东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-12220及其靶基因的表达谱

东方蜜蜂微孢子虫Nosema ceranae侵染成年蜜蜂导致蜜蜂微孢子虫病。本研究旨在验证东方蜜蜂微孢子虫nce-miR-12220的存在和表达,并检测nce-miR-12220及其靶基因在病原侵染意大利蜜蜂Apis mellifera ligustica (简称意蜂)工蜂过程的表达谱。Stem-loop RT-PCR和Sanger测序结果显示nce-miR-12220真实存在和表达。靶向预测结果显示nce-miR-12220共靶向KRAB-A和γ tubulin等15个基因。上述靶基因可注释到19个GO条目和3条KEGG通路。RT-qPCR结果显示,相较于接种后1 d (1 day post infection,1 dpi),nce-miR-12220在2 dpi上调表达,而在3-12 dpi阶段总体表现出显著下调表达的趋势。类似地,与1 dpi相比,靶基因KRAB-A在2 dpi上调表达,而在3-12 dpi阶段总体呈下调表达的趋势。另外,与1 dpi相比,靶基因γ tubulin在2-12 dpi阶段总体表现出显著下调表达的趋势。上述结果表明nce-miR-12220与KRAB-A和γ tubulin之间存在潜在的靶向结合和正向调控关系;东方蜜蜂微孢子虫通过下调表达nce-miR-12220抑制KRAB-A的表达进而促进增殖;意蜂工蜂可能通过抑制东方蜜蜂微孢子虫的γ tubulin表达抵御病原侵染。研究结果明确了nce-miR-12220及其靶基因KRAB-A和γ tubulin在东方蜜蜂微孢子虫侵染意蜂工蜂过程中的动态表达规律,为深入探究nce-miR-12220在病原侵染中的功能及调控机制提供了理论和实验依据。     

东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程中nce-miR-10660及其靶基因表达谱

【目的】本研究旨在为探究nce-miR-10660调控东方蜜蜂微孢子虫Nosema ceranae侵染的作用机制提供理论和实验依据。【方法】采用Stem-loop RT-PCR对前期鉴定到的东方蜜蜂微孢子虫nce-miR-10660进行表达验证,再通过Sanger测序验证nce-miR-10660的序列。利用相关生物信息学软件预测和分析nce-miR-10660的靶基因。通过RT-qPCR检测nce-miR-10660及其靶基因在东方蜜蜂微孢子虫侵染意大利蜜蜂Apis mellifera ligustica工蜂过程中中肠中的表达谱。【结果】Stem-loop RT-PCR和Sanger测序结果分别证实了nce-miR-10660在东方蜜蜂微孢子虫孢子中的表达和真实存在。靶向预测结果显示nce-miR-10660共靶向RRDRP和RCDP 42等9个基因;分别有2和6个靶基因可被分别注释到KEGG数据库中的4条通路和GO数据库中的23个条目。RT-qPCR结果显示,相较于东方蜜蜂微孢子虫侵染后1 d时意大利蜜蜂工蜂中肠中nce-miR-10660的表达量,东方蜜蜂微孢子虫侵染后2 d时意...     

东方蜜蜂微孢子虫侵染意大利蜜蜂工蜂过程的nce-miR-23928及其靶基因表达谱

【背景】东方蜜蜂微孢子虫(Nosema ceranae)专性侵染成年蜜蜂中肠上皮细胞而导致的微孢子虫病给养蜂业造成严重损失。【目的】检测东方蜜蜂微孢子虫nce-miR-23928及其靶基因在侵染意大利蜜蜂(Apis mellifera ligustica)工蜂过程的表达谱,为深入探究nce-miR-23928在东方蜜蜂微孢子虫侵染中的功能及调控机制提供依据。【方法】通过RNAhybrid、miRanda和TargetScan软件预测nce-miR-23928的靶基因。使用BLAST工具将上述靶基因比对到基因本体论(geneontology,GO)、京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)、Nr和Swiss-Prot数据库以获得相应注释。采用实时荧光定量PCR(realtimequantitativePCR,RT-qPCR)技术检测nce-miR-23928及其靶基因在东方蜜蜂微孢子虫侵染意蜂工蜂过程中的相对表达量。【结果】相较于接种后1 d (1 day post infection, 1 dpi),nce-...     

