内共生菌Wolbachia在蓖麻粉虱内的分布

【目的】在粉虱类害虫生物防治的载体植物系统中,蓖麻粉虱Trialeurodesricini是较好的替代寄主。Wolbachia是节肢动物中分布最广的细胞内共生菌,对昆虫的生殖和发育具有重要影响,明确蓖麻粉虱中Wolbachia的分布可为蓖麻粉虱Wolbachia的深入研究奠定基础,为粉虱类害虫的生物防治提供新的理论依据和思路。【方法】通过内共生菌Wolbachia的wsp基因A大组和B大组特异性引物对蓖麻粉虱的室内饲养种群及蓖麻粉虱与丽蚜小蜂同笼饲养20代的种群逐代进行PCR检测,对扩增到的基因进行测序并构建系统发育树,进行相关分析。【结果】蓖麻粉虱种群中只有A大组Wolbachia分布,感染率为40%,系统发育分析表明该类群的Wolbachia属于A大组中的Dro亚组,与报道的其他寄生蜂种群感染的Wolbachia同属一组。丽蚜小蜂种群中的Wolbachia属B大组,蓖麻粉虱与丽蚜小蜂混合饲养20代,逐代检测发现Wolbachia并未在蓖麻粉虱与丽蚜小蜂种群间发生水平传播。【结论】蓖麻粉虱的室内饲养种群中Wolbachia的感染率仅为40%,说明Wolbachia能够在蓖麻粉虱种群中稳定遗传,但在蓖麻粉虱个体间水平传播的能力较弱。短时间内Wolbachia无法在蓖麻粉虱与丽蚜小蜂种群间水平传播。     

柑橘木虱两地理种群的内共生菌群落组成及传菌能力

柑橘黄龙病（Huanglongbing, HLB）是经柑橘木虱Diaphorina citri传播的最主要柑橘病害之一, 危害严重时能对柑橘产业造成毁灭性的破坏。为了鉴定福建和海南2个地理种群柑橘木虱的内共生菌群落组成, 本研究对16S rRNA部分保守序列进行PCR扩增, 并利用特异性引物对不同内共生菌进行了感染率检测; 另外, 还通过人工接虫的方法, 探索柑橘木虱成虫在带黄龙病菌蕉柑Citrus reticulata cv. Tankan上的获菌能力, 以及带菌柑橘木虱成虫对黄岩蜜橘C. reticulata cv. Subcompressa的传菌能力。研究发现, 这2个地理种群的柑橘木虱含有相同的内共生菌组成, 包括α-Proteobacteria, Wolbachia spp., γ-Proteobacteria, mycetocyte symbionts, β-Proteobacteria, Oxalobacter和β-Proteobacteria, Herbaspirillum, 而且这2个地理种群柑橘木虱的4种内共生菌的携带率均在95%以上。柑橘木虱成虫在带菌蕉柑上饲菌28 d后, 带菌率可达到82%, 而带菌柑橘木虱成虫在黄岩蜜橘上传菌75 d后, 可导致橘树整体带菌。本研究为柑橘木虱的进一步研究和防虫治病途径提供了一些理论依据。     

