The separation of Liriomyza huidobrensis (Diptera: Agromyzidae) from related indigenous and non-indigenous species encountered in the United Kingdom using cellulose acetate electrophoresis

The non-indigenous pest leaf miner Liriomyza huidobrensis and its close relatives L. bryoniae and L. strigata belong to a natural group. These species can be separated from one another by means of protein electrophoresis on a cellulose acetate membrane. The protocol for this diagnostic assay involves staining for two enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and leucine-glycine peptidase (PEP). Other leaf miner species that might also be found under glass, Chromatomyia syngenesiae, C. horticola and the non-indigenous L. trifolii and L. sativae , are clearly distinguishable from the L. huidobrensis group of species by this assay. The effect of parasitism on L. huidobrensis by Dacnusa sibirica was investigated and shown unlikely to cause misidentification of the leaf miner host. Results obtained from practical use of the assay to identify unknown specimens from ports and nurseries are compared with those obtained from flies kept in laboratory culture and used during development of the assay. No new variation was found for G6PDH. New PEP variation was found for both L. huidobrensis and L. bryoniae but this did not affect the integrity of the assay. A biochemical key to these agromyzid leaf miners is presented.     

两种外来斑潜蝇实验种群生物学特征比较

美洲斑潜蝇LiriomyzasativaeBlanchard和南美斑潜蝇Liriomyzahuidobrensis(Blanchard)是我国的2种重要入侵性害虫。本实验室在标准化流程下饲养了48(L.huidobrensis)-160(L.sativae)个世代的稳定实验种群。美洲斑潜蝇的实验种群需要较大的繁殖规模(>100对)才能保持群体的稳定性。2种斑潜蝇4个发育阶段的形态特征和幼虫为害特征差别明显。南美斑潜蝇的发育历期比美洲斑潜蝇长,总产卵量低于美洲斑潜蝇,但产卵历期明显长于美洲斑潜蝇。这可能是南美斑潜蝇较之容易繁殖的重要原因。该实验种群的建立为物种驯化、寄主选择性、抗性筛选及其它生理学、生态学等诸多研究打下基础。     

Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. Langei (Diptera: Agromyzidae) applied to three recent leafminer invasions.

A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frick and L. huidobrensis (Blanchard). This method requires polymerase chain reaction (PCR) amplification of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. Spel cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costly than DNA sequencing,which is currently the only other way to differentiate these two species. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and find that the invasive leafminer in all three locations is L. huidobrensis.     

重大潜在入侵害虫番茄潜叶蛾的SS-CoI快速检测技术

【背景】番茄潜叶蛾是源自南美洲的一种最具毁灭性的世界性入侵害虫,其适生区范围广,且与其他潜叶类昆虫难以准确、快速识别。【方法】以田间常见的其他6种潜叶类害虫为对照,采用基于mtDNACOl基因的种特异性SS—COI方法,研究番茄潜叶蛾快速分子检测技术。【结果】种特异性检测结果显示,SS-COl引物只对番茄潜叶蛾mtDNA具有扩增能力,对美洲斑潜蝇、南美斑潜蝇、三叶草斑潜蝇、葱斑潜蝇、豌豆潜叶蝇、桃潜叶蛾等其他潜叶类害虫不具有扩增效果。该引物不仅对成虫和单粒卵具有很好的扩增效能,对单根触角、头部、胸部、腹部以及翅和足等成虫残体亦具有同样的扩增能力,其最低检出阈值为312．5Pg·μL^-1。【结论与意义】该方法在有效防范番茄潜叶蛾侵入我国,保障农业生产安全和生态环境安全中意义重大。     

Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy

The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome.     

