Mitochondrial DNA Diversity and Genetic Differentiation of the Honeybee (Apis cerana) in Thailand

Genetic diversity of the honeybee (Apis cerana) in Thailand collected from north, northeast, the central region, peninsular Thailand, and Samui Island (n = 181) was examined by PCR–RFLP of ATPase6–ATPase8. Interestingly, 78 individuals (43.09%) of the southern-latitude bees exhibited length heteroplasmy of the PCR product. The gel-eluted ATPase6–ATPase8 (825 bp) of each bee was restricted with TaqI, SspI, and VspI, respectively. Eight mitotypes were generated and revealed biogeographic differentiation between conspecific samples of A. cerana. AAA, ACA, AAD, BAA, ADA, and ABA were found only in the north-to-central samples (north, northeast, and central region); BBB and BBC were found in the southern-latitude bees; and BBC was restrictively found in the Samui sample. Large genetic distances were observed between each of the north-to-central samples and peninsular Thailand and Samui samples, but lower levels of genetic distance were found within each region. Geographic heterogeneity and phylogenetic analyses indicated that Thai A. cerana could be genetically differentiated into northern Thailand, peninsular Thailand, and Samui Island populations.     

中华蜜蜂工蜂视叶胚后发育过程中的细胞凋亡

本研究通过形态解剖和原位末端标记法(TUNEL),对中华蜜蜂Apis cerana cerana视叶胚后发育过程中的细胞凋亡进行了研究,结果表明:视叶内的细胞程序性死亡开始出现在1龄幼虫末期,随后凋亡细胞数量逐渐增加;在视叶的胚后发育过程中,细胞凋亡经历了3个高峰期,即2龄幼虫、5龄幼虫和蛹发育的第2天;在视叶3个部分的发育中,视髓层中细胞凋亡的数量远远多于视小叶和视神经节层,而视神经节层最少,说明了细胞凋亡的数量和位置与各部分结构发育的时间以及神经投射有关。广泛的细胞凋亡是蜜蜂视叶发育过程中的一个显著特征。     

Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis

Variability of mitochondrial DNA (mtDNA) of the honey bee Apis mellifera L. has been investigated by restriction and sequence analyses on a sample of 68 colonies from ten different subspecies. The 19 mtDNA types detected are clustered in three major phylogenetic lineages. These clades correspond well to three groups of populations with distinct geographical distributions: branch A for African subspecies (intermissa, monticola, scutellata, andansonii and capensis), branch C for North Mediterranean subspecies (caucasica, carnica and ligustica) and branch M for the West European populations (mellifera subspecies). These results partially confirm previous hypotheses based on morphometrical and allozymic studies, the main difference concerning North African populations, now assigned to branch A instead of branch M. The pattern of spatial structuring suggests the Middle East as the centre of dispersion of the species, in accordance with the geographic areas of the other species of the same genus. Based on a conservative 2% divergence rate per Myr, the separation of the three branches has been dated at about 1 Myr BP.     

浙江省金华市中华蜜蜂线粒体DNA遗传多样性研究

为了解浙江省金华市中华蜜蜂的遗传多样性及遗传分化现状,基于mtDNA tRNA1eu-COⅡ序列对金华市9个区县市共81个中蜂样本进行序列测定,分析了金华市中蜂群体的遗传多样性、遗传结构和遗传分化情况.结果共发现14个单倍型,其中主体单倍型JH1为9个地理群体所共享,与已报道的福建省中蜂主体单倍型以及浙江省丽水市的中蜂...     

