Isolation and characterization of nine microsatellite loci from Aphelinus mali (Hymenoptera: Aphelinidae), a parasitoid of Eriosoma lanigerum (Hemiptera: Aphididae)

Abstract Nine novel microsatellite markers are presented for Aphelinus mali, a parasitoid of the woolly apple aphid Eriosoma lanigerum. Loci were characterized for 28 individuals from a single orchard in central Chile. Seven loci were polymorphic within A. mali (3–6 alleles per locus; observed heterozygosity ranging from 0.04 to 0.57) and are therefore useful for population genetic studies within this species.     

入侵性害虫——苹果绵蚜田间种群数量的调查方法

2007年6月中旬到10底在山东省莱阳市对苹果绵蚜Eriosoma lanigerum(Hausmann)的田间发生数量进行调查研究。在田间定点调查5株苹果树上苹果绵蚜虫落数量和虫落面积,并在其它苹果树上随机选择20个虫落,计数每个虫落中苹果绵蚜的活体数,从而计算单位面积虫落中苹果绵蚜的平均数量。分析指出,以计算法求得的虫落中苹果绵蚜的实际数量为指标,既考虑了苹果绵蚜虫落的数量和大小,也考虑了单位面积虫落中苹果绵蚜活体数,并且把被苹果绵蚜蚜小蜂寄生的僵蚜排除在外,能较客观地反映出田间实际情况,是一种比较准确的表示苹果绵蚜田间数量的方法。     

Genetic diversity of woolly apple aphid Eriosoma lanigerum (Hemiptera: Aphididae) populations in the Western Cape, South Africa

The woolly apple aphid Eriosoma lanigerum (Hausmann) is one of the most damaging apple pests in South Africa. Information on its genetic diversity is lacking and this study, in which the genetic structure of parthenogenetic E. lanigerum populations was characterized in the Western Cape Province of South Africa, represents the first local study of its kind. A total of 192 individuals from four different regions were collected and analysed using amplified fragment length polymorphism (AFLP). Using five selective AFLP primer pairs, 250 fragments were scored for analysis. Results indicated that a low level of genetic variation was apparent in E. lanigerum populations in the Western Cape (H = 0.0192). Furthermore, populations collected from geographically distant regions were very closely related, which can partly be explained by the fact that agricultural practices were responsible for dissemination of populations from a common ancestor to geographically distant areas. The low level of variation found indicated that the possibility of controlling E. lanigerum in the Western Cape using host plant resistance is favourable. This is the first report of AFLP being used to characterize the genetic structure of an aphid species. Results indicate that this marker may be useful for analysis of other aphid species.     

苹果绵蚜在不同寄主果园中的种群数量动态比较

【背景】在我国,苹果绵蚜对苹果等寄主的危害日益严重,发生面积不断扩大,而品种抗性是害虫治理的一种重要手段。【方法12007—2009年于山东莱阳地区系统调查了苹果绵蚜在不同寄主果园的发生动态规律,比较了不同寄主对苹果绵蚜的抗性差异。【结果】苹果绵蚜在红富士、乔纳金、新红星苹果园和西府海棠果园中1a有2个发生高峰,5月底到7月下旬是第1个高峰,也是全年发生的最高峰,9月初到10月底是全年发生的第2个高峰,而7月底到8月底是苹果绵蚜发生的低谷期。苹果绵蚜在红富士苹果上的发生数量显著高于在乔纳金、新红星苹果和西府海棠上的发生数量。在3a的调查时间内,苹果绵蚜在红富士、乔纳金、新红星苹果上每次平均发生量分别为153．2、31．6、30．3头·株^-1。越冬前调查表明,苹果绵蚜对红富士的为害率显著高于青香蕉、乔纳金、新红星和小国光,小国光的被害率以及虫落数量最低。【结论与意义】红富士是苹果绵蚜的感虫品种,而小国光、乔纳金和新红星相对抗虫。文中讨论了苹果绵蚜抗性品种的筛选方法,为掌握不同寄主对苹果绵蚜的抗性差异以及制定苹果绵蚜的抗性治理策略提供了依据。     

