Daily number of bee louse (Braula coeca) in honey bee (Apis mellifera carnica and A. m. syriaca) colonies maintained under semi‐arid conditions

Experimental work was conducted at two apiaries located in Irbid district and in Shuna North, Jordan, during the years 2004–2006. The aims of these investigations were to estimate the seasonal changes in the infestation rates of the bee louse (Braula sp.) and to develop an easy and rapid method of estimating the infestation rate on workers with bee Braula. Two major honey bee subspecies are reared in Jordan; Apis mellifera carnica and Apis mellifera syriaca were used in this study. The results showed that the infestation rate began to increase rapidly in May, reaching the season's maximum rate of 16.2%, 15.8% and 17.4% for A. m. carnica and 22.6%, 23.9% and 22.9% for A. m. syriaca in December of 2004, 2005 and 2006, respectively. The maximum adult numbers of bees were found in April and June, whereas the minimum for the year was in January in both honey bee subspecies colonies during the study period. The actual population of the bee louse could be estimated by counting the daily dropped lice and multiplying by a factor of 158. This factor is valid for the experimental colonies of both subspecies kept for 3 years under semi‐arid Mediterranean conditions.     

Fatty acid composition of the parasitic mite Varroa destructor and its host the worker prepupae of Apis mellifera

The present study analyzes the fatty acid (FA) profile of lipids isolated from Varroa destructor Anderson & Trueman, a parasitic mite of the honey bee (Apis mellifera L.), uninfected and infected worker prepupae of the Carnolian subspecies Apis mellifera carnica Pollmann, and bee bread fed to the worker brood. Significant differences are observed in the FA profiles of lipids isolated from parasites, hosts and bee bread. Parasitism by V. destructor (henceforth, varroosis) induces visible changes in the lipid profile of worker prepupae. In infected prepupae, the percentage of total saturated FAs is lower and the percentage of unsaturated FAs is higher than in uninfected insects. These differences result from significant changes in the percentages of FAs that are most abundant in the evaluated groups (i.e. C16:0, C18:1 9c, C18:2n‐6 and C18:3n‐3 FAs). In mites and in uninfected and infected prepupae, the predominant FAs are oleic acid (41.07 ± 2.26%, 42.79 ± 1.21% and 45 ± 0.20%, respectively) and palmitic acid (22.62 ± 0.87%, 39.48 ± 0.43% and 36.84 ± 0.22%, respectively). Highly significant differences in FA composition are noted between bee bread and worker brood. The results suggest specific mechanisms of FA uptake, accumulation and metabolism in the food chain of this parasitic association, beginning from the food processed by nurse bees for larval feeding, through host organisms (worker brood) to V. destructor mites.     

The impact of pollen consumption on honey bee (Apis mellifera) digestive physiology and carbohydrate metabolism

Carbohydrate‐active enzymes play an important role in the honey bee (Apis mellifera) due to its dietary specialization on plant‐based nutrition. Secretory glycoside hydrolases (GHs) produced in worker head glands aid in the processing of floral nectar into honey and are expressed in accordance with age‐based division of labor. Pollen utilization by the honey bee has been investigated in considerable detail, but little is known about the metabolic fate of indigestible carbohydrates and glycosides in pollen biomass. Here, we demonstrate that pollen consumption stimulates the hydrolysis of sugars that are toxic to the bee (xylose, arabinose, mannose). GHs produced in the head accumulate in the midgut and persist in the hindgut that harbors a core microbial community composed of approximately 10⁸ bacterial cells. Pollen consumption significantly impacted total and specific bacterial abundance in the digestive tract. Bacterial isolates representing major fermentative gut phylotypes exhibited primarily membrane‐bound GH activities that may function in tandem with soluble host enzymes retained in the hindgut. Additionally, we found that plant‐originating β‐galactosidase activity in pollen may be sufficient, in some cases, for probable physiological activity in the gut. These findings emphasize the potential relative contributions of host, bacteria, and pollen enzyme activities to carbohydrate breakdown, which may be tied to gut microbiome dynamics and associated host nutrition.     

