismutase, MnSOD）及过氧化氢酶（catalase, CAT）基因mRNA表达量的变化。实验分别在幼虫、 蛹、 成虫3个阶段5个时间点（产卵后5 d的幼虫, 产卵后14 d的蛹, 出房后1 d, 12 d, 32 d的成虫）取样。结果表明, 3个抗氧化酶基因在各个发育阶段均有表达, 其中CuZnSOD和MnSOD mRNA的表达量在幼虫到蛹期和出房后12-32 d两个阶段呈下降趋势, 而都以出房后1 d时表达量最大, 蛹期表达量最小, 且显著低于其他4个时期(P<0.05)。CuZnSOD基因 mRNA的表达量在成虫阶段的下降趋势不明显, MnSOD mRNA的表达量则显著下降(P<0.05); 而CAT基因的mRNA表达量以蛹期最低, 且其表达量随蜜蜂的生长发育稳定上升, 在出房后12 d和32 d时表达量显著高于其他3个时期。这些研究结果在分子水平上揭示了抗氧化酶基因在蜜蜂不同发育时期的作用。     

Analysis of nuclear copies of mitochondrial sequences in honeybee (Apis mellifera) genome

At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.     

Phylogenetic analysis of Starmerella apis in honey bees (Apis mellifera)

Honey bees are among the most effective pollinators that promote plant reproduction. Bees are highly active in the pollen collection season, which can lead to the transmission of selected pathogens between colonies. The clade Starmerella comprises yeasts that are isolated mainly from bees and their environment. When visiting plants, bees can come into contact with Starmerella spp. The aim of this study was to determine the prevalence and phylogenetic position of S. apis in bee colonies. Bee colonies were collected from nine apiaries in three regions. Ten colonies were sampled randomly from each apiary, and pooled samples were collected from the central part of the hive in each colony. A total of 90 (100%) bee colonies from nine apiaries were examined. Starmerella apis was detected in 31 (34.44%) samples, but related species were not identified. The 18S rRNA amplicon sequences of S. apis were compatible with the GenBank sequences of Starmerella spp. from India, Japan, Syria, Thailand, and the USA. The amplicon sequences of S. apis were also 99.06% homologous with the sequences deposited in GenBank under accession numbers JX515988 and NG067631 .This is the first study to perform a phylogenetic analysis of S. apis in Polish honey bees.     

蜜蜂蜂王与工蜂级型分化研究进展

蜜蜂ApismekiferaL .是典型的社会性昆虫 ,蜂王和工蜂都是由受精卵发育而来的二倍体成蜂 ,但是在形态、生理、行为等方面有明显的差异 ,属于不同的级型。蜂王和工蜂的级型分化的关键时期发生在幼虫的 4龄末至 5龄止。分化是由分化基因调控的 ,幼虫期食物的质和量是分化的外部决定因子。JH对两级型中卵巢的分化有非常重要的调控作用。蜜蜂脑或其它组织中可能有分泌调控CA的咽侧体调节激素 ,它们通过对CA中JH的合成和分泌的调控而参与了分化的调控。章鱼胺等生物胺也参与了分化调控过程。