基于small RNA组学分析揭示意大利蜜蜂响应东方蜜蜂微孢子虫胁迫的免疫应答机制

【目的】通过深度测序和组学分析在small RNA组学层面揭示意大利蜜蜂(Apis mellifera ligustica,简称意蜂)响应东方蜜蜂微孢子虫(Nosema ceranae)的免疫应答机制。【方法】利用small RNA-seq技术对正常及N. ceranae胁迫7 d和10 d的意蜂工蜂中肠(Am7CK、Am7T、Am10CK和Am10T)进行深度测序。利用相关生物信息学软件对测序数据进行质控、已知micro RNA (mi RNA)鉴定、新mi RNA预测及mi RNA的结构特征分析。通过Stem-loop RT-PCR验证新mi RNA的表达。按照|log2(Fold change)|≥1和P≤0.05的标准筛选出Am7CKvs.Am7T和Am10CKvs.Am10T比较组的差异表达mi RNA(differentially expressed mi RNA,DEmi RNA)。利用软件预测DEmi RNA靶向结合的m RNA并进行GO和KEGG数据库注释,根据注释信息对细胞和体液免疫相关通路及富集靶m RNA进行统计和分析。根据靶向结合关系构建DEmi RNA和免疫通路相关差异表达m RNA (differentially expressed m RNA,DEm RNA)的调控网络。采用RT-q PCR对数据的可靠性和DEmi RNA的差异表达进行验证。【结果】共获得165895574条原始读段和132028990条有效序列标签,各组的组内Pearson相关性平均在87.92%及以上。共鉴定到928个已知mi RNA和56个新mi RNA。这些mi RNA的长度介于18–28nt,多数的长度为18nt和22nt且首位碱基主要偏向U。验证了12个新mi RNA的真实表达。Am7CKvs.Am7T比较组包含48个上调mi RNA和36个下调mi RNA;Am10CK vs. Am10T比较组包含56个上调mi RNA和51个下调mi RNA。两个比较组的DEmi RNA可分别靶向结合9827个和10720个m RNA。这些靶m RNA可分别注释到50和47条功能条目,以及138和135条KEGG通路。DEmi RNA与免疫通路相关靶m RNA的调控网络分析结果显示,Am7CK vs. Am7T中有26个DEmi RNA靶向与内吞作用等免疫通路相关的10个DEm RNA;Am10CK vs. Am10T中有15个DEmi RNA靶向与MAPK信号通路等免疫通路相关的10个DEm RNA。验证了测序数据和4个DEmi RNA差异表达的可靠性。【结论】研究结果揭示了宿主DEmi RNA可能通过调控物质和能量代谢、细胞和体液免疫对N. ceranae产生应答,但DEmi RNA不参与抗菌肽基因的表达调控;mi R-1-z可能参与宿主的细胞增殖、细胞凋亡和免疫进程;氧化磷酸化通路可能在宿主免疫应答及宿主-病原互作中发挥特殊作用。     