南方部分地区烟粉虱及其寄生蜂内共生菌Cardinium的检测及其系统发育关系

【目的】研究我国南方四个省份6个烟粉虱Bemisia tabaci种群和2个蚜小蜂种群内共生菌Cardinium的感染率及其系统发育关系。【方法】利用PCR技术与DNA测序技术检测来自广州、肇庆、南宁、昆明、厦门烟粉虱种群以及广州2个蚜小蜂种群内Cardinium的感染情况,通过基于mt COⅠDNA序列和16S r RNA序列建立烟粉虱和Cardinium系统进化树,分析烟粉虱、寄生蜂和共生菌Cardinium的协同进化关系。【结果】广州的Asia II1与Asia II7烟粉虱种群、肇庆与南宁的MEAM1烟粉虱种群、昆明与厦门的Mediterranean烟粉虱种群以及广州的丽蚜小蜂Encarsia formosa、双斑蚜小蜂Encarsia bimaculata种群体内都有Cardinium的感染,且以Mediterranean与土著的Asia II1、Asia II7感染率较高(65.2%~92.5%),寄生蜂体内的感染率较低(16.7%~18.6%)。通过进化树对比分析发现,Cardinium与烟粉虱寄主之间无协同进化现象,但是Cardinium在烟粉虱不同地理种群之间,烟粉虱与寄生蜂之间的同源性超过98%。【结论】Cardinium在两种寄主昆虫之间进行相互感染与传播的概率较大,寄生蜂可能是Cardinium在不同烟粉虱种群之间水平传播的桥梁之一。     

7种内共生病毒对柑橘木虱生殖力的影响

[目的]柑橘木虱吸食柑橘和九里香等芸香科植物,更是柑橘黄龙病的主要传播媒介。了解柑橘木虱共生微生物对虫体产卵量的影响,可为该虫的生态调控提供理论依据。[材料]应用Illumina HiSeq技术对柑橘木虱进行转录组和小RNA (siRNA)高通量测序,对拼接的序列进行功能注释。通过单雌单苗的饲养方法,分析通过测序揭晓的7种内共生病毒对柑橘木虱的影响,包括单雌总产卵量、单雌日产卵量和单雌寿命的变化。[结果]通过高通量测序共获得unigenes序列22429条,共有17673条unigenes注释至NR、NT、Pfam等数据库。根据siRNA分析数据,获得了多条柑橘木虱内共生病毒的序列。生物学验证试验发现,柑橘木虱呼肠孤病毒、Hubei tick virus 2、褐飞虱呼肠孤病毒、Mal de Rio Cuarto virus、Synechococcus phage S-RSM4、Tokyovirus A1 DNA和Diaphorina citri picorna-like virus isolate BR1 7种内共生病毒显著降低了雌虫群体总产卵量,但是对单雌日产卵量和单雌寿命无显著影响。[结论]研究结果为柑橘木虱的共生微生物多样性提供证据,也为进一步探索内共生病毒对宿主昆虫的意义提供了思路和理论基础,为后续柑橘木虱-内共生微生物的互作研究提供数据支持。     

亚洲不同地理种群褐飞虱内共生菌Wolbachia的分子检测

褐飞虱Nilaparvata lugens Stl是水稻的重要害虫,而Wolbachia是其重要的次生共生细菌之一。为了解褐飞虱种群感染Wolbachia的情况,本研究对采自世界褐飞虱主要分布区的22个地理种群感染Wolbachia的情况进行了检测和分析。qPCR结果表明,除杭州2009年种群没有检测出Wolbachia外,其它21个种群中都有不同程度的Wolbachia感染。对Wolbachia的wsp基因序列的比较和进化分析表明,褐飞虱各地理种群Wolbachia都属于B群,并可进一步分为Ori和Con两个亚群。其中18个褐飞虱种群中的Wolbachia属于Ori亚群,广东清远和浙江桐乡褐飞虱种群中的Wolbachia则属于Con亚群,而菲律宾Iloilo褐飞虱种群分别检测到同时有2个亚群Wolbachia感染。本结果可为进一步研究褐飞虱与共生菌的共生关系、褐飞虱致害性变异和迁飞路线分析及利用Wolbachia防治褐飞虱提供依据。     