美洲斑潜蝇SS-PCR检测技术研究

【背景】美洲斑潜蝇是一种严重威胁瓜果蔬菜、烟草、棉花等经济作物和花卉生产的入侵性害虫。由于潜叶蝇类害虫体型较小、生活方式隐蔽、形态相似,本文针对其难以快速准确地进行形态鉴别的问题,以美洲斑潜蝇为研究对象,以菜田常见的4种潜叶蝇类害虫为参照,采用种特异性PCR方法（species-specific PCR,SS-PCR）,研究其快速分子检测鉴定技术。【方法】调用GenBank中一段936bp的美洲斑潜蝇线粒体DNA（mtDNA）细胞色素氧化酶亚基Ⅰ基因（COⅠ）的序列（Gen-Bank登录号为EU219613）,并根据此基因片段的碱基序列设计引物1对,其扩增片段大小为294bp。【结果】种特异性检验结果显示,该引物只对美洲斑潜蝇的COⅠ基因具有扩增能力,对其他种类如南美斑潜蝇、三叶斑潜蝇、葱斑潜蝇、豌豆潜叶蝇等没有扩增能力。该引物不仅对成虫具有良好的扩增效果,对蛹、幼虫以及单粒卵也具有同样的扩增效果,其最低检出阈值为1/3840头成虫。【结论与意义】SS-PCR技术体系可用于美洲斑潜蝇的鉴定识别与检测监测,对阻止其进一步扩散蔓延具有重要意义。     

入侵害虫南美斑潜蝇在西藏首次发现及其寄生蜂调查

【目的】南美斑潜蝇Liriomyza huidobrensis为我国重要的入侵害虫,我们于2017年7月首次发现其已扩散至西藏。本研究旨在了解西藏地区南美斑潜蝇的发生现状及其寄生蜂种类。【方法】2017和2018年6月上旬-9月中旬对西藏拉萨、林芝、山南和日喀则4个地区的南美斑潜蝇的发生危害及其寄生蜂组成进行了调查采样。【结果】调查的西藏这4个地区均发现南美斑潜蝇分布和危害;温室发生时间为6月上旬-9月中旬,而露地的发生高峰在7月下旬-8月上旬。其寄主植物有9科21种,主要有豆科(Fabaceae)、茄科(Solanaceae)和伞形科(Umbelliferae)等,主要嗜好经济作物有豇豆Vigna unguiculata, 扁豆Lablab purpureus , 莴笋Lactuca sativa var. angustana和白菜Brassica rapa ssp. pekinensis。其寄生蜂包括姬小蜂科(Eulophidae)、茧蜂科(Braconidae)和瘿蜂科(Cynipidae)共计3科9属16种,分别为双斑潜蝇姬小蜂Diglyphus bimaculatus, 豌豆潜蝇姬小蜂Diglyphus isaea, 小斑潜蝇姬小蜂Diglyphus minoeus, 万氏潜蝇姬小蜂Diglyphus wani, Diglyphus sp., 纵纹瑟姬小蜂Cirrospilus vittatus, Hemiptarsenus sp., 芙新姬小蜂Neochrysocharis formosa, 底比斯姬小蜂Chrysocharis pentheus, 普金姬小蜂Chrysocharis pubicornis, Chrysocharis sp., 潜蝇柄腹姬小蜂Pediobius metallicus, Pnigalio sp., 西伯利亚离颚茧蜂Dacnusa sibirica, Dacnusa sp.和Gronotoma sp.;其中优势种为西伯利亚离颚茧蜂、双斑潜蝇姬小蜂和豌豆潜蝇姬小蜂,其个体数量占比分别为总采集个体数的48.7%, 22.1%和12.9%。【结论】西藏南美斑潜蝇的寄生蜂种类丰富,发挥着重要的自然控制作用。建议进一步加强优势寄生蜂生物学、控害潜力评价以及利用研究,为南美斑潜蝇的绿色防控提供指导。     

我国入侵害虫西部喙缘蝽的溯源分析

本文对我国外来入侵物种西部喙缘蝽Leptoglossus occidentalis Heidemann, 1910进行了溯源分析,依据对国内种群的观察和标本简要描述了其形态特征和生活习性。通过野外采集国内种群标本,提取扩增COI基因序列,结合原产地和国外其他入侵种群的公共序列,构建西部喙缘蝽COI基因数据集,以中国入境口岸对木材的检疫情况为参考,分析西部喙缘蝽的入侵来源。全世界目前已知10种单倍型,中国存在4种。山东青岛种群有H＿8、H＿9和H＿10三种单倍型,其中H＿8与韩国共享,H＿9和H＿10为中国特有单倍型,青岛种群可能部分来自韩国;山东烟台两个采集点的种群都属于H＿2,可能来自北美东部,与青岛种群的入侵来源不同。以上分析表明,我国西部喙缘蝽种群可能来源于多次独立入侵事件,同时在国内发生了小范围的扩散迁移。本研究首次对我国入侵害虫西部喙缘蝽做了溯源分析,为更好地制定规制措施,开展检疫防治工作提供了科学依据。     

Host range and genetic strains of leafminer flies (Diptera: Agromyzidae) in eastern Brazil reveal a new divergent clade of Liriomyza sativae

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Influence of Liriomyza species (Diptera: Agromyzidae) and their host plants, on oviposition by Opius dissitus females (Hymenoptera: Braconidae)

Two factors, among others, influence the oviposition of Opius dissitus Muesebeck. The first was the host species, the second the plant upon which the pest host larvae develop. O. dissitus females prefer Liriomyza trifolii (Burgess) larvae over those of L. huidobrensis (Blanchard). O. dissitus females were more attracted to the larvae of L. trifolii or L. huidobrensis when these were present on courgette rather than tomato or lettuce.     

Inferring modes of colonization for pest species using heterozygosity comparisons and a shared-allele test

Long-range dispersal of a species may involve either a single long-distance movement from a core population or spreading via unobserved intermediate populations. Where the new populations originate as small propagules, genetic drift may be extreme and gene frequency or assignment methods may not prove useful in determining the relation between the core population and outbreak samples. We describe computationally simple resampling methods for use in this situation to distinguish between the different modes of dispersal. First, estimates of heterozygosity can be used to test for direct sampling from the core population and to estimate the effective size of intermediate populations. Second, a test of sharing of alleles, particularly rare alleles, can show whether outbreaks are related to each other rather than arriving as independent samples from the core population. The shared-allele statistic also serves as a genetic distance measure that is appropriate for small samples. These methods were applied to data on a fruit fly pest species, Bactrocera tryoni, which is quarantined from some horticultural areas in Australia. We concluded that the outbreaks in the quarantine zone came from a heterogeneous set of genetically differentiated populations, possibly ones that overwinter in the vicinity of the quarantine zone.     

Toxicity of chemicals commonly used in Indonesian vegetable crops to Liriomyza huidobrensis populations and the Indonesian parasitoids Hemiptarsenus varicornis, Opius sp., and Gronotoma micromorpha, as well as the Australian parasitoids Hemiptarsenus varicornis and Diglyphus isaea

Liriomyza huidobrensis (Blanchard) and Liriomyza sativae (Blanchard) are important pests of vegetable crops in Indonesia and are likely to spread to neighboring countries. Three pesticides (dimehypo, abamectin, and cyromazine) are currently used to control these pests, but there is little information on their effectiveness against field populations and on their impact on parasitoids controlling Liriomyza species. The toxicity of these chemicals to L. huidobrensis and three common parasitoids (Hemiptarsenus varicornis Gerault, Opius sp., and Gronotoma micromorpha Perkins) was therefore evaluated in Indonesia with mortality laboratory assays. All three chemicals were effective against larvae of three populations of L. huidobrensis with different histories of chemical exposure. Dimehypo caused mortality in adult Opius sp., G. micromorpha, and H. varicornis, whereas abamectin was toxic only at concentrations substantially higher than the field rate. Cyromazine did not influence survival of the parasitoids. A commonly used fungicide, mancozeb, had no impact on parasitoid mortality. Trials were repeated with a strain of H. varicornis from Australia and a different parasitoid (Diglyphus isaea) recently found in Australia. Neither parasitoid was influenced by mancozeb or cyromazine. Abamectin applied at field rates caused some mortality among the adults of both species, but was less toxic than chlorpyrifos. Abamectin produced lower LC50s against Australian H. varicornis than against Indonesian H. varicornis. These results suggest that cyromazine can be incorporated into Liriomyza control programs in Indonesia that conserve parasitoids, whereas dimehypo and abamectin need to be used cautiously. Local Australian parasitoids should help control L. huidobrensis as long as only cyromazine and nontoxic fungicides are applied.     

Foliar Application of the Entomopathogenic Nematode Steinernema feltiae Against Leafminers on Vegetables

Data from a comparative study of the efficacy of Steinernema feltiae for the control of three species of leafminer formed the basis of an application schedule which successfully suppressed an outbreak of the statutory leafminer pest, Liriomyza huidobrensis . All three instars of Liriomyza bryoniae and L. huidobrensis were similarly susceptible to S. feltiae at 20 o C and > 90% relative humidity (RH). Although all larval instars of Chromatomyia syngenesiae were susceptible to S. feltiae , mortality was lower than for L. bryoniae . Repeat applications of S. feltiae to L. bryoniae and C. syngenesiae indicated that a nematode treatment to the second/early third instar larvae was more effective than applying higher rates of nematodes when humidities were less than 90% RH. In a trial on lettuce at a commercial glasshouse a mean L. huidobrensis mortality of 82 +/- 5% was recorded after an S. feltiae application, significantly higher than the chemical treatment, heptenophos.     

华东地区园林叶螨的初步研究（蜱螨目：叶螨总科）

笔经过数年的调查研究,查知华东园林植物上叶螨种类90种,分属3科22属。章报道了这些种类及其新的寄主植物和分布,研究发现,酢酱草如叶螨,柑桔全爪螨、构始叶螨、截形如螨,神泽叶螨,柏小爪螨,合肥埃须螨,俄勒冈小扁螨,刘氏短须螨和卵形短须螨等为园林植物上的主要种类,二点叶螨和朱砂叶螨是世界性分布的重要害螨。通常被认为是主要种类,但在华东园林植物上分布尚有限,加强检疫防止它们的传播扩散是十分必要的,章讨论了其它有关问题,所有标本均存放于上海农学院园环系农螨研究室。     

Molecular phylogeny of the Sphaerophorus globosus species complex

The Sphaerophorus globosus complex (Lecanorales, lichenized Ascomycota) shows a large morphological variation, and three relatively distinct morphotypes can be distinguished in parts of the distribution area. Here, we utilize a multigene‐based maximum‐parsimony approach (nITS+ LSU rDNA, mtSSU rDNA, β‐tubulin, and actin) to investigate whether these morphotypes constitute distinct species. The results show that there are at least two well‐supported monophyletic groups that we interpret as phylogenetic species within the S. globosus complex. These species do not completely correspond to the predefined morphotypes. One group, an apparently undescribed species, contains noncoralloid specimens from the North American Pacific Northwest. The other group, S. globosus, consists of two well‐supported monophyletic groups: one contains coralloid epiphytic specimens from the North American Pacific Northwest that are morphologically indistinguishable from epiphytic specimens from Europe and are presently interpreted as belonging to the same species and the other is morphologically variable and contains terrestrial specimens from Europe, North America, and southernmost South America and coralloid epiphytic and epilithic specimens from Europe. The results suggest that the population in southernmost South America originated by long‐distance dispersal from arctic populations in the Northern Hemisphere.     

Appropriateness of probit-9 in the development of quarantine treatments for timber and timber commodities

Following the increasing international phasing out of methyl bromide for quarantine purposes, the development of alternative treatments for timber pests becomes imperative. The international accreditation of new quarantine treatments requires verification standards that give confidence in the effectiveness of a treatment. Probit-9 mortality is a standard for treatment effectiveness that has its origin in fruit fly research, and has been adopted by the United States Department of Agriculture for fruit flies and several other pests. Following this, the probit-9 standard has been adopted as a benchmark for many quarantine treatments worldwide. This article discusses aspects of the application of this concept for a range of timber pests. Problematic issues include the often small pest populations available for testing, the limits of modeling pest responses to a treatment in the absence of sufficient numbers for treatment verification, the species diversity of pests and host materials and the physical and chemical conditions of host material or treatment conditions. Where treatment verification by killing large numbers of individuals is impossible, data collected from small populations or under specific conditions must be interpreted with caution. We discuss possible alternative approaches to probit-9 as a treatment efficacy standard.     

Taxonomic relationships among Turkish water frogs as revealed by phylogenetic analyses using mtDNA gene sequences

We assessed taxonomic relationships among Turkish water frogs through estimation of phylogenetic relationships among 62 adult specimens from 44 distinct populations inhabiting seven main geographical regions of Turkey using 2897 bp sequences of the mitochondrial Cytb, 12S rRNA and 16S rRNA genes with equally-weighted parsimony, likelihood, and Bayesian methods of inference. Monophyletic clade (Clade A) of the northwesternmost (Thrace) samples is identified as Pelophylax ridibundus. The other clade (Clade B) consisted of two monophyletic subclades. One of these contains specimens from southernmost populations that are regarded as an unnamed species. The other subclade consists of two lineages, of which one corresponds to P. caralitanus and another to P. bedriagae. Taxonomic relationships of these two species are discussed and recognition of P. caralitanus as a subspecies of P. bedriagae is proposed.     

中国大陆一新纪录种——美洲棘脊蓟马(缨翅目,蓟马科)及其分布和寄主植物

报道中国大陆1新纪录种,美洲棘脊蓟马Echinothrips americanus Morgan,1913(缨翅目,蓟马科,蓟马亚科),这也是棘蓟马属Echinothrips Moulton,1911在中国大陆的首次纪录.目前该种仅在我国北京海淀区的辣椒上发现.棘蓟马属以头、前胸背板和后胸背板具网状刻纹与针蓟马亚科Panchaetothripinae特征十分相近,但以发达的中胸内叉骨刺与之相区别.美洲棘蓟马系检疫性害虫,本文列出了它的世界分布和寄主植物种类.实证标本保存在浙江大学昆虫科学研究所.     

Host specificity testing and suitability of the parasitoidMicroctonus hyperodae (Hym.: Braconidae,Euphorinae) as a biological control agent ofListronotus bonariensis (Col.: Curculionidae) in New Zealand

The behaviour of the parasitoidMicroctonus hyperodae Loan was studied under quarantine conditions to determine its likely host range in New Zealand. The species was imported from South America as a potential biological control agent of Argentine stem weevil,Listronotus bonariensis (Kuschel). The study involved systematic evaluation of the parasitoid's behaviour when exposed to 24 non-host weevil species; all but three of these were native to New Zealand. Of those tested, four were found to sustain someM. hyperodae development. However, further examination showed that in all but one species,Irenimus aequalis (Broun), parasitoid development was impeded, with up to 50% of the larvae becoming encapsulated. Overall, those weevil species that were attacked produced only 19% of the parasitoids derived fromL. bonariensis controls. As an adjunct to this quarantine study, a review of the habitats of the native weevil and target pest populations indicated that refugia would probably exist for native alpine species. I. aequalis was not considered to be threatened byM. hyperodae as this weevil has benefited from the advent of European agricultural systems to the extent that it is now recognised as a minor pest. In view of its relatively oligophagous behaviour, the parasitoid was recommended as suitable for release.      

Loop-Mediated Isothermal Amplification Combined with PCR for Rapid Identification of the Ethiopian Fruit Fly (Diptera: Tephritidae)

Nowadays, with increasing trend of trans-boundary transportation of agricultural products and higher probability of introduction of many invasive species into new areas, fast and precise species diagnosis is of great significance particularly at the port of entry, where morphological identification often requires adult insect specimens especially with specialist insects. The cucumber fruit fly, Dacus ciliatus Loew (Diptera: Tephritidae), ranks as one of the most destructive agricultural pests attacking mainly fruits of Cucurbitaceae. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although cucumber fruit fly adults can usually be identified and distinguished from the other species by morphological keys, it is often difficult or impossible to distinguish this species from the other tephritids that share host plants by using material from other stages of development. In such situations, using a quick and robust alternative species diagnostic tool would be valuable. In this study, we assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for the rapid detection and discrimination of cucumber fruit fly DNA from some other common tephritid species attacking Cucurbitaceae, using material from different stages of development. The described method was species-specific and sensitive and provided a rapid diagnostic tool to detect D. ciliaus even by non-experts.