意大利蜜蜂工蜂中肠的环状RNA及其调控网络分析

【目的】环状RNA(circRNA)在可变剪接、转录调控和来源基因的表达调控等方面具有重要功能。本研究旨在分析意大利蜜蜂Apis mellifera ligustica工蜂中肠circRNA的数量、种类、结构特征和作用,并通过构建和分析circRNA的调控网络探索circRNA的调控功能。【方法】在实验室条件下人工饲养意大利蜜蜂工蜂,利用circRNA-seq技术对意大利蜜蜂7和10日龄成年工蜂中肠样品进行深度测序。利用find_circ软件从质控后的数据中预测circRNA。通过BLAST比对GO和KEGG数据库,对circRNA的来源基因进行功能和代谢通路注释。利用TargetFinder软件预测circRNA靶向结合的miRNA及miRNA靶向结合的mRNA,通过Cytoscape v.3.2.1软件对circRNA-miRNA和circRNA-miRNA-mRNA调控网络进行构建及可视化。通过设计背靠背引物和线性扩增引物RT-PCR对预测出的circRNA进行验证。【结果】意大利蜜蜂工蜂中肠样品的测序平均得到136 463 071条clean reads,去除rRNA后各样品的anchor reads均在136 779 122条及以上。共预测出10 833个circRNA,长度主要介于15~1 000 nt;上述circRNA的类型丰富,其中已注释的外显子circRNA数量最多,分布在西方蜜蜂1号染色体的circRNA数量最多,其次为8号染色体。CircRNA的来源基因可注释到包括结合、细胞进程和细胞在内的45个GO条目,以及包括内吞作用、内质网蛋白加工及核糖体在内的121条KEGG代谢通路,表明circRNA在意大利蜜蜂工蜂中肠的生长、发育、新陈代谢和细胞生命活动等生物学过程中发挥重要作用。进一步构建circRNA-miRNA和circRNA-miRNA-mRNA调控网络,分析结果显示部分circRNA可能作为竞争性内源RNA吸附结合microRNA,从而调控基因的表达水平。最后,对随机选择的3个circRNA的RT-PCR结果验证了其真实存在。【结论】本研究对意大利蜜蜂工蜂中肠中的circRNA进行预测、分析及鉴定。研究结果提供了中肠circRNA的数量、种类、结构特征、作用和调控网络的信息,揭示了circRNA可能通过作用于来源基因和作为竞争性内源RNA在意大利蜜蜂工蜂中肠的生长发育和免疫防御中发挥作用,为深入研究circRNA在意大利蜜蜂中肠发育及胁迫响应过程中的功能奠定了基础。     

东方蜜蜂微孢子虫孢子中微小RNA的鉴定与分析

【目的】丰富东方蜜蜂微孢子虫Nosema ceranae的微小RNA(microRNA, miRNA)信息,并为深入探究miRNA在病原孢子和病原侵染中的功能提供理论和实验依据。【方法】基于已获得的small RNA-seq数据,利用生物信息学软件对东方蜜蜂微孢子虫的纯净孢子中的miRNA进行鉴定和分析。采用茎环反转录PCR(stem-loop RT-PCR)检测已鉴定的miRNA的表达;通过分子克隆与Sanger测序验证miRNA的序列。使用TargetFinder软件预测这些miRNA的靶基因,并对靶基因进行数据库注释。根据miRNA与靶基因的靶向结合关系构建调控网络,再利用Cytoscape软件进行可视化。【结果】在东方蜜蜂微孢子虫孢子中共鉴定到10个miRNA;这些miRNA的长度分布介于21~25 nt,首位碱基表现出U偏向性,每一位碱基的偏向性差异明显。Stem-loop RT-PCR检测结果表明这10个miRNA均真实表达;Sanger测序结果证实了随机选取的其中2个miRNA的序列真实性。共预测出249个靶基因,其中分别有249, 118, 136和3个靶基因可注释到Nr,Swiss-Prot, KOG和eggNOG数据库。此外,分别有134和71个靶基因可分别注释到GO数据库的30个功能条目和KEGG数据库的54条通路。【结论】本研究揭示了东方蜜蜂微孢子虫孢子中miRNA的存在和表达;这些miRNA通过调控潜在靶基因的表达参与孢子的生命活动。     