烟粉虱及其优势寄生蜂内共生菌的种类及系统发育分析

为了研究入侵我国的2个主要烟粉虱隐种Bemisia tabaci MEAM1和MED及其3种优势寄生蜂(浅黄恩蚜小蜂Encarsia sophia、丽蚜小蜂E.formosa、海氏桨角蚜小蜂Eretmocerus hayati)体内感染内共生菌的种类丰度,并进一步探讨其系统发育关系,本文利用分子生物学手段对昆虫体内细菌的16S rRNA基因序列进行扩增、测序和分析,并采用邻接法(Neighbor-Joining,NJ)和最大似然法(MaximumLikehood,ML)分别构建优势内共生菌的系统发育树。结果表明,烟粉虱2个隐种内共生菌的种类丰度大于其3种优势寄生蜂,3种优势寄生蜂中丽蚜小蜂内共生菌的种类丰度最高;同源性分析发现烟粉虱和寄生蜂所携带的Rickettsia基因同源性达到99%,属于Rickettsia bellii种,进一步的进化树分析也发现所研究物种的Rickettsia和Hamiltonella均可各自聚为同一进化分支。烟粉虱及其优势寄生蜂体内含有种类丰富的内共生菌,其中优势内共生菌Rickettsia和Hamiltonella各自亲缘关系很近,说明内共生菌在烟粉虱和寄生蜂间可能进行水平传播。     

烟粉虱内共生菌16S rDNA的变异与系统发生

对 5年连续饲养在不同种寄主植物上的B型烟粉虱北京种群的内共生菌 1 6SrDNA基因进行了PCR扩增和测序。结合已知序列 ,构建了不同寄主植物烟粉虱初生内共生菌约 1 0 0 0bp的 1 6SrDNA及次生内共生菌约1 2 5 0bp的 1 6SrDNA的分子系统树。结果表明 ,中国北京不同寄主植物的B型烟粉虱内共生菌及世界其它地区烟粉虱内共生菌可能是同一种的不同生态型 ,内共生菌在其宿主分化后进行了选择 ,之后与其宿主长期共同进化、共同适应 ,为宿主对不同生境的适应提供了一定的基础     

海南地区螺旋粉虱三类次级内共生菌的检测

螺旋粉虱Aleurodicus dispersus Russell是一种重要的农林害虫。本研究分别利用次级内共生菌Cardinium和Arsenophonus的16S rDNA和Wolbachia wsp基因对海南省16地区的螺旋粉虱的3种次级内共生菌Cardinium, Arsenophonus和Wolbachia感染情况及相关基因序列进行了测定和分析。3种次级内共生菌Cardinium, Arsenophonus和Wolbachia检测结果表明, Cardinium和Arsenophonus均可感染海南地区的螺旋粉虱, 其中乐东、 陵水和澄迈3个地区所有寄主上的螺旋粉虱的Arsenophonus感染率为100%, 三亚、 琼中和临高部分寄主上的螺旋粉虱的Arsenophonus感染率为66.7%, 而儋州、 五指山和万宁3个地区的螺旋粉虱未发现被Arsenophonus感染; 三亚的番石榴上的螺旋粉虱的Cardinium感染率为100%, 琼海白沙的印度紫檀上螺旋粉虱的Cardinium感染率为100%, 其他寄主上的感染率均小于66.7%; 在所检测的43个螺旋粉虱种群中, 40和31个种群中分别检测出有Arsenophonus 和Cardinium感染, 种群感染率分别为93.0%和72.1%; 在所有检测的个体中, 120个个体中有105个感染Arsenophonus, 93个个体中有70个感染Cardinium, 个体感染率分别为87.5% 和75.3%; 在检测的所有样本中, 只有三亚印度紫檀上的螺旋粉虱检测到Wolbachia, 种群感染率为2.3%, 个体感染率仅为0.8%。这些检测结果表明, 海南地区螺旋粉虱次级内共生菌Arsenophonus的感染率高于Cardinium的感染率, Wolbachia的感染率极低。序列分析表明, 海南不同螺旋粉虱种群的Cardinium的16S rDNA序列一致, 而且与烟粉虱的Cardinium 16S rDNA序列一致性很高, 为97.6%; 不同螺旋粉虱种群的Arsenophonus的16S rDNA序列也完全一致, 其与西班牙加那利群岛螺旋粉虱的Arsenophonus的16S rDNA序列一致性较高, 为85.1%。此外, Wolbachia wsp基因序列分析表明, 海南螺旋粉虱的Wolbachia为B组, 这是国内螺旋粉虱感染Wolbachia的首次报道。     