苹果绵蚜在中国适生区预测及发生影响因子

苹果绵蚜是苹果上一种重要的检疫性害虫,每年给苹果产区造成严重的经济损失.预测苹果绵蚜适生性区域和影响其定殖扩散的环境因子,能够开展苹果绵蚜分布区域测报、制定有效的检疫措施并为防治决策提供依据.本研究基于最大熵算法的生态位模型MaxEnt和地理信息系统软件ArcGIS对苹果绵蚜进行适生区分析及预测,用受试者工作特征曲线(ROC)对预测模型和预测结果进行评估,并用Jackknife方法分析影响绵蚜发生的重要因子.结果表明,苹果绵蚜在我国适生范围很广,其中,辽宁、山东、河南、河北、安徽、江苏、陕西等省的适生性指数较高,对苹果绵蚜发生具有重要影响的是与温度相关的环境因子.     

基于EPG的三种刺吸式害虫在苹果苗上的取食行为比较

【目的】苹果绵蚜Eriosoma lanigerum、绣线菊蚜Aphis citricola和梨网蝽Stephanitis nashi是苹果园的一类重要害虫,它们以刺吸式口器对苹果树造成不同程度的危害。本研究旨在明确这3种刺吸式昆虫在苹果树上取食行为差异。【方法】利用刺吸电位(electrical penetration graph, EPG)技术对苹果绵蚜和绣线菊蚜成虫在苹果苗韧皮部和非韧皮部上的EPG指标,以及苹果绵蚜、绣线菊蚜和梨网蝽成虫苹果苗上的取食行为进行了比较分析,分析了这3种害虫的成虫在苹果苗上取食8 h各种波形平均持续时间的占比。【结果】结果表明,苹果绵蚜和绣线菊蚜成蚜在苹果苗上均产生6种取食波形,即非刺探波(np)、路径波(C)、意外穿刺细胞非主动取食细胞波(pd)、木质部取食波(G)、韧皮部唾液分泌波(E1)和韧皮部取食波(E2);而梨网蝽成虫取食过程中只产生非刺探波(np)、表皮刺穿波(A)、叶肉细胞取食波(Gc)和木质部取食波(E)4种波形。从蚜虫在苹果苗非韧皮部上取食的EPG指标看,苹果绵蚜成蚜pd波平均时间显著高于绣线菊蚜的,而刺探次数、np波总时间和pd波次数均显著低于绣线菊蚜的。从蚜虫在苹果苗韧皮部上取食的EPG指标来看,除第1次出现E2波的时间外,各指标没有显著差异。从3种害虫在苹果苗上取食8 h各波形的占比来看,梨网蝽成虫的np波总时间所占比例最高(53%),其次是绣线菊蚜成虫的(24%),苹果绵蚜成虫的最低(为1%)。同时,苹果绵蚜和绣线菊蚜成虫取食波为E2波,所占总时间比例分别为35%和25%,而梨网蝽的取食波为Gc波(占总时间比例为36%)和E波(占总时间比例为11%)。【结论】本研究阐释了苹果园刺吸式口器害虫的生态位分离和取食行为学机制,为果园刺吸式口器害虫的综合治理提供了理论依据。     

苹果绵蚜发生危害特点及防治对策

苹果绵蚜主要在我国辽东半岛、胶东半岛和昆明一带局部发生 ,每年 5～ 6月和 9～ 1 0月为害虫发生盛期。近几年来 ,危害逐年加重 ,并不断扩展蔓延 ,对我国苹果的产量和品质构成了严重威胁。该文介绍了苹果绵蚜在我国的危害情况和发生规律 ,分析了其发生与危害的特点 ,并提出了有效防治对策     

苹果绵蚜的生物学特性及其综合治理

苹果绵蚜是一种重要的外来人侵性害虫,在世界的各苹果种植区均有分布,对苹果造成严重危害。本文主要介绍了苹果绵蚜的生物学特性、发生危害特点以及各种防治措施,包括植物检疫、农业防治、生物防治、化学防治和抗性育种等,为在我国广大果区对苹果绵蚜进行综合治理提供依据。     