东方蜜蜂微孢子虫传染病学分析

东方蜜蜂微孢子虫病是一种由东方蜜蜂微孢子虫(Nosema ceranae)引起的蜜蜂传染病,已经蔓延到全球。蜜蜂感染东方蜜蜂微孢子虫后会导致早衰、哺育能力下降、生产力和繁殖能力降低,严重时可直接导致蜂群瓦解。本文从传染病学角度出发,对近10年东方蜜蜂微孢子虫病原学、流行病学和防治方法等方面进行总结,以此提高对微孢子虫的认识,为微孢子虫防治提供新思路。     

意大利蜜蜂工蜂中肠piRNA的鉴定与分析

【目的】Piwi蛋白互作RNA(PIWI-interacting RNA, piRNA)在昆虫的发育和免疫等重要生物学过程发挥重要的调控作用。本研究旨在丰富西方蜜蜂Apis mellifera的piRNA信息,并为进一步探究差异表达piRNA(differentially expressed piRNA, DEpiRNA)调控意大利蜜蜂Apis mellifera ligustica工蜂中肠发育的分子机理提供基础。【方法】基于已获得的意大利蜜蜂7日龄(Am7)和10日龄(Am10)工蜂中肠的small RNA(sRNA)组学数据,对piRNA进行预测和分析。将质控后的sRNA数据比对西方蜜蜂参考基因组,再将比对上的序列标签(tags)进一步比对数据库以滤除rRNA和tRNA等小非编码RNA(non-coding RNA,ncRNA),进而根据piRNA的长度特征鉴定piRNA。采用TPM(tags per million)算法对piRNA表达量进行计算和归一化处理。根据|log₂ fold change|≥1且P≤0.05的标准筛选Am7 vs Am10 比较组的DEpiRNA。通过相关软件预测DEpiRNA的靶mRNA并进行GO和KEGG数据库注释。利用Cytoscape软件对DEpiRNA-mRNA调控网络进行可视化。通过Stem-loop RT-PCR对随机选取的Am7与Am10两组共有的6个piRNA的表达进行验证。利用RT-qPCR对随机挑选的6个DEpiRNA的表达趋势进行验证。【结果】从意大利蜜蜂工蜂中肠中共鉴定到596个piRNA,长度介于24~33 nt,且Am7和Am10组中不同长度分布范围的piRNA的首位碱基偏向性具有明显差异。在Am7 vs Am10比较组共筛选出41个DEpiRNA,其中piR-ame-11093, piR-ame-1111451,piR-ame-190949和piR-ame-932156可分别靶向1 195, 1 018, 4 040和1 063条mRNA。上述靶mRNA可分别注释到45个功能条目和45条通路。Stem-loop RT-PCR验证结果显示6个piRNA(piR-ame-1084826, piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500和piR-ame-774987)均真实表达。RT-qPCR结果显示共有6个DEpiRNA (piR-ame-1084826,piR-ame-11093, piR-ame-14476, piR-ame-24995, piR-ame-39500和piR-ame-774987)的表达趋势与测序数据中的表达趋势一致,证实了本研究中sRNA-seq数据的可靠性和piRNA差异表达趋势的真实性。【结论】本研究从意大利蜜蜂工蜂中肠中鉴定到596个piRN,长度介于24~33 nt。不同长度分布范围的piRNA的首位碱基偏向性具有明显差异;piR-ame-11093, piR-ame-1111451,piR-ame-190949和piR-ame-932156具有靶向调控基因表达参与工蜂中肠发育过程的潜力。     