中国部分地区柑桔木虱体内感染Wolbachia的检测及其系统发育分析

Wolbachia是一种广泛分布于节肢动物体内的共生细菌, 该共生菌经寄主卵的细胞质传播并参与多种寄主生殖活动调控, 对节肢动物的物种进化有着重要意义并可能应用于害虫的生物防治。本研究以柑桔黄龙病（Huanglongbing, HLB）的主要传播媒介——柑桔木虱Diaphorina citri为研究对象, 通过Wolbachia的16S rDNA、 细胞分裂基因（ftsZ）、 表面蛋白基因（wsp）的特异引物对来自于中国广西北海、 广西柳州、 广东深圳、 广东阳春、 福建福州5个地区的柑桔木虱进行PCR扩增, 并对扩增产物进行克隆测序, 与GenBank中相关序列进行比对分析, 建立了柑桔木虱共生Wolbachia的系统发育树。结果显示上述5个地区的柑桔木虱均存在Wolbachia感染, 通过Wolbachia的16S rDNA, ftsZ基因和wsp基因DNA序列的系统发育分析表明, 5个地区的柑桔木虱体内的Wolbachia均属于B组; 基于wsp基因的系统发育分析进一步表明5个地区的亚洲柑桔木虱体内的Wolbachia属于B组的Con亚组, 且不同地区柑桔木虱体内的Wolbachia的16S rDNA, ftsZ基因和wsp基因序列差异不明显。研究结果为认识柑桔黄龙病传播媒介昆虫的进化及其综合防治提供了重要的参考依据。     

烟粉虱内共生菌16S rDNA的特性研究

对烟粉虱内共生菌16SrDNA的酶切结果及部分生物体16SrDNA的(G C)%分析结果表明:烟粉虱初生内共生菌的16SrDNA能够被EcoRI酶切成两个片段、而不能够分别被BamHI与SacI酶切;烟粉虱次生内共生菌的16SrDNA没有BamHI内切酶位点、而能够分别被EcoRI或Sac I酶切成大小不同的两个片段。(G C)mol%与菌的分类地位有关,同时还与菌的可培养能力有关。Proteobacteriaγ亚纲的烟粉虱初生内共生菌与α亚纲的Rickettsia、线粒体的16SrDNA相似,富含(A T)mol%,具低的(G C)mol%。而γ亚纲的次生内共生菌及大肠杆菌与β亚纲的mealybugs初生内共生菌的16SrDNA相似,富含(G C)mol%。说明初生内共生菌可能与烟粉虱同时发生,并且形成一种非常紧密的共生关系,次生内共生菌与烟粉虱关系松散一些,其特性近似于自由生活的细菌,更有可能获得纯培养体。16SrDNA的系统进化树表明,烟粉虱次生内共生菌属于Proteobacteriaγ-3亚纲,而初生内共生菌属于Proteobacteriaγ亚纲的另一分支。     

The biology and phenology of the citrus whitefly, Dialeurodes citri, on citrus in the Coastal Plain of Israel

The citrus whitefly, Dialeurodes citri (Ashmead) (Homoptera: Aleyrodidae) (CWF), is a polyphagous citrus pest, originating in S. E. Asia, which was first discovered in the Western Galilee region of Israel in 1975. The larval instars of CWF secrete honeydew, and the sooty mould fungus, which develops on the honeydew, causes damage to the tree and its fruit. In observations made in Israel between 1987 and 1991 CWF usually completed three generations per year, although a small proportion of the population had two or four generations. From October to April a combination of shortened photoperiod and reduced temperature arrest the development of CWF in the fourth larval instar. The average length of the life cycle from egg to adult emergence was 65 days in the first generation (April–June), 52 days in the second generation (July–August) and 8 months in the third generation (September–April). Under laboratory conditions of L16: D8 and 25 °C, the mean developmental time was 54 days, and the duration of the egg, first larval instar, second larval instar, third larval instar, fourth larval instar and putative pupa (red eyes) were 12.1, 6.5, 5.5, 7.6, 18, and 4.7 days, respectively. Adults prefer to lay their eggs on young, fully developed citrus leaves, but also oviposit on older leaves. The inner region and shaded sides of the tree are preferred for oviposition.     