Resistance of the honey bee, Apis mellifera to the acarian parasite Varroa destructor: behavioural and electroantennographic data

Abstract. One way in which Apis mellifera honey bees resist Varroa destructor is by detection and elimination of nestmates. This study uses behavioural tests and electroanntennography to assess the role of chemostimuli in recognition by honey bees of this acarian ectoparasite. Behavioural tests using living or dead parasites involved observation of honey bee grooming activity (antennation) under controlled conditions in Petri dishes, and removal behaviour (uncapping and elimination of parasitized and unparasitized control brood cells) under natural conditions. Some bees from colonies with both small and large parasite populations showed aggressive behaviour (biting). No difference was observed according to whether the mite was dead or alive. Under natural conditions, bees uncapped more parasitized cells than control cells. Electroantennographic tests were performed to measure sensitivity to various Varroa extracts at three concentrations (10, 20 and 30 Varroa Equivalents). Only 30 Varroa Equivalent methanol extracts made from Varroa collected from brood cells elicited significantly greater antennal response than controls (pure solvent). All three methanol extracts elicited significantly greater antennal response than controls. No response was observed using Varroa extracts made with acetone or hexane. These findings suggest that polar products may act as chemostimuli for recognition of V. destructor by honey bees. Further study will be necessary to determine which polar products are involved in this recognition and assess grooming and removal behaviour using these products.     

新一代测序技术在植物转录组研究中的应用

随着DNA测序技术的发展,新一代测序技术以其高通量、低成本的特点,成为越来越多的生物学研究者在开展工作时的首选。在所有的新一代测序技术中,454测序系统是最早实现商业化且发展相对成熟的一种,目前被广泛的应用于各个领域的生物学研究中。文章以454测序系统为例,综述了新一代测序系统的原理、优缺点,及其在植物转录组研究中的应用,并对其在植物研究领域中可能的发展应用方向进行了展望。     

Patterns in Varroa destructor depend on bee host abundance,availability of natural resources,and climate in Mediterranean apiaries

1. Varroa destructor Linnaeus (Acari: Varroidae) is one of the greatest threats to apiculture. This study examines the role of host density, natural resource availability for bees, the management and climate in driving spatial and annual variability in the abundance of Varroa, and the occurrence of colony losses, recorded in apiaries across a Mediterranean island over a 2‐year period, using a hierarchical generalised linear model framework. 2. The seasonal abundance of Varroa showed a bimodal pattern with two peaks, the first one being in spring and larger than the second one located in summer/autumn. In contrast, bee colony losses were mainly concentrated in autumn/winter. 3. The abundance patterns of Varroa were best explained by models combining host, climate, and resource availability. A key novel finding was that low availability of natural flowering resources leads to high levels of infestation of Varroa, highlighting the importance of preserving natural resources around apiaries for the maintenance of pollination services in the landscape. Varroa abundance was also found to increase as bee density increased, probably as a result of the greater brood availability. Moreover, Varroa abundance increased as temperatures decrease and decreases as relative humidity increases, which is consistent with previous studies. Anti‐varroa treatments were only found to impact Varroa levels in the second 6 months of the year. Organic treatments outperformed synthetic treatments. 4. Empirical research on optimal seasonal timing and combinations of treatments, as well as impacts of climate and resource availability on natural dynamics of bees and Varroa in different climate zones, is urgently required.     

Effect of a lactic acid treatment during winter in temperate climate upon Varroa jacobsoni Oud. and the bee (Apis mellifera L.) colony

Three groups of bee colonies were treated with lactic acid, the pesticide Perizin or lactic acid and Perizin in order to validate the applicability of lactic acid in Varroa mite control. The lactic acid treatment was conducted during winter. Eight ml of lactic acid (15%) per comb side were applied with a dosage gun. The treatment was highly efficient and 94.2%–99.8% of the mites in a colony were killed. Due to precise dosage the lactic acid treatment caused less bee mortality than a treatment with the pesticide Perizin. A lactic acid treatment at-0.2°C caused bee mortality comparable to a Perizin treatment. The number of queen losses after lactic acid treatment and after Perizin treatment was comparable. The number of bees, the size of the brood area, the amount of stored honey and Nosema infestation rates were not significantly different in lactic acid treated colonies and Perizin treated colonies in spring after treatment.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