基于微卫星标记的苹果绵蚜种群遗传结构动态

【目的】苹果绵蚜是我国重要的入侵害虫,对苹果生产造成了严重危害。近年来,苹果绵蚜扩散面积增大,危害加重。了解苹果绵蚜入侵过程中的分子生态变化,可为该虫的综合防控提供依据。【方法】利用微卫星标记技术,选择6个微卫星位点对山东省6个地区(烟台、威海、青岛、潍坊、聊城、泰安)2012—2015年苹果绵蚜种群遗传结构变化规律进行分析。【结果】2012—2015年,山东省6个地区的苹果绵蚜遗传多样性随时间推移逐渐降低。其中,2012年的遗传多样性极显著高于2013—2015年;2013—2015年之间虽然差异不显著,但随时间推移,等位基因观测值(Na)和期望杂合度(He)等遗传多样性指数有逐渐降低的趋势。通过无限等位基因模型、双相突变模型和逐步突变模型分析发现,6个地区的苹果绵蚜均经历了瓶颈效应,是遗传多样性降低的主要原因。对6个微卫星位点分析发现,Erio20、Erio75和Erio78扩增到的苹果绵蚜等位基因数量以及这3个位点的多样性指数随时间推移逐渐降低。【结论】Erio20、Erio75和Erio78是引起苹果绵蚜遗传多样性降低的主要多态位点。苹果绵蚜可能进化出了"超级克隆"基因型,因此,其可在我国适应不同生态环境并增大扩散范围。     

Mutation and evolution of microsatellite loci in Neurospora

The patterns of mutation and evolution at 13 microsatellite loci were studied in the filamentous fungal genus Neurospora. First, a detailed investigation was performed on five microsatellite loci by sequencing each microsatellite, together with its nonrepetitive flanking regions, from a set of 147 individuals from eight species of Neurospora. To elucidate the genealogical relationships among microsatellite alleles, repeat number was mapped onto trees constructed from flanking-sequence data. This approach allowed the potentially convergent microsatellite mutations to be placed in the evolutionary context of the less rapidly evolving flanking regions, revealing the complexities of the mutational processes that have generated the allelic diversity conventionally assessed in population genetic studies. In addition to changes in repeat number, frequent substitution mutations within the microsatellites were detected, as were substitutions and insertion/deletions within the flanking regions. By comparing microsatellite and flanking-sequence divergence, clear evidence of interspecific allele length homoplasy and microsatellite mutational saturation was observed, suggesting that these loci are not appropriate for inferring phylogenetic relationships among species. In contrast, little evidence of intraspecific mutational saturation was observed, confirming the utility of these loci for population-level analyses. Frequency distributions of alleles within species were generally consistent with the stepwise mutational model. By comparing variation within species at the microsatellites and the flanking-sequence, estimated microsatellite mutation rates were approximately 2500 times greater than mutation rates of flanking DNA and were consistent with estimates from yeast and fruit flies. A positive relationship between repeat number and variance in repeat number was significant across three genealogical depths, suggesting that longer microsatellite alleles are more mutable than shorter alleles. To test if the observed patterns of microsatellite variation and mutation could be generalized, an additional eight microsatellite loci were characterized and sequenced from a subset of the same Neurospora individuals.     

Optimal profit of the parasitation by Aphelinus mali in an IPM complementary strategy for the control of Eriosoma lanigerum

During summer the parasitoid Aphelinus mali may certainly reduce the infestation of woolly apple aphid (Eriosoma lanigerum), but studies on the single interaction rarely indicate sufficient biological control in the period May-June. In this period chemical control by spirotetramat or pirimicarb remains indispensable in order to anticipate on dense migration waves and subsequent colonization of extension shoots by E. lanigerum. The limited parasitation by A. mali around flowering is linked with a delayed emergence from diapause and with a slower reproduction rate than its host. In 2010 and 2011 the first adult flights monitored on yellow sticky traps corresponded perfectly with the currently used prediction models for A. mali. Further accurate monitoring all along the season enabled also to determine a well defined endo-parasitic phase of A. mali occurring after the small peak observed around flowering. During this endo-parasitic phase A. mali larvae reside inside their mummified host. Compounds with higher acute toxicity on A. mali adults, like chloronicotinyl insecticides (CNI's), are preferably positioned here. Selectivity in the time can then be claimed. Respecting this principle, the further parasitation potential of A. mali in summer is not hampered. Preservation of the first peak of flights of A. mali in the pre-flowering period is essential for an exponential flight increase. This is essential for the parasitation of E. lanigerum in summer, which constitutes a valuable complement in the integrated control strategy.     

Estimating gene flow between refuges and crops: a case study of the biological control of Eriosoma lanigerum by Aphelinus mali in apple orchards

Parasitoid disturbance populations in agroecosystems can be maintained through the provision of habitat refuges with host resources. However, specialized herbivores that feed on different host plants have been shown to form host-specialized races. Parasitoids may subsequently specialize on these herbivore host races and therefore prefer parasitizing insects from the refuge, avoiding foraging on the crop. Evidence is therefore required that parasitoids are able to move between the refuge and the crop and that the refuge is a source of parasitoids, without being an important source of herbivore pests. A North-South transect trough the Chilean Central Valley was sampled, including apple orchards and surrounding Pyracantha coccinea (M. Roem) (Rosales: Rosacea) hedges that were host of Eriosoma lanigerum (Hemiptera: Aphididae), a globally important aphid pest of cultivated apples. At each orchard, aphid colonies were collected and taken back to the laboratory to sample the emerging hymenopteran parasitoid Aphelinus mali (Hymenoptera: Aphelinidae). Aphid and parasitoid individuals were genotyped using species-specific microsatellite loci and genetic variability was assessed. By studying genetic variation, natural geographic barriers of the aphid pest became evident and some evidence for incipient host-plant specialization was found. However, this had no effect on the population-genetic features of its most important parasitoid. In conclusion, the lack of genetic differentiation among the parasitoids suggests the existence of a single large and panmictic population, which could parasite aphids on apple orchards and on P. coccinea hedges. The latter could thus comprise a suitable and putative refuge for parasitoids, which could be used to increase the effectiveness of biological control. Moreover, the strong geographical differentiation of the aphid suggests local reinfestations occur mainly from other apple orchards with only low reinfestation from P. cocinnea hedges. Finally, we propose that the putative refuge could act as a source of parasitoids without being a major source of aphids.     

苹果绵蚜蚜小蜂——对苹果绵蚜有控制潜能的寄生蜂

苹果绵蚜蚜小蜂是苹果绵蚜的重要寄生蜂。本文综述了苹果绵蚜蚜小蜂的研究概况,包括该寄生蜂的引种与分布、寄主范围、生物学特性、控制效果与影响因素、重寄生蜂及合理利用等,指出应加快苹果绵蚜蚜小蜂的规模化生产,充分发挥其对苹果绵蚜的控制作用。     

Population genetics of the potentially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Tephritidae)

A set of 10 microsatellite markers was used to survey the levels of genetic variability and to analyse the genetic aspects of the population dynamics of two potentially invasive pest fruit fly species, Ceratitis rosa and C. fasciventris, in Africa. The loci were derived from the closely related species, C. capitata. The degree of microsatellite polymorphism in C. rosa and C. fasciventris was extensive and comparable to that of C. capitata. In C. rosa, the evolution of microsatellite polymorphism in its distribution area reflects the colonization history of this species. The mainland populations are more polymorphic than the island populations. Low levels of differentiation were found within the Africa mainland area, while greater levels of differentiation affect the islands. Ceratitis fasciventris is a central-east African species. The microsatellite data over the Uganda/Kenya spatial scale suggest a recent expansion and possibly continuing gene flow within this area. The microsatellite variability data from C. rosa and C. fasciventris, together with those of C. capitata, support the hypothesis of an east African origin of the Ceratitis spp.     

Misalignment-mediated DNA polymerase beta mutations: comparison of microsatellite and frame-shift error rates using a forward mutation assay

Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA](10), [TC/AG](11), and [TTCC/AAGG](9). DNA templates contained the microsatellites inserted in-frame into the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase beta (polbeta) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polbeta error frequencies within the dinucleotide sequences were (2-9) x 10(-3), 14-72-fold higher than the ssDNA template frequencies. The polbeta error frequencies within the tetranucleotide sequences were (4-6) x 10(-3), a 4-13-fold increase over background. Strand biases were observed for the [TC/AG](11) and [TTCC/AAGG](9) alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.     