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and huanglongbing disease do not exist in the Stapleton Station area of the Northern Territory of Australia

Abstract  A series of specimens of the Asian citrus psyllid, Diaphorina citri , collected from the Northern Territory (NT) in 1915 was recently rediscovered in the Natural History Museum, London. Surveys were conducted in 2002 on suitable hosts in the locality of the 1915 collections to see if the infestation had persisted. These failed to detect either D. citri or the bacterium that it transmits and that causes huanglongbing disease in citrus. It is presumed that D. citri was eradicated fortuitously by the removal of all citrus plants above latitude 19°S during an eradication program for citrus canker in the NT from 1916 until 1922.     

The effects of conspecific and heterospecific interactions on foraging and oviposition behaviours of two parasitoids of Diaphorina citri

Direct observation was used to construct activity budgets for Diaphorencyrtus aligarhensis (Shafee, Alam & Agarwal) (Hymenoptera: Encyrtidae) and Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae), foraging for nymphs of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Two populations of D. aligarhensis were used in experiments: a biparental population from Pakistan and a uniparental population from Taiwan. Parasitoids were provisioned with 20 D. citri nymphs, second through fifth instar; female parasitoids from all three populations foraged alone, in conspecific pairs, or in heterospecific pairs. Walking occupied approximately 40% of the activity budget of D. aligarhensis and ovipositor probing occupied 10–20%. In contrast, probing and walking each accounted for approximately 30% of the activity budget of T. radiata. The presence of conspecific females affected the activity budget of D. aligarhensis females, such that the focal female was observed walking less often and probing more frequently. Heterospecific females did not affect D. aligarhensis activity. These results suggest mutual interference may occur between D. aligarhensis females foraging on the same host patch. The behaviour of T. radiata was not significantly affected by the presence of conspecific or heterospecific competitors. These results are discussed with respect to the release of multiple species of natural enemies in the classical biological control programme targeting D. citri in California.     

Twelve polymorphic microsatellite loci from the Asian citrus psyllid,Diaphorina citri Kuwayama,the vector for citrus greening disease,huanglongbing

Twelve polymorphic microsatellite markers were developed from microsatellite‐enriched DNA libraries and mined from an expressed sequence tags library of Diaphorina citri, the vector of the citrus greening disease (huanglongbing). Analysis of 288 individuals from Florida, Texas, and Brazil showed that allelic diversity ranged from three to eight alleles per locus and observed and expected heterozygosities ranged from 0.014 to 0.569 and from 0.052 to 0.653, respectively. These variable microsatellite loci can provide means for assessing overall genetic variation and migration patterns for this agriculturally important pest species. This information can be used to aid in developing successful management strategies.     

Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus

Abstract  The Asiatic citrus psyllid ( Diaphorina citri Kuwayama [Hemiptera: Sternorrhyncha: Psyllidae]) is a vector of huanglongbing (citrus greening), a devastating disease of citrus caused by phloem-limited bacteria. Growing guava ( Psidium guajava ) as an intercrop appears to be a successful means of reducing psyllid numbers within citrus orchards; however, the mechanism by which such a reduction is achieved is unknown. To determine the repellent effect of guava leaf and factors attributed to this activity, responses of adult psyllids to guava leaf and its odor were evaluated in cage tests and Y-tube olfactometer test. The results showed that guava leaf possessed a repellent effect against the adult citrus psyllids. Fewer psyllids were found on citrus leaves in the presence of guava foliage than in its absence. Young and old guava leaf showed equal repellent activity. By covering the guava shoots with net cloth, it was revealed that the repellent effect of guava leaf against adult psyllids on citrus was attributed to the volatile compounds, rather than physical factors. The olfactometer response of adult psyllids to guava leaf odor was dosage-dependent. Between guava odor and control, only 35.00%, 25.00% and 16.25% of the psyllids moved toward guava odor when presented with 5.0, 10.0 and 15.0 g of guava shoots, respectively. The olfactometer experiments also showed that both male and female psyllids responded similarly to the guava leaf odor.     

