线粒体细胞色素氧化酶I (mt COI) 序列分析揭示扶桑绵粉蚧复合种存在隐存谱系

过去约20年间扶桑绵粉蚧Phenacoccus solenopsis Tinsley在许多国家造成严重危害。近年来该害虫传入我国, 对农业生产构成严峻威胁。外来入侵物种的快速准确鉴定是科学研究及其管理的基础。本研究对来自海南三亚和陵水地区的扶桑绵粉蚧的线粒体细胞色素氧化酶I （mt COI）测序并与美国佛罗里达州的扶桑绵粉蚧序列进行了比较。系统发育树表明该物种存在两个遗传支系, 即海南支系与佛罗里达支系; 这两个支系的遗传距离约3%。这是利用mt COI分子标记对我国扶桑绵粉蚧的首次分子鉴定. 这些结果提示：（1）扶桑绵粉蚧可能是至少含有两个隐存谱系或姊妹种的复合种; （2）新入侵我国海南三亚和陵水地区的扶桑绵粉蚧是该复合种内的一个隐存谱系或物种, 且海南三亚和陵水种群可能不是来自美国佛罗里达州。扶桑绵粉蚧隐存谱系的发现对于该物种的分子鉴定及其入侵生态学研究具有重要意义。     

基于转录组数据高通量发掘扶桑绵粉蚧微卫星引物

【目的】扶桑绵粉蚧Phenacoccus solenopsis Tinsley是我国重要的检疫性害虫。简单重复序列(simple sequence repeat,SSR)研究在遗传图谱和物理图谱的构建、分子标记辅助育种、品种鉴定、基因定位、遗传多样性、动植物分类和进化等方面具有重要意义。筛选的SSR引物将为扶桑绵粉蚧遗传多样性分析、进化分析及入侵生物学等奠定基础。【方法】利用高通量搜索的方法对扶桑绵粉蚧转录组中28 120条unigenes的数据进行搜索。【结果】共找到1 781个SSR位点。扶桑绵粉蚧转录组中SSRs的主要重复类型是单核苷酸重复,占SSR总数的89.44%;其次是三核苷酸重复,占SSR总数的7.52%。单核苷酸重复里主要是A/T基序,占了总量的87.42%。基于筛选的SSRs,运用Primer 3软件进行引物的批量设计,共有481个unigenes成功设计引物,共设计出1 228对引物。【结论】研究表明利用扶桑绵粉蚧转录组数据开发SSR标记是可行的,本研究开发的引物将为扶桑绵粉蚧遗传多样性分析、进化分析及入侵生物学等奠定基础。     

基于形态特征和线粒体COI基因探讨扶桑绵粉蚧物种的有效性并记述一体色变异型扶桑绵粉蚧

扶桑绵粉蚧(Phenacoccus solenopsis)于2008年首次在广东发现,到目前为止,浙江、广西、云南等10多个省市均有其入侵的报道。为探讨入侵中国的扶桑绵粉蚧是否存在两大隐存谱系或姊妹种的复合种,作者对浙江的该物种进行了调查。在调查过程中,发现体色浅橘黄色、背部具三对黄色斑点的体色变异型个体,通过形态特征比较和线粒体COI基因部分序列的分析,证实该体色变异型粉蚧为扶桑绵粉蚧。同时对中国、巴基斯坦、美国的扶桑绵粉蚧COI基因序列进行碱基差异比较、遗传距离(genetic distance)分析,发现所扩增的浙江省内6个地点7种不同寄主植物上的25条扶桑绵粉蚧COI基因(694bp)可以分成3种单倍型,这3种单倍型与中国海南、中国广州、巴基斯坦和美国加州的扶桑绵粉蚧遗传分歧较小(0–1.0%),而与美国佛罗里达州的遗传分歧较大(3–3.6%);但两者遗传距离小于绵粉蚧属内物种之间的遗传距离(13.0–17.2%)。综合形态特征和COI基因数据的分析结果显示,扶桑绵粉蚧可能没有达到种间分化。基于碱基差异所构建的网络关系图、遗传分歧差异和系统发生关系分析,扶桑绵粉蚧存在两个进化支系,至于是否是复合种,目前尚难作结论,还有待更多证据。     

利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧

扶桑绵粉蚧Phenacoccus solenopsis Tinsley是我国近年新发现的一种严重威胁农林业生产的重要外来入侵害虫。针对扶桑绵粉蚧与其他粉蚧类昆虫难以准确快速识别且适生区广泛的问题, 以扶桑绵粉蚧为靶标, 以我国常见的其他7种粉蚧为参照, 采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ (mtDNA COI) 基因序列的种特异性(species-specific COI, SS-COI) PCR方法, 研究其快速分子检测技术。通过已知粉蚧的COI基因序列设计通用型引物1对, 获得扶桑绵粉蚧及其他7种粉蚧包括康氏粉蚧Pseudococcus comstocki Kuwana、 南洋臀纹粉蚧Planococcus lilacius Cockerell、 木槿曼粉蚧Maconellicoccus hirsutus (Green)、 甘蔗红粉蚧Saccharicoccus sacchari (Cockerell)、 新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley、 番石榴粉蚧Planococcus minor Maskel和石蒜绵粉蚧Phenacoccus solani Ferris的COI基因序列, 根据测序结果及数据库中已知粉蚧的COI基因序列设计SS COI引物1对(PSZTF1/PSZTR1), 其扩增片段大小为546 bp。种特异性检验结果表明, 该引物只对扶桑绵粉蚧的COI基因具有扩增能力, 对其他7种粉蚧不具有扩增效果; 该引物不仅对成虫具有良好的扩增能力, 对不同虫态的扶桑绵粉蚧以及来自我国不同省市的14个地理种群和口岸截获的来自巴基斯坦的扶桑绵粉蚧亦具有同样的扩增效能。这些结果表明, 该技术体系完全可用于扶桑绵粉蚧的准确识别及其检测监测, 对有效阻截其进一步扩张蔓延意义重大。     

云南省扶桑绵粉蚧的分布和危害

【背景】扶桑绵粉蚧是一种危险的外来入侵害虫,自2008年在我国首次发现以来,其发生范围急剧扩大。但有关云南省扶桑绵粉蚧的发生情况缺乏系统的调查报道。【方法】2010年3月~2013年3月,对云南省16个州（市）72个区（市、县）的扶桑绵粉蚧的分布与危害情况进行了系统的调查,并对其天敌情况进行了初步调查。【结果】扶桑绵粉蚧在云南文山州富宁县,西双版纳州景洪市、勐海县,德宏州芒市,红河州元阳县、蒙自市,保山市隆阳区,丽江市华坪县,楚雄州永仁县,怒江州泸水县8个州（市）共10个市（县）有零星分布。其寄主植物共14科18属19种,包括园林观赏植物3种、经济作物1种、蔬菜作物2种、粮食作物1种、杂草11种,其中有12种植物在我国其他疫区未见报道;调查过程中发现其天敌3种。【结论与意义】扶桑绵粉蚧在云南省呈零星点状分布、疫区间受害情况有差异,未对经济作物造成严重损害。本研究可为云南省开展扶桑绵粉蚧的检疫防控提供参考。     

转换寄主前后扶桑绵粉蚧取食行为的EPG分析

【目的】分析扶桑绵粉蚧Phenacoccus solenopsis在发生寄主转移前后的取食行为,以据此评价其寄主适应性。【方法】利用刺吸电位（EPG）技术,量化扶桑绵粉蚧转换寄主植物前后的取食行为并进行对比分析。【结果】寄主转换后,扶桑绵粉蚧的取食行为受到显著的影响,需要耗费更多的时间到达韧皮部,总取食时间显著变短,取食效率降低。但寄主转换后,下一代成虫的取食效率提高,总刺探数、取食刺探数、总刺探时间以及到达韧皮部的效率与时间均与寄主转换前的扶桑绵粉蚧个体不存在显著差异。【结论】扶桑绵粉蚧的取食行为存在高度可塑性,这种特性利于扩大其寄主植物范围,有可能是促其成为重要农业入侵生物的原因之一。     

扶桑绵粉蚧与长角立毛蚁的互惠关系及其对寄主棉花叶片叶绿素荧光特性的影响

【背景】草食动物对寄主植物的取食或损伤会诱导改变植物的光合作用,从而直接影响植株的健康生长。产蜜昆虫与蚂蚁的互惠关系是物种相互促进的一种重要的生态学现象,能够促进产蜜昆虫的种群数量,然而这种互惠关系及其对寄主植物光合生理的影响还知之甚少。【方法】在室内条件下,运用叶绿素荧光动力学技术研究了外来入侵害虫扶桑绵粉蚧与长角立毛蚁的互惠对寄主棉花叶片叶绿素荧光特性的影响。【结果】随着扶桑绵粉蚧危害时间的延续,寄主植物上蚂蚁和扶桑绵粉蚧的数量均呈现显著上升的趋势,而在危害后期,蚂蚁存在情况下扶桑绵粉蚧的数量要明显低于无蚂蚁处理;在扶桑绵粉蚧取食寄主棉花20 d后,有、无蚂蚁存在的棉花叶片的光合利用率α值较无虫处理分别下降了53.5%和37.0%;存在蚂蚁或扶桑绵粉蚧危害后期对棉花叶片最大相对电子传递效率r ETRmax有显著影响,然而扶桑绵粉蚧单独取食或与蚂蚁互作的情况下未显著影响棉花叶片对强光的耐受能力(Ek)。【结论与意义】研究明确了扶桑绵粉蚧与长角立毛蚁的互惠关系对寄主棉花叶片的光合生理产生了一定的负面效应,为进一步解释扶桑绵粉蚧入侵、扩散及暴发的生态学过程提供了科学依据。     

饥饿对扶桑绵粉蚧存活率和产卵量的影响

【背景】扶桑绵粉蚧是近年来入侵我国的重要害虫,食物是影响其生长发育及种群发展的重要因素。【方法】在室内条件下研究了饥饿对扶桑绵粉蚧不同龄期若虫和初羽化雌成虫存活率和雌成虫产卵量的影响。【结果】扶桑绵粉蚧不同虫态在饥饿条件下存活的时间存在显著差异,表现为雌成虫〉2龄若虫=3龄若虫〉1龄若虫。扶桑绵粉蚧各龄若虫和雌成虫随着饥饿时间的延长,存活率逐渐下降。其中,雌成虫存活率下降速度较慢,完全饥饿8d后存活率仍有50％左右;2、3龄若虫50％个体死亡需要饥饿的时间约为6d,1龄若虫约需5．5d。饥饿会显著降低1龄若虫存活率,但对2、3龄若虫没有显著影响。扶桑绵粉蚧雌成虫饥饿4d对其产卵前期、平均每头雌虫一生的产卵量没有显著影响,但寿命显著低于对照。【结论与意义】扶桑绵粉蚧耐饥力较强,这有助于它在野外建立种群。该结果为进一步评价扶桑绵粉蚧的入侵潜能提供了依据。     

扶桑绵粉蚧转录组分析

【目的】建立扶桑绵粉蚧Phenacoccus solenopsis Tinsley的转录组数据库,揭示扶桑绵粉蚧转录组的整体表达特征。【方法】采用Illumina Hi SeqTM40`00测序平台开展对扶桑绵粉蚧雌成虫转录组测定,对原始数据进行过滤和组装。【结果】共获得58 322 258条序列读取片段(reads),共9.48 Gb(Gen Bank登录号:SAMN06130426)57 422 032条有效转录组数据。进一步组装拼接后,共获得94 475个单基因簇(unigene),平均长度为700 bp。将unigene与数据库中的序列进行BLASTX比对,成功注释20 949个unigenes,其中,Nr注释的unigenes与豌豆蚜Acyrthosiphon pisum unigenes同源性最高,达18.69%。扶桑绵粉蚧转录组unigenes根据GO功能注释大致可分为细胞组分、分子功能和生物过程三大类55个分支,与结合活性、催化活性、细胞进程和代谢进程相关的unigenes较多。此外,本研究还筛选到20条与脂类代谢相关的途径和与性信息素代谢相关的序列。并通过与Nr和Swiss-prot蛋白质数据库比对,获得了15 037条编码序列(CDS)片段。【结论】本研究初步阐明扶桑绵粉蚧雌成虫转录组的整体表达模式,为进一步研究扶桑绵粉蚧的基因功能及性信息素的代谢途径奠定了基础。     

班氏跳小蜂对新寄主资源的寄生适合度

【目的】班氏跳小蜂Aenasius bambawalei是扶桑绵粉蚧Phenacoccus solenopsis的伴迁性天敌,迄今已知该寄生蜂只能寄生扶桑绵粉蚧,但是我们发现该寄生蜂还能成功寄生另一种外来有害生物——石蒜绵粉蚧P. solani。本研究旨在明确班氏跳小蜂对该新寄主资源的寄生适合度,为今后充分开发、利用该寄生蜂提供科学依据。【方法】在实验室条件下,以马铃薯Solanum tuberosum块茎作为粉蚧的寄主植物,测定了班氏跳小蜂对不同龄期石蒜绵粉蚧的寄生适合度,并在非选择条件下测定了上述两种寄主上僵蚧形成时间以及该寄生蜂羽化时间、寿命、后足胫节长度、寄生率、羽化率及子代性比。【结果】班氏跳小蜂可寄生石蒜绵粉蚧的雌成虫及2龄和3龄若虫,但只有寄生雌成虫才能正常羽化出蜂。分别以石蒜绵粉蚧与扶桑绵粉蚧为寄主时,最适合的蜂蚧比分别为2∶15和2∶20。不同寄主对僵蚧形成时间及班氏跳小蜂的羽化时间无显著影响,但显著影响班氏跳小蜂的寿命以及雌蜂后足胫节长度,尤其是扶桑绵粉蚧上羽化的寄生蜂的寿命比以石蒜绵粉蚧上的长约20 d。而且,班氏跳小蜂对扶桑绵粉蚧的寄生率(54.0%)显著高于对石蒜绵粉蚧的(25.3%),但班氏跳小蜂在这两种寄主上的羽化率均在90%以上,差异不显著;寄生扶桑绵粉蚧的雌蜂比例高于寄生石蒜绵粉蚧的雌蜂比例。【结论】班氏跳小蜂能寄生石蒜绵粉蚧,且只在雌成虫上完成世代发育;与在更适寄主扶桑绵粉蚧上比较,石蒜绵粉蚧上羽化的班氏跳小蜂在寿命及雌蜂个体大小上有劣势。     

Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST‐derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high‐quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST‐based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.     

寄主植物对扶桑绵粉蚧体型及体内能源物质含量的影响

[目的]研究寄主植物对扶桑绵粉蚧雌成虫体型和体内能源物质含量的影响,为扶桑绵粉蚧的寄主适应性和风险分析提供科学参考.[方法]在室内用8种寄主植物连续饲养扶桑绵粉蚧5代后,测量7日龄雌成虫体长、体宽和体重,并测定体内能源物质(脂肪、可溶性蛋白质、可溶性糖)含量,分析上述指标在不同寄主间的差异.[结果]取食不同寄主后,扶桑...     

Effects of the invasive plant Eupatorium adenophorum on the exotic mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae) and their natural enemies

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The potential distribution of an invasive mealybug Phenacoccus solenopsis and its threat to cotton in Asia

• 1 In recent years, an invasive mealybug Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) has attacked cotton (Gossypium hirsutum L.) in Pakistan and India, causing severe economic losses. This polyphagous pest was probably introduced accidentally from North America. Infestations have broken out suddenly and spread rapidly.
• 2 Seasonal and annual population growth data of P. solenopsis from nine locations in its native range in the U.S.A., and the distribution of the mealybug worldwide, were analyzed using the CLIMEX model. This indicated that tropical regions worldwide were highly suitable for P. solenopsis.
• 3 Its potential distribution was limited by cold in high latitudes and altitudes, and dryness in northern Africa, inland Australia and parts of the Middle East. CLIMEX was used to predict where P. solenopsis might establish, and to estimate the potential threat to cotton yield in Asia. The key limiting factors were low precipitation as well as minimum temperatures in northern areas.
• 4 When irrigation was factored into the simulation, the potential distribution of P. solenopsis expanded dramatically, indicating that P. solenopsis presents a great economic threat to cotton in Asia and other parts of the world.

     

基于宏基因组测序的扶桑绵粉蚧内共生菌多样性研究

[目的] 扶桑绵粉蚧是一种重要的入侵害虫,也被列为我国的检疫性有害生物。内共生菌的群落结构及与扶桑绵粉蚧关系的研究,将为研究扶桑绵粉蚧的入侵生物学和防治提供新的思路和参考。[方法] 利用宏基因组测序技术对室内饲养扶桑绵粉蚧内共生菌的物种组成、丰度和多样性进行了研究。[结果] 共鉴定获得29个门,47个纲,105个目,178个科,245个属,299个种。变形菌门、γ-变形菌纲、肠杆菌目、肠杆菌科、伯克氏菌属、Candidatus分别为各分类阶元的优势种群。共预测了36505个基因,其中77.27%的基因分布在200~499 nt的范围内。基因集KEGG注释表明,扶桑绵粉蚧内共生菌代谢相关基因最多,达384个,占49.48%;环境信息处理基因最少,仅8个,占1.03%;代谢相关基因最多的为氨基酸代谢基因,为64个,占28.19%。基因集COG注释表明,翻译、核糖体结构和生物发生的基因最多,其次为氨基酸转运和代谢基因,分别占27.64%和12.31%。[结论] 初步确定了扶桑绵粉蚧内共生菌菌群的群落结构和优势菌群,并初步分析了这些内共生菌的功能基因情况,为进一步研究内共生菌与扶桑绵粉蚧之间的关系提供了基础,以期从内共生菌的角度为该虫的防控提供新的策略。     

Comparison of Phenacoccus solenopsis specimens from different regions of Pakistan using COI molecular barcoding (Hemiptera: Pseudococcidae)

Because correct identification of insects is crucial for pest management involving chemical or biological control agents, we have used a molecular approach to identify and characterize specimens of the cotton pest Phenacoccus solenopsis Tinsley (Sternorrhyncha: Pseudococcidae) present in different regions of Pakistan. The specimens were analyzed through DNA sequence analysis of their mitochondrial COI (mtCOI) gene using an improved procedure that could distinguish between the pest and its associated parasitoid. Our analysis showed no variation among the mealybug specimens from different geographical locations of Pakistan and confirmed that this is the same species and haplotype that is infesting cotton plants in other parts of Asia. This information will assist in the development of biological control programs against P. solenopsis in Pakistan and other Asian countries.     

广西及越南农业外来有害生物入侵现状

【背景】广西地理环境独特,与越南水陆相连,气候、作物等条件相似,边境贸易往来频繁,致使其成为我国遭受外来有害生物入侵最严重的地区之一。【方法】通过本底调查,分析总结了广西农业外来有害生物入侵的现状。【结果】广西主要入侵动物26种,入侵微生物18种,入侵植物21种,高风险入侵物种包括辣椒实蝇、木薯绵粉蚧、扶桑绵粉蚧、稻水象甲、黄瓜绿斑驳花叶病毒等;来自越南的潜在危险性有害生物共38种。【结论与意义】广西受外来有害生物入侵的风险加剧,并有进一步扩散蔓延的趋势。提出应对入侵生物的综合防控对策,为广西外来入侵有害生物的防控提供参考。     