Methionine as a methyl donor regulates caste differentiation in the European honey bee (Apis mellifera)

Nutrition contributes to honey bee caste differentiation, but the role of individual nutrients is still unclear. Most essential amino acid contents, except that of methionine (Met), are greater in royal jelly than worker jelly. After ∼3.5 d, the Met content in the latter was slightly greater than in the former. Met is the major raw material used in the synthesis of S-adenosyl-L-methionine, an active methyl donor for DNA methylation, which is an epigenetic driver of caste differentiation. Here, we tested whether Met regulates caste differentiation in honey bees by determining its effects on the caste development of bees receiving four diets: the basic, basic + 0.2% Met, basic + 0.2% Met + 20 mg/kg 5-azacytidine, and basic + 20 mg/kg 5-azacytidine. The presence of Met decreased the adult bee body length and the numbers of ovarioles, indicating that Met may direct the development of female larvae toward worker bees. The upregulated expression of SAMS, Dnmt1, and Dnmt3 caused by Met exposure in 4-d-old larvae indicated that the worker-inductive effects of Met may occur through the promotion of DNA methylation. We investigated the co-effects of Met and glucose on bee development, and found that the effects of an increased glucose level on the number of ovarioles and body length did not strengthen the worker-inductive effects caused by Met. Our results contribute to caste development theory and suggest that Met—as a methyl donor—plays a regulatory, but not decisive, role in caste differentiation.     

贵州越夏期与越冬期中华蜜蜂病毒病发生的调查

蜜蜂是重要的传粉昆虫,在农业生产和生态平衡中起着重大作用。近年来蜜蜂数量大幅下降,由于农药使用、生态污染、气候变化等原因,以及受到天敌如胡蜂Vespidae的影响,更重要的是受到病原如真菌、细菌以及各种病毒的危害。其中蜜蜂病毒病是造成蜜蜂数量减少的重要原因之一。为了调查贵州省越夏期和越冬期中华蜜蜂感染病毒病的情况,利用RT-PCR技术对贵州省兴义市、息烽县、台江县、龙里县采集的样本进行检测。结果显示：贵州省越夏期以红火蚁Solenopsis invicta Buren病毒Sindbis virus（SINV）为主要流行病毒,而越冬期以囊状幼虫病毒Sacbrood virus（SBV）和黑蜂王台病毒Black queen cell virus（BQCV）为主要流行病毒。本研究初步调查了贵州省主要中蜂饲养区在两个重要饲养阶段发生蜜蜂病毒病情况,这将在一定程度上为防治蜜蜂病毒病提供理论依据。     

中国甘肃东北部地区东方蜜蜂(Apis cerana)形态学研究

本研究采集甘肃东北部地区泾川蜂场、庄浪蜂场和平凉蜂场的东方蜜蜂Apis cerana 43群,采样点地貌包括山地、丘陵和平原.运用蜜蜂形态学研究方法对东方蜜蜂40个形态特征指标进行测量分析.运用SPSS13.0对蜜蜂形态数据进行方差分析、主成分分析、聚类分析及环境因子相关性分析.研究结果表明:甘肃东北部地区不同采样点东方蜜蜂形态特征指标存在显著差异,而且与环境因素显著相关.     

Uncovering the novel characteristics of Asian honey bee,Apis cerana,by whole genome sequencing

Background

The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana.

Results

Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes.

Conclusions

This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-16-1) contains supplementary material, which is available to authorized users.     

T-RFLP analysis of bacterial communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand

This study investigated bacterial community structures in the midguts of Apis mellifera and Apis cerana in Thailand to understand how bacterial communities develop in Apis species. The bacterial species present in replicate colonies from different locations and life stages were analysed. PCR amplification of bacterial 16S rRNA gene fragments and terminal restriction fragment length polymorphism analyses revealed a total of 16 distinct terminal restriction fragments (T-RFs), 12 of which were shared between A. mellifera and A. cerana populations. The T-RFs were affiliated to Beta- and Gammaproteobacteria, Firmicutes and Actinomycetes. The Gammaproteobacteria were found to be common in all stages of honey bee, but in addition, the Firmicutes group was found to be present in the worker bees. Bacterial community structure showed no difference amongst the replicate colonies, but was affected to some degree by geographical location, life stage and species of honey bees.     

The genomic basis of adaptation to high‐altitude habitats in the eastern honey bee (Apis cerana)

The eastern honey bee (Apis cerana) is of central importance for agriculture in Asia. It has adapted to a wide variety of environmental conditions across its native range in southern and eastern Asia, which includes high‐altitude regions. eastern honey bees inhabiting mountains differ morphologically from neighbouring lowland populations and may also exhibit differences in physiology and behaviour. We compared the genomes of 60 eastern honey bees collected from high and low altitudes in Yunnan and Gansu provinces, China, to infer their evolutionary history and to identify candidate genes that may underlie adaptation to high altitude. Using a combination of F_ST‐based statistics, long‐range haplotype tests and population branch statistics, we identified several regions of the genome that appear to have been under positive selection. These candidate regions were strongly enriched for coding sequences and had high haplotype homozygosity and increased divergence specifically in highland bee populations, suggesting they have been subjected to recent selection in high‐altitude habitats. Candidate loci in these genomic regions included genes related to reproduction and feeding behaviour in honey bees. Functional investigation of these candidate loci is necessary to fully understand the mechanisms of adaptation to high‐altitude habitats in the eastern honey bee.     

Genetic pollution and number of matings in a black honey bee (Apis mellifera mellifera) population

Summary In the south-east of France, local honey bees possess only the B allele at the MDH locus, whereas the races which are usually imported into this area do not have this allele. The proportion of non-B genes in a sample of drones was used to measure the genetic pollution in the local population. Within the course of a breeding scheme of local bees, 99 queens, whose genotypes are BB, were naturally mated between April 25 and June 10, 1985 at la Tave (Gard, France). Twenty daughters-workers of each queen were analysed at the MDH locus. The frequency of the B allele in drones that mated with these queens is estimated by the proportion of workers with genotype BB and the genetic pollution by the cumulated frequency of the other alleles. The sampling variances of these frequencies involve a coefficient which is a function of the average number of drones mated with a queen. This latter parameter is estimated through the maximum likelihood method. In addition to the three well-known alleles, a rare allele (frequency=0.0055), possibly equivalent to the S1 allele described by Badino et al. (1983), has been found in three different colonies. Cumulating the frequencies of the non-B alleles results in an estimation of the genetic pollution equal to 0.0394 (±0.0071). This low value allows us to proceed to the next step of the selection project. The mean number of drones mated to a queen is 12.4 with a (10.4–19.3) confidence interval at the 90% level.     

Non-destructive sources of DNA used to genotype honey bee (Apis mellifera) queens

We describe a method for genotyping honey bee queens Apis mellifera L. (Hymenoptera: Apidae), using biological materials that are normally cast off during development (larval and pupal exuviae), or can be removed without apparent damage to queen longevity or acceptability to workers (wing clippings). Highly polymorphic microsatellite loci were successfully amplified from DNA from all of these sources, although with differing degrees of success. DNA was extracted using a simple Chelex 100® boiling procedure. Four microsatellite primers were used to amplify the DNA, and the PCR products were visualized on an ALFexpress Automated Sequencer. Genotypes created from these sources were consistent with those originating from tarsal tissue. Successful retrieval and amplification of DNA from the exuviae from immature queens allows potential breeding individuals to be genotyped and selected before they become adults. This procedure may therefore have value as DNA marker‐assisted breeding programs are developed for honey bees.     

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M _W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.     

Signatures of selection in the Iberian honey bee (Apis mellifera iberiensis) revealed by a genome scan analysis of single nucleotide polymorphisms

Understanding the genetic mechanisms of adaptive population divergence is one of the most fundamental endeavours in evolutionary biology and is becoming increasingly important as it will allow predictions about how organisms will respond to global environmental crisis. This is particularly important for the honey bee, a species of unquestionable ecological and economical importance that has been exposed to increasing human‐mediated selection pressures. Here, we conducted a single nucleotide polymorphism (SNP)‐based genome scan in honey bees collected across an environmental gradient in Iberia and used four F_ST‐based outlier tests to identify genomic regions exhibiting signatures of selection. Additionally, we analysed associations between genetic and environmental data for the identification of factors that might be correlated or act as selective pressures. With these approaches, 4.4% (17 of 383) of outlier loci were cross‐validated by four F_ST‐based methods, and 8.9% (34 of 383) were cross‐validated by at least three methods. Of the 34 outliers, 15 were found to be strongly associated with one or more environmental variables. Further support for selection, provided by functional genomic information, was particularly compelling for SNP outliers mapped to different genes putatively involved in the same function such as vision, xenobiotic detoxification and innate immune response. This study enabled a more rigorous consideration of selection as the underlying cause of diversity patterns in Iberian honey bees, representing an important first step towards the identification of polymorphisms implicated in local adaptation and possibly in response to recent human‐mediated environmental changes.     