转录组分析揭示东方蜜蜂微孢子虫侵染意大利蜜蜂的分子机制

【目的】本研究旨在通过差异表达基因(differentially expressed gene, DEG)分析以及毒力因子和其他侵染相关因子分析,在转录组水平揭示东方蜜蜂微孢子虫Nosema ceranae侵染意大利蜜蜂Apis mellifera ligustica的分子机制。【方法】基于前期已获得高质量的东方蜜蜂微孢子虫纯化孢子(NcCK)及侵染意大利蜜蜂工蜂7和10 d的东方蜜蜂微孢子虫(分别为NcT1和NcT2)转录组数据,根据P≤0.05且|log₂(Fold change)|≥1的标准,通过比较分析筛选出NcCK vs NcT1, NcCK vs NcT2和NcT1 vs NcT2比较组的DEG。通过相关生物信息学软件对上述DEG进行Venn分析、GO分类和KEGG代谢通路富集分析。根据Nr和KEGG数据库注释信息和相关文献进行对东方蜜蜂微孢子虫的毒力因子和侵染相关因子的统计和分析。通过RT-qPCR验证转录组数据及DEG表达趋势。【结果】从NcCK vs NcT1, NcCK vs NcT2和NcT1 vs NcT2比较组分别鉴定出1 397, 1 ...     

基于中肠代谢组学意大利蜜蜂工蜂枣花病的发病机理研究

【目的】利用代谢组学分析患枣花病意大利蜜蜂Apis mellifera ligustica工蜂中肠内代谢物变化,挖掘出与蜜蜂枣花病相关的重要代谢通路,旨在揭示蜜蜂枣花病发病机制,为枣花病靶向药物的研发提供理论依据。【方法】分别利用液相色谱-质谱联用(liquid chromatography-mass spectrometry, LC-MS)和气相色谱-质谱联用(gas chromatography-mass spectrometry, GC-MS)技术对患枣花病和健康意大利蜜蜂工蜂进行中肠非靶向代谢组检测;通过主成分分析(principal component analysis, PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA),以变量投影重要性(variable projection importance, VIP)>1.0,差异倍数(fold change, FC)>2.0和P<0.05为标准筛选差异代谢物,并对其进行KEGG注释和通路富集;利用生...     

Low natural levels of Nosema ceranae in Apis mellifera queens

Queens are the primary female reproductive individuals in honey bee colonies and, while they are generally free from Nosema ceranae infection, they are nevertheless susceptible. We sought to determine whether queens are naturally infected by N. ceranae, as these infections could be a factor in the rapid spread of this parasite. Queens were analyzed using real-time PCR and included larval queens, newly emerged, and older mated queens. Overall, we found that all tissues we examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis. The infection of the ovaries and spermatheca suggests the possibility of vertical transmission of N. ceranae.     

意大利蜜蜂幼虫肠道的miRNAs的生物信息学预测及分析

【目的】蜜蜂肠道是食物消化和营养吸收的主要部位,同时也是抵御病原侵染的重要场所。本研究旨在对意大利蜜蜂Apismelliferaligustica(简称意蜂)幼虫肠道的microRNAs(miRNAs)及其靶基因进行深入分析,进而解析miRNAs在幼虫肠道发育和生长过程中的作用。【方法】利用small RNA-seq(sRNA-seq)技术对意蜂4、5和6日龄幼虫肠道进行测序,通过相关生物信息学软件对意蜂幼虫肠道的miRNAs进行预测及分析。利用TargetFinder软件预测miRNAs的靶基因,然后将靶基因通过BLAST软件进行GO和KEGG数据库的功能注释。利用Cytoscape软件构建miRNAs与其靶向结合的mRNAs的调控网络。【结果】意蜂幼虫肠道样品的测序共获得96 329 456条有效标签序列(Clean tags),预测出560个miRNAs,包括45个novelmiRNAs。上述miRNAs的长度主要分布在17-27nt之间,且不同长度的miRNAs的首位碱基偏向性具有明显差异。表达量聚类分析结果显示,331个miRNAs为4、5和6日龄幼虫肠道所共有且表达量较为稳定,随着发育时间延长,特有miRNAs的表达水平呈总体增加的趋势。利用TargetFinder软件共预测出16479个意蜂幼虫肠道的靶基因,其中有8132和3361个可分别注释到GO和KEGG数据库,进一步分析发现有224个靶基因注释在Wnt信号通路,分别有2个和27个靶基因与保幼激素和蜕皮激素的调控密切相关。【结论】本研究在全基因组水平对意蜂幼虫肠道的miRNAs进行预测和分析,研究结果不仅丰富了意蜂的miRNAs信息,也为深入研究意蜂幼虫肠道的生长发育机理打下了初步基础。     

意大利蜜蜂工蜂中肠响应Nosema ceranae胁迫的高表达基因分析

东方蜜蜂微孢子虫Nosema ceranae是专性寄生蜜蜂中肠上皮细胞的单细胞真菌,对意大利蜜蜂(意蜂)具有较强的侵染性。本研究利用RNA-seq技术对正常10 d (Apis mellifera ligustica control group,Am CK)和N. ceranae胁迫10 d的意蜂工蜂中肠(Apis mellifera ligustica treatment group,Am T)进行测序,共得到160 847 237 100条原始读段,过滤后得到1 066 955 298条有效读段。主成分分析结果显示Am CK与Am T测序样品的组内重复性较好,组间的基因表达模式差异明显。GO分类结果显示,Am CK与Am T的前100位高表达基因(HEGs)分别分布于32和33个GO terms,基因富集数最多的均为代谢进程。KEGG代谢通路(pathway)富集分析结果显示,Am CK的前100位HEGs富集在21个pathways,基因富集数最多的是内吞作用、信号通路和嘌呤代谢; Am T的前100位HEGs富集在26个pathways,基因富集数最多的是内吞作用、RNA转运和蛋白质的内质网加工。前100位HEGs的Venn分析结果显示Am CK与Am T的共有HEGs为87个,特有HEGs分别均为13个。研究结果揭示了N. ceranae胁迫意蜂工蜂中肠过程中的基因表达谱信息,也为解析N. ceranae致病的分子机理提供了有益信息和线索。     

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.     

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera)

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.     

Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.     

Prevalence of the microsporidian Nosema ceranae in honeybee (Apis mellifera) apiaries in Central Italy

Nosema ceranae and Nosema apis are microsporidia which play an important role in the epidemiology of honeybee microsporidiosis worldwide. Nosemiasis reduces honeybee population size and causes significant losses in honey production. To the best of our knowledge, limited information is available about the prevalence of nosemiasis in Italy. In this research, we determined the occurrence of Nosema infection in Central Italy. Thirty-eight seemingly healthy apiaries (2 to 4 hives each) were randomly selected and screened from April to September 2014 (n = 11) or from May to September 2015 (n = 27). The apiaries were located in six areas of Central Italy, including Lucca (n = 11), Massa Carrara (n = 9), Pisa (n = 9), Leghorn (n = 7), Florence (n = 1), and Prato (n = 1) provinces. Light microscopy was carried out according to current OIE recommendations to screen the presence of microsporidiosis in adult worker honeybees. Since the morphological characteristics of N. ceranae and N. apis spores are similar and can hardly be distinguished by optical microscopy, all samples were also screened by multiplex polymerase chain reaction (M-PCR) assay based on 16S rRNA-gene-targeted species-specific primers to differentiate N. ceranae from N. apis. Furthermore, PCR-positive samples were also sequenced to confirm the species of amplified Nosema DNA. Notably, Nosema spores were detected in samples from 24 out of 38 (63.2%, 95% CI: 47.8–78.5%) apiaries. Positivity rates in single provinces were 10/11, 8/9, 3/9, 1/7, or 1/1 (n = 2). A full agreement (Cohen's Kappa = 1) was assessed between microscopy and M-PCR. Based on M-PCR and DNA sequencing results, only N. ceranae was found. Overall, our results highlighted that N. ceranae infection occurs frequently in the cohort of honeybee populations that was examined despite the lack of clinical signs. These findings suggest that colony disease outbreaks might result from environmental factors that lead to higher susceptibility of honeybees to this microsporidian.