Genetic structure of the endangered species <Emphasis Type="Italic">Pinna nobilis</Emphasis> (Mollusca: Bivalvia) inferred from mtDNA sequences

This study examines the population genetic structure of the endangered bivalve Pinna nobilis (Mollusca: Bivalvia), based on novel mtDNA sequences (partial COI and 16S rDNA mtDNA genes). The analyzed nucleotide sequences of COI were 729 bp in size, coding for a 243 amino acid peptide, while the analyzed nucleotide sequences of 16S rDNA were 489 bp in size. These sequences of P. nobilis were the first DNA sequences of the species submitted to any Genetic Data Base. Population samples from four geographic regions from Greece, as well as a population sample of Atrina fragilis (as an outgroup) were used. High values of haplotypic diversity were found in the population samples of P. nobilis, based on the COI sequences. A single base in the analyzed 16S rDNA sequences was different in all analyzed individuals from a single population sample (Chios island) differentiating it from the other ones. These mtDNA sequences could be informative for further genetic analyses of the endangered species, contributing in conservation plans for its protection and/or aquaculture investigations.     

Effects Of Tending By Solenopsis Invicta (Hymenoptera: Formicidae) On The Sugar Composition And Concentration In The Honeydew Of An Invasive Mealybug,Phenacoccus Solenopsis (Hemiptera: Pseudococcidae)

Mutualistic interactions between ants and hemipterans are mediated by the honeydew produced by the hemipterans. Previous works have demonstrated that the invasive mealybug Phenacoccus solenopsis produces abundant honeydew and attracts a large number of workers of the fire ant Solenopsis invicta. Mealybugs exhibit higher fecundity when tended by fire ants. The honeydew produced by P. solenopsis plays an important role in interactions between these two species. However, relatively few studies have focused on whether there is a cost to P. solenopsis mealybugs of being tended by S. invicta through changes in their excretion behavior and the quantity of honeydew produced. Our results indicated that the honeydew of P. solenopsis contains xylose, fructose, sucrose, trehalose, melezitose, and raffinose. The sugar concentration in the mealybug honeydew changed in an ant‐tended treatment. When tended by fire ants, the mealybugs generated honeydew with a significantly decreased xylose concentration. In contrast, the droplets showed a considerable increase in the melezitose concentration. P. solenopsis excreted honeydew more frequently when tended by S. invicta, but the weights of the droplets excreted by the ant‐tended mealybugs were significantly lower. In addition, S. invicta exhibited a significant preference for different sugars. Melezitose was visited more intensively than the other sugars in two choice tests. These results may suggest that, to attract more tending ants, mealybugs adjust their carbohydrate metabolism.     

Does the host plant affect the benefits from mutualisms? The invasive mealybug and ghost ant association

1. The benefits to trophobionte hemipterans are affected by the ant tending level, which is a widely accepted statement. The ant tending level is closely related to multiple factors. It is clear that the ant tending level can be affected by the temporal factor, age‐specific, the density of the hemipterans, and quantity and quality of honeydew produced by hemipterans. 2. Few studies of ant–hemipteran mutualisms have reported the patterns of host plants‐dependent effects, and whether host plants influence the ant tending level that is also unclear. As such, laboratory experiments were conducted to test whether the colony growth rate of an invasive mealybug Phenacoccus solenopsis Tinsley, parasitism of Aenasius bambawalei Hayat, an dominant parasitoid of P. solenopsis, are affected by tending by ghost ants (Tapinoma melanocephalum(Fabricius)], host plants (tomato and cotton), and interactions between the two factors. The difference in the ant tending level between the host plants was also determined. 3. The results showed that mealybug colony growth and parasitism were significantly affected by ant tending and host plant separately. There were significant interactions between the independent factors on the mealybug colony growth rate and percentage parasitism. These results suggest that benefits to mealybugs are host plant‐dependent.