基于分子标记的宏基因组16S rRNA基因高变区选择策略

随着新一代DNA测序技术出现,人们能够同时对多个DNA样本的宏基因组进行并行分析,尤其是以16S rRNA基因高变区为分子标记的测序已经成为微生物多样性研究最为简洁有效的方法. 目前二代高通量测序的读长不能覆盖16S rRNA基因的全长,需要选择一个有效的高变区进行测序.十多年来,对于16S rRNA基因高变区的选择策略没有统一的标准.本文分析了常用的高变区选择策略,指出不同环境条件是影响高变区选择的重要因素之一.在此基础上,提出了高变区选择的参考准则,同时建议应对选择的高变区进行有效评估.     

Five hundred and fifty microsatellite markers for the study of the honeybee (Apis mellifera L.) genome

Microsatellites are currently considered the most useful genetic markers with wide applications in genomics, quantitative and population genetics. We present here the structure of the core sequence of 552 microsatellites, together with the sequences of the primers and the length of the sequenced allele. These microsatellites were isolated from several libraries constructed from either fractions of total genomic DNA or from clones of a bacterial artificial chromosome (BAC) library. All 552 loci are polymorphic in the honeybee. Many of them were also successfully amplified in three other species of Apis: A. cerana (58%), A. dorsata (59%) and A. florea (38%). A summary of the variability of 36 loci in the three main evolutionary lineages of A. mellifera is given.     

Genomics and proteomics of Apis mellifera filamentous virus isolated from honeybees in China

Apis mellifera filamentous virus (AmFV) is a large DNA virus that is endemic in honeybee colonies. The genome sequence of the AmFV Swiss isolate (AmFV CH–C05) has been reported, but so far very few molecular studies have been conducted on this virus. In this study, we isolated and purified AmFV (AmFV CN) from Chinese honeybee (Apis mellifera) colonies and elucidated its genomics and proteomics. Electron microscopy showed ovoid purified virions with dimensions of 300–500×210–285 nm, wrapping a 3165×40 nm filamentous nucleocapsid in three figure-eight loops. Unlike AmFV CH–C05, which was reported to have a circular genome, our data suggest that AmFV CN has a linear genome of approximately 493 kb. A total of 197 ORFs were identified, among which 36 putative genes including 18 baculoviral homologs were annotated. The overall nucleotide similarity between the CN and CH–C05 isolates was 96.9%. Several ORFs were newly annotated in AmFV CN, including homologs of per os infectivity factor 4 (PIF4) and a putative integrase. Phylogenomic analysis placed AmFVs on a separate branch within the newly proposed virus class Naldaviricetes. Proteomic analysis revealed 47 AmFV virion-associated proteins, of which 14 had over 50% sequence coverage, suggesting that they are likely to be main structural proteins. In addition, all six of the annotated PIFs (PIF-0–5) were identified by proteomics, suggesting that they may function as entry factors in AmFV infection. This study provides fundamental information regarding the molecular biology of AmFV.     

新一代测序技术应用于microRNA检测

MicroRNAs(miRNAs)是一类在进化上高度保守的非编码小分子单链RNA(~22nt),在基因转录后调控中发挥至关重要的作用。越来越多的证据表明,miRNAs参与很多重要的生理和病理过程,例如发育、器官形成、调亡、细胞增殖、肿瘤发生等。近年来飞速发展的新一代测序技术在miRNA检测方面具有重要的应用。文章简要介绍了新一代测序技术3大平台的基本步骤和原理,测序数据的生物信息学分析方法以及新一代测序技术在miRNA方向的主要应用。相比于传统的miRNA检测方法,新一代测序技术具有通量高、对遗传物质检测完全且准确度高,可重复性好等优点,在探索新miRNA、miRNA互补链、miRNA编辑、miRNA异构体检测以及miRNA靶基因检测等方面具有巨大优势。随着新一代测序技术的不断发展,测序成本不断降低,在未来几年,新一代测序技术的使用率或将大大增加。新一代测序技术的不断应用将进一步促进人类对于miRNA在各种生理病理过程中的功能和调控的认识。     

意大利蜂营养腺的形态结构研究

研究了意大利蜂分泌蜂王浆的主要腺体──营养腺的形态结构及腺体随工蜂日龄增加而变化的规律。结果表明,工蜂羽化后,在各个日龄段随其职能分工的不同,营养腺形态结构也有相应的变化。从羽化出房的幼峰到哺育蜂到采集蜂,其营养腺要经历发育─→饱满发达─→衰退萎缩的过程。分泌细胞中的粗面内质网变化更为明显。     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

Discovering and sequencing new plant viral genomes by next‐generation sequencing: description of a practical pipeline

Small‐scale sequencing has improved substantially in recent decades, culminating in the development of next‐generation sequencing (NGS) technologies. Modern NGS methods have helped the discovery of many new plant viruses. Nevertheless, there is still a need to establish solid assembly pipelines targeting small genomes characterised by low identities to known viral sequences. Here, we describe and discuss the fundamental steps required for discovering and sequencing new plant viral genomes by NGS. A practical pipeline and standard alternative tools used in NGS analysis are presented.     

Behavioural responses of Varroa destructor (Acari: Varroidae) to extracts of larvae, cocoons and brood food of worker and drone honey bees, Apis mellifera (Hymenoptera: Apidae)

Abstract. Varroa destructor is a parasitic mite of the honey bee species Apis cerana Fabr . and A. mellifera L. Mature females reproduce on the immature stages of their hosts, producing more viable female offspring on drone hosts than on worker hosts. Thus, immature drones are more likely to be infested with mites than immature workers. To investigate the hypothesis that differences in host chemistries underlie the biased distribution of mites between worker and drone brood, the arrestment responses of mites to solvent extracts of a number of stimuli normally encountered by a mite during its life cycle were measured. Mites were arrested by cuticular extracts of worker and drone larvae obtained at 0, 24 and 48 h prior to the time when cell capping is completed. Mites were also arrested by extracts of worker and drone, brood food and cocoons, and by a blend of synthetic fatty acid esters previously shown to be active in the host acquisition process. In a wind tunnel bioassay, mites were attracted to odours from living fifth-instar worker and drone larvae, but not to volatiles from cocoons, brood food or a blend of fatty acid esters. The sex of the host was not an important factor affecting the behavioural responses of the mites in any assay. We conclude that host kairomones play a role in the host acquisition process, but we found no evidence to support the hypothesis that mites use these substances to differentiate between worker and drone brood.     

西方蜜蜂表皮蛋白基因apd-like转录起始位点的定位及cDNA序列的分析

Apidermin蛋白家族是根据蜜蜂表皮蛋白apidermin 1-3（APD 1-3）而命名的一个新型的昆虫结构性表皮蛋白家族。为了鉴定西方蜜蜂Apis mellifera基因组序列上毗邻基因簇apd 1-3的一个预测基因座LOC727145是否为一个新的apd基因,本研究在用5′LongSAGE标签定位该基因的转录起始位点（TSS）的基础上,利用其中的3条5′LongSAGE标签序列作为上游引物,通过RT-PCR方法克隆了该基因的cDNA序列（GenBank登录号： GU358197, GU358199, GU358198）。生物信息学分析发现,基因座LOC727145含有2个外显子和1个“GT-AG”型内含子,其cDNA序列富含GC（70％）,可编码一条长152 aa残基的高度疏水性多肽。此多肽序列的氨基酸组成与蜜蜂APD 1-3表皮蛋白类似, 富含Ala, Gly, Pro, Leu 和Val 5种氨基酸（占77％）, 其中Ala残基含量最高（29％）。该多肽序列与蜜蜂APD-1表皮蛋白序列的相似性为50％, 且其N末端的预测信号肽序列与APD 蛋白的信号肽序列类似。5′LongSAGE标签的基因组定位结果显示,基因座LOC727145在雄蜂头部中表达丰度很高,RNA PolⅡ可从6个不同的TSS上以不同效率起始转录,其中由一个优势TSS上起始了90％的转录。本研究为apidermin表皮蛋白家族增添了一个新成员, 命名为apidermin-like (apd-like)。