三种提取苹果绵蚜基因组DNA方法的比较

参考国内外昆虫基因组DNA提取的常用方法,选取KAc法、氯仿-异戊醇法和盐析法3种方法对苹果绵蚜(Eriosoma lanigerum)基因组DNA进行提取.通过基因组DNA直接琼脂糖凝胶电泳、SSR引物扩增产物的琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳检测,对3种方法所提基因组DNA的质量进行了比较,并综合分析了提取所需时间及所用试剂的毒性大小等.结果表明,盐析法操作程序较简便快捷,节时省工,可得较好质量的DNA样本,且对试验操作人员无伤害,是一种值得推广应用的基因组DNA提取方法.     

Evidence for complex mutations at microsatellite loci in Drosophila.

Fifteen lines each of Drosophila melanogaster, D. simulans, and D. sechellia were scored for 19 microsatellite loci. One to four alleles of each locus in each species were sequenced, and microsatellite variability was compared with sequence structure. Only 7 loci had their size variation among species consistent with the occurrence of strictly stepwise mutations in the repeat array, the others showing extensive variability in the flanking region compared to that within the microsatellite itself. Polymorphisms apparently resulting from complex nonstepwise mutations involving the microsatellite were also observed, both within and between species. Maximum number of perfect repeats and variance of repeat count were found to be strongly correlated in microsatellites showing an apparently stepwise mutation pattern. These data indicate that many microsatellite mutation events are more complex than represented even by generalized stepwise mutation models. Care should therefore be taken in inferring population or phylogenetic relationships from microsatellite size data alone. The analysis also indicates, however, that evaluation of sequence structure may allow selection of microsatellites that more closely match the assumptions of stepwise models.     

Microsatellite markers for the ectomycorrhizal basidiomycete Lactarius mammosus

We report the characterization of polymorphic microsatellite markers in the ectomycorrhizal fungus Lactarius mammosus Fr. Two enrichment protocols were used to isolate microsatellite loci and polymorphism was explored within 31 sporocarps originating from a forest site in northern Sweden. We found nine variable microsatellite loci with the number of alleles per locus varying between 2 and 5, and expected heterozygosities ranging from 0 to 0.84. These loci are available for the analysis of genetic structure and gene flow in L. mammosus populations.     

Breakdown of phylogenetic signal: a survey of microsatellite densities in 454 shotgun sequences from 154 non model eukaryote species

Microsatellites are ubiquitous in Eukaryotic genomes. A more complete understanding of their origin and spread can be gained from a comparison of their distribution within a phylogenetic context. Although information for model species is accumulating rapidly, it is insufficient due to a lack of species depth, thus intragroup variation is necessarily ignored. As such, apparent differences between groups may be overinflated and generalizations cannot be inferred until an analysis of the variation that exists within groups has been conducted. In this study, we examined microsatellite coverage and motif patterns from 454 shotgun sequences of 154 Eukaryote species from eight distantly related phyla (Cnidaria, Arthropoda, Onychophora, Bryozoa, Mollusca, Echinodermata, Chordata and Streptophyta) to test if a consistent phylogenetic pattern emerges from the microsatellite composition of these species. It is clear from our results that data from model species provide incomplete information regarding the existing microsatellite variability within the Eukaryotes. A very strong heterogeneity of microsatellite composition was found within most phyla, classes and even orders. Autocorrelation analyses indicated that while microsatellite contents of species within clades more recent than 200 Mya tend to be similar, the autocorrelation breaks down and becomes negative or non-significant with increasing divergence time. Therefore, the age of the taxon seems to be a primary factor in degrading the phylogenetic pattern present among related groups. The most recent classes or orders of Chordates still retain the pattern of their common ancestor. However, within older groups, such as classes of Arthropods, the phylogenetic pattern has been scrambled by the long independent evolution of the lineages.     

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an approximately 25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.