Nocturnal dispersal by femaleAcarapis woodi in honey bee (Apis mellifera) colonies

Comparisons were made between the infestation levels of the honey bee tracheal miteAcarapis woodi (Rennie) in newly emerged honey bees (Apis mellifera L.) exposed for 12 h during the daytime or nighttime in mite-infested bee colonies. Bees exposed during the night harbored a significantly higher number of mites (718) when compared with the daytime bees (88 mites) (n=14 day/night cycles utilizing 33 colonies). On 4 days of an 8-day study, three test colonies were closed during the daytime to eliminate foraging flights. Thus equal numbers of bees were present in the colonies during the day and night sample periods. These 4 flightless days were compared to 4 free-flight days and mite dispersal rates were not significantly different. Additionally, the movement of bees on the combs of four glass-walled observation hives was quantified on 10 days at 0800, 1200, 1600, 2000, 2400 and 0400 h. Bee movement at 2400 and 0400 h was significantly lower than the other observation times. Movement of host bees may be one factor involved in the increased nighttime mite dispersals. These findings do not support the hypothesis that the absence of foraging bees during the day reduces the bee to bee contact time, thus reducing mite dispersals between host bees. Differential diurnal activity levels between host bees and mite parasites was demonstrated. The exact role of host-bee behavior and/or mite behavior in the nighttime dispersal patterns observed, remains for further investigation.     

中国东方蜜蜂的形态分化(英文)

【目的】遗传分化研究是认识蜜蜂形态多样性和适应性进化的重要环节,是确定蜜蜂资源管理单位和保护单位的前提,有助于保护蜜蜂的遗传资源。本研究通过分析形态分化,研究中国东方蜜蜂Apiscerana在中国地理环境下的遗传分化和遗传资源分布。【方法】从我国所有东方蜜蜂分布区102个采样点共采集6 147头东方蜜蜂工蜂,每一采样点10~20群中取60头工蜂进行解剖,测定与翅、个体大小、后足和体色相关的33个形态特征,进行多变量形态统计分析,划分形态类群。同时对分出的不同东方蜜蜂类群的形态特征及分布模式进行分析。【结果】根据判别分析和主成分分析的聚类结果,我国东方蜜蜂分为14个形态类群。有5个类群具有较小的个体大小。海南类群个体最小,其次为滇南类群,台湾类群,南方类群和北方类群。这5个类群之间在吻长、前翅长、前翅第3亚缘室结构、体色和蜡镜长上存在显著差异。长白类群具有最大的肘脉指数、蜡镜和第5背板绒毛带宽。而西藏波密类群具有全国最小的第五背板绒毛带宽。西北类群具有最长的后足。川西高原的5个形态类群具有个体大、体色黑等特点。其中,巴塘类群肘脉指数(3.0169)全国最小,个体大小全国最大。阿坝类群具有仅次于长白类群的肘脉指数,且翅长和第七腹板最大。德荣类群体色最黑。雅江类群具有独特的翅脉角 (A4, N23, E9和J10最小,B4最大)。川滇类群在川西高原个体最小。【结论】本研究在全面收集我国所有东方蜜蜂分布区样本,尤其是西藏波密、台湾省和川西高原的珍贵样本的基础上,进行了东方蜜蜂形态测量学分析。在我国共发现东方蜜蜂14个形态类群：海南类群、滇南类群、长白类群、台湾类群、波密类群、阿坝类群、巴塘类群、德荣类群、雅江类群、川滇类群、川贵类群、西北类群、南方类群以及北方类群。研究结果为中国东方蜜蜂遗传资源的保护和遗传资源的开发利用提供理论基础。