Spatial dispersion and sampling of citrus whitefly,Dialeurodes citri,for control decisions in a citrus orchard

1 Monitoring the citrus whitefly Dialeurodes citri (Ashmead) in citrus orchards in Israel is necessary for control decisions in IPM in the period before the establishment of natural enemies. The main damage caused by D. citri is due to sooty mould, which colonizes the sweet secretions of this pest. 2 Using Taylor’s Power Law to describe the variance–mean relationship, the dispersion of the citrus whitefly was found to be aggregated at all developmental stages. The action threshold was estimated using regression of sooty mould level on egg or larval counts. 3 Two-stage sampling plans were constructed in different pest generations, for eggs and larval stages, on the basis of the variance between leaves on a tree and the variance between trees in a plot. 4 Fixed sample size plans were prepared, with possible numbers of trees and the numbers of leaves per tree to be sampled, in order to attain a given precision level. 5 Sequential sampling plans were also drawn up, on the basis of four leaves per tree, where trees are sampled until a decision can be reached. The maximum number of trees to be sampled was set according to the corresponding fixed sample size plan. 6 Simulation of the sequential sampling plans showed them to be highly reliable in terms of the decision reached with economy in sample size for high and low infestation levels.     

A Survey of the bacteriophage WO in the endosymbiotic bacteria Wolbachia

Bacteriophages are common viruses infecting prokaryotes. In addition to their deadly effect, phages are also involved in several evolutionary processes of bacteria, such as coding functional proteins potentially beneficial to them, or favoring horizontal gene transfer through transduction. The particular lifestyle of obligatory intracellular bacteria usually protects them from phage infection. However, Wolbachia, an intracellular alpha-proteobacterium, infecting diverse arthropod and nematode species and best known for the reproductive alterations it induces, harbors a phage named WO, which has recently been proven to be lytic. Here, phage infection was checked in 31 Wolbachia strains, which induce 5 different effects in their hosts and infect 25 insect species and 3 nematodes. Only the Wolbachia infecting nematodes and Trichogramma were found devoid of phage infection. All the 25 detected phages were characterized by the DNA sequence of a minor capsid protein gene. Based on all data currently available, phylogenetic analyses show a lack of congruency between Wolbachia or insect and phage WO phylogenies, indicating numerous horizontal transfers of phage among the different Wolbachia strains. The absence of relation between phage phylogeny and the effects induced by Wolbachia suggests that WO is not directly involved in these effects. Implications on phage WO evolution are discussed.     

Biology of Diaphorina citri (Hem., Psyllidae) on different hosts and at different temperatures

Abstract:  The objective of this work was to study the effect of different hosts and temperatures on Diaphorina citri Kuwayama (Hem., Psyllidae) biology. Citrus limonia (Rangpur lime), Murraya paniculata (orange jessamine) and Citrus sunki (Sunki mandarin) were used as hosts. Measurements included duration and viability of the egg and nymphal stages, sex ratio, fecundity and longevity. In order to verify the effect of temperature on D. citri biology, the duration and viability of its developmental stages and biological cycle were compared at seven different temperature conditions. Durations of the embryonic and nymphal stages were similar for Rangpur lime, orange jessamine and mandarin. However, the nymphal viability obtained on mandarin was lower than the values obtained on the other hosts. Fecundity was higