基于Illumina HiSeq测序技术研究吡虫啉对意大利蜜蜂雄蜂的影响

[目的]雄蜂对蜂群繁衍有着非常重要的作用.本研究旨在探究吡虫啉对意大利蜜蜂Apis mellifera Ligustica雄蜂生长发育和基因表达产生的影响.[方法]以意大利蜜蜂雄蜂为研究对象,分别以0.00001、0.0001和0.001 μg/μL浓度的吡虫啉对雄蜂幼虫进行连续饲喂处理.每天观察并记录幼虫的发育形态及死亡率,在雄蜂幼虫后期(移虫后6d)测量幼虫体重.利用Illumina HiSeq测序技术对经吡虫啉处理的雄蜂进行转录组测序,进而对差异表达基因进行深入分析.[结果]取食吡虫啉后的雄蜂幼虫,体重低于正常雄蜂,当浓度高于0.0001μg/μL时差异显著;雄蜂幼虫取食吡虫啉后出现死亡现象,且死亡率随吡虫啉浓度的升高而增大;差异表达基因分析结果上调与下调基因数量分别为390个和130个.GO富集分析结果上调基因共分布于55个GO条目,富集基因数量最多的是细胞进程、细胞、细胞组件、细胞膜、细胞膜组件、结合,下调基因共分布于48个GO条目,富集基因数量最多的是细胞进程、细胞、细胞组件.富集在有关生殖功能的差异表达基因中,上调基因数量为21个,下调基因数量为5个.KEGG代谢通路富集分析结果上调基因富集在159个通路上,其中富集基因数最多的是蛋白质消化吸收和神经活性配体-受体相互作用通路.下调基因富集在71个通路上,其中富集基因数最多的是溶酶体、胰液分泌、神经活性配体-受体相互作用通路.[结论]吡虫啉能抑制意大利蜜蜂雄蜂的生长发育,甚至造成幼虫死亡,同时,可以影响雄蜂的神经系统、代谢系统和生殖系统等.本研究结果为蜜蜂资源保护提供理论依据.     

亚致死剂量吡虫啉胁迫对意大利蜜蜂工蜂嗅觉学习行为的影响及其脑部转录组分析

[目的]分析吡虫啉处理对意大利蜜蜂Apis mellifera ligustica嗅觉学习行为及脑部基因转录的影响,为新烟碱类杀虫剂对蜜蜂的负面影响提供依据.[方法]实验室条件下一次性饲喂意大利蜜蜂成年工蜂含有4 ng吡虫啉的50％蔗糖溶液,以饲喂不合吡虫啉的50％蔗糖溶液为对照,通过伸吻反应(proboscis ex...     

蜂王上颚腺信息素对中华蜜蜂、意大利蜜蜂雄蜂选择行为的影响

【目的】分析蜂王上颚腺信息素(Queen mandibular pheromone,QMP)对中华蜜蜂Apis cerana cerana(简称中蜂)雄蜂与意大利蜜蜂Apismelliferaligustica(简称意蜂)雄蜂行为反应的变化,探索两蜂种雄蜂之间婚飞的干扰机理。【方法】本试验通过室内Y型嗅觉仪检测QMP及其主要成分反式-9-氧代-2-癸烯酸[(E)-9-oxodec-2-enoicacid,9-ODA]对飞行、爬行状态下中蜂雄蜂与意蜂雄蜂选择行为的差异进行研究。【结果】飞行或爬行状态下的中蜂雄蜂和意蜂雄蜂,均对3.5、7.0和14.0μg/μL的9-ODA没有显著趋向性反应(P 0.05);飞行中蜂雄蜂、飞行与爬行意蜂雄蜂均对0.04、0.2、1.0及7.0μg/μL的QMP具有显著趋避反应(P 0.05),而QMP对爬行中蜂雄蜂无显著影响(P 0.05);在中蜂雄蜂和意蜂雄蜂共存时的试验中发现:飞行、爬行意蜂雄蜂均对7.0μg/μL QMP存在显著趋避反应(P 0.05),而7.0μg/μL QMP对飞行或爬行中蜂雄蜂均无显著影响(P 0.05)。【结论】在室内环境下,QMP对中蜂、意蜂雄蜂有驱避作用。     

吡虫啉与环境温度协同作用对意大利蜜蜂的影响

本研究以吡虫啉为例,旨在探讨不同环境温度下亚致死浓度农药对意大利蜜蜂Apis mellifera ligustica急性毒性、免疫基因和解毒酶系活性的影响.通过人工饲喂法确定不同温度(25和35℃)下吡虫啉对1日龄蜜蜂的经口急性毒性,并根据毒力曲线计算得到25℃环境的LC10为4.032 mg/L.人工饲喂蜜蜂2μL该...     

吡虫啉对意大利蜜蜂脑乙酰胆碱受体分布的影响

蜜蜂是自然界主要的授粉昆虫; 新烟碱类杀虫剂(neonicotinoid insecticide)通过结合害虫体内乙酰胆碱受体（nAChR）使害虫致死, 是目前广泛用于田间害虫防控的杀虫剂。本研究以意大利蜜蜂Apis mellifera ligustica和新烟碱类杀虫剂的代表品种吡虫啉为材料, 应用免疫组织化学的方法, 研究了正常成年蜜蜂脑内蘑菇体及视叶nAChR-α7的表达和分布; 分析了亚致死剂量新烟碱类杀虫剂吡虫啉对nAChR-α7表达和分布的影响。结果表明, nAChR-α7在正常蜜蜂脑蘑菇体和视叶中均可检测到, 在蘑菇体中分布相对较少, 但在视叶分布丰富。吡虫啉对nAChR-α7在视叶的表达和分布有显著抑制作用, 但对蘑菇体nAChR-α7的表达没有显著影响。结果提示, 新烟碱类杀虫剂吡虫啉除了文献报道的抑制nAChR的表达外, 还能抑制nAChR-α7的表达量, 这是新烟碱类杀虫剂作用机制的新发现。     

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。     

实验室饲养对中华蜜蜂雄蜂精液及转录水平基因表达的影响

为探索中华蜜蜂Apis cerana cerana Fabricius种用雄蜂的高效培育方法,本研究利用实验室和蜂群条件培育中华蜜蜂性成熟雄蜂,比较各实验组雄蜂的体重、可采集精液比例及精子活力,利用转录组测序技术解析实验室饲养对雄蜂基因表达的影响。结果表明,与蜂群饲养组相比,实验室饲养组中华蜜蜂性成熟雄蜂的精子活力无显著差异,但雄蜂的体重和可采集精液比例显著降低。转录组测序共筛选获得实验室饲养组雄蜂的差异表达基因602个,其中上调表达186个,下调表达416个。上调的差异表达基因显著富集于蛋白质结合、核质、淘汰的核质组分和胞内细胞器等19个GO功能分类;下调的差异表达基因显著富集于ATP合成耦合质子转运、线粒体内膜、呼吸体和碳水化合物代谢过程等22个GO功能分类,以及氧化磷酸化、碳代谢、脂肪酸降解和三羧酸循环等6条KEGG通路。表明实验室饲养可作为培育中华蜜蜂种用雄蜂的备选方法进行深入研究。实验室饲养可能通过抑制机体能量代谢和干扰线粒体功能影响雄蜂的生长和精子发生。研究结果为进一步研发中华蜜蜂种用雄蜂的工厂化培育技术提供了科学参考,有助于加速我国本土蜜蜂的良种选育进程。     

意大利蜜蜂幼虫防御素Defensin-2在吡虫啉胁迫下的表达分析

防御素在昆虫免疫抵抗农药危害中具有重要作用,但蜜蜂防御素Defensin在幼虫抵御吡虫啉毒性中的作用鲜有报道。生物信息学鉴定意大利蜜蜂Apis mellifera ligustica防御素Defensin-2,分析其在吡虫啉暴露下蜜蜂幼虫中的表达模式,探讨其在幼虫抵御吡虫啉中的作用。生物信息学方法分析Defensin-2的氨基酸序列特征、功能域、高级结构、互作靶标、系统进化聚类关系;RT-qPCR分析幼虫口服暴露吡虫啉后Defensin-2基因的表达特征。结果显示,Defensin-2蛋白由104个氨基酸残基组成,相对分子质量为11.9 kDa,等电点PI为8.54,具有信号肽和跨膜结构域。多重序列比对显示Defensin-2属于防御素样超家族。系统进化分析表明,Defensin-2聚类于蜜蜂家族;RT-qPCR分析表明,在半致死浓度吡虫啉暴露下,Defensin-2基因在24 h时显著上调表达后在72 h时下调表达,但在环境浓度吡虫啉暴露后72 h,Defensin-2表达量呈显著上调。Defensin-2可能在意大利蜜蜂幼虫抵御吡虫啉毒性中发挥着重要作用。     

中国东方蜜蜂雄蜂的形态特征

为探讨雄蜂形态特征应用于中国东方蜜蜂分类区划的价值及可能性,测定全国16个省市地区东方蜜蜂的28个与雄蜂职能相关的形态学指标,利用SPSS19.0将获得的形态指标数据进行均数分析、ANOVA分析、主成分分析和聚类分析。结果显示,雄蜂样本中三亚个体最小,工蜂样本中白沙个体最小,二者同属海南地区,均数分析结果基本一致;全部28个形态指标各样点雄蜂之间均存在显著性差异(p0.01),与对照组工蜂ANOVA分析结果完全一致(p0.01);利用形态指标的主成分分析发现雄蜂组全部10个长度指标和肘脉a及3个重量指标聚集为一个独立区域,与对照组工蜂反映出主要信息基本一致;聚类分析中基于雄蜂形态指标的聚类结果对比目前公认的东方蜜蜂分类结论,可以大致区分各地的东方蜜蜂种群。因此,利用雄蜂形态特征分类中国东方蜜蜂具有一定可能性和参考价值,本研究为东方蜜蜂资源的区划提供了新思路和基础数据。     

吡虫啉对意大利蜜蜂乙酰胆碱酯酶的亚致死效应

【目的】确定吡虫啉对意大利蜜蜂Apis mellifera的致死中浓度,探究吡虫啉对意大利蜜蜂乙酰胆碱酯酶的亚致死效应。【方法】本文采用药膜法、点滴法和饲喂法测定吡虫啉对意大利蜜蜂的毒力曲线,及蜜蜂接触吡虫啉后头部乙酰胆碱酯酶活性的变化。应用RT-q PCR技术研究饲喂LC5浓度吡虫啉后,蜜蜂乙酰胆碱酯酶基因Ace1和Ace2的m RNA相对表达量。【结果】饲喂法、点滴法和药膜法3种方法测定的吡虫啉对意大利蜜蜂的致死中浓度分别是7.15 mg/L、0.078 ng/蜂和51 ng/cm2。3种作用方式下,吡虫啉均抑制了乙酰胆碱酯酶活性;随着浓度增加,ACh E酶活性处于下降状态,但降低较少。LC5浓度的吡虫啉对蜜蜂ACh E活性具有明显影响,24 h内ACh E活性呈现增强-抑制-增强的趋势。饲喂蜜蜂LC5亚致死浓度的吡虫啉后,Ace1和Ace2被诱导表达,但在1、2和16 h与对照无明显差异。【结论】亚致死浓度的吡虫啉对蜜蜂乙酰胆碱酯活性具有抑制作用,并且存在明显的剂量效应和时间效应,对Ace1和Ace2具有诱导效应,酶活性水平和m RNA相对表达水平不一致。     

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.     

西方蜜蜂对氟吡呋喃酮和吡虫啉胁迫的响应

因新烟碱类农药对蜜蜂高毒,作为其潜在替代品的氟吡呋喃酮（Flupyradifurone,FPF）受到广泛关注。由于FPF被认为对“蜜蜂安全”,可在作物开花期使用,因而蜜蜂可能存在长期接触FPF的现象。农药胁迫下蜜蜂机体生理指标的响应是解析农药慢性毒性的基础。本文比较了FPF和吡虫啉（Imidacloprid,IMI）对蜜蜂存活率、解毒和氧化应激相关酶类以及免疫和凋亡相关基因表达的影响,结果表明,两种农药对哺育蜂的慢性经口毒性相近,均显著降低存活率,导致中肠组织损伤和细胞死亡,并且引起氧化应激、解毒和免疫反应。此外,幸存蜜蜂可能通过抑制过氧化物酶活性和下调细胞凋亡基因表达耐受农药胁迫。以上结果为解析两种农药慢性毒性和抗农药蜂种选育提供一定的理论依据。     

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

Background

The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases.

Results

We provide quantifications and evidence for GC bias, error rates, error sequence context, effects of quality filtering, and the reliability of quality values. By combining different filtering criteria we reduced error rates 7-fold at the expense of discarding 12.5% of alignable bases. While overall error rates are low in HiSeq data we observed regions of accumulated wrong base calls. Only 3% of all error positions accounted for 24.7% of all substitution errors. Analyzing the forward and reverse strands separately revealed error rates of up to 18.7%. Insertions and deletions occurred at very low rates on average but increased to up to 2% in homopolymers. A positive correlation between read coverage and GC content was found depending on the GC content range.

Conclusions

The errors and biases we report have implications for the use and the interpretation of Illumina sequencing data. GAIIx and HiSeq data sets show slightly different error profiles. Quality filtering is essential to minimize downstream analysis artifacts. Supporting previous recommendations, the strand-specificity provides a criterion to distinguish sequencing errors from low abundance polymorphisms.     

Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and Genome Analyzer systems

Background  

The generation and analysis of high-throughput sequencing data are becoming a major component of many studies in molecular biology and medical research. Illumina's Genome Analyzer (GA) and HiSeq instruments are currently the most widely used sequencing devices. Here, we comprehensively evaluate properties of genomic HiSeq and GAIIx data derived from two plant genomes and one virus, with read lengths of 95 to 150 bases.     

Honeybees (Apis mellifera) modulate dance communication in response to pollution by imidacloprid

Imidacloprid, one of the most commonly used insecticides, is highly toxic to honeybees and other beneficial insects. Imidacloprid is a chloronicotinyl insecticide, which has a highly specific affinity to the nicotinic acetylcholine receptors (nAChRs) in the honeybee’s nervous system. So it may interfere with dance behavior and memory formation. We found the waggle dances were modulated in honeybees fed sucrose water containing imidacloprid (pesticide group) compared to those fed normal sucrose water (control group). In our data, dancers of the pesticide group significantly increased the variance of divergence angle and the return phases in waggle dances than the control group. And the dance followers in pesticide group significantly increased the variance of crop content than the control group. Furthermore, four learning and memory related genes were significantly regulated at the gene expression levels between pesticide and control group. Our data revealed that the sub-lethal dose of imidacloprid impaired the honeybees’ learning and memory and resulted in cognitive disorder. The dancers may adjust their recruitment behavior leading to the observed reduced number of followers. We conclude that modulation of in-hive communication serves to protect the colony from foraging toxic food.     

Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones

The current study aimed to investigate the important reproductive biology and morphology of A.m. jemenitica queens and drones through measuring the weight of virgin and mated queens, size and weight of spermathecae, weight of ovaries, number of ovarioles, quantity and viability of semen in queen and drones. Accordingly, the average weights of 0.139 ± 0.01 g and 0.143 ± 0.013 g recorded for virgin and mated queens respectively. The sizes of spermathecae were 1.248 ± 0.103 mm and 1.25 ± 0.022 mm for virgin and mated queens respectively. The mean weight of ovaries was 0.013 ± 0.003 g and the numbers of ovarioles varied from 124 to 163 with the mean of 142.9 ± 9.47 and with no significant difference between virgin and mated queens. The average number of stored sperm per spermathecae of mated queen was estimated to be 4.202 ± 0.613 million with the viability of 80.39%. The average number of sperm per drone recorded was 8,763,950 ± 1,633,203.15 with viability of 79.54 ± 6.70%. In general, the current study revealed that the values recorded for reproductive biology and morphological characters of A. m. jemenitica queens and drones were relatively lower than values recorded for other Apis mellifera races. This mainly could be associated with the body size of the race which is known to be the smallest race among A. mellifera races. Moreover, the harsh environmental conditions of the regions, high temperature, low humidity and limited resources may have contributed for the smaller biological and morphological values. The information will serve as a base in future selection and breeding of program of the race.     

Antennal malformations in light ocelli drones of Apis mellifera (Hymenoptera, Apidae)

Malformed antennae of Apis mellifera light ocelli drones were drawn, dissected and mounted permanently on slides containing Canada balsam, in order to count the olfactory discs present in each segment, in comparison with the number of those structures in normal antennae of their brothers. Some drones presented morphological abnormalities in a single segment of the right or left antenna, but others had two or more malformed segments in a same antenna. Drones with malformations in both antennae were also observed. The 4th and 5th flagellum segments were the most frequently affected. In a low number of cases the frequency of olfactory discs in malformed segments did not differ from that one recorded for normal segments. However, in most cases studied, the antennal malformations brought about a significant reduction in the number of olfactory discs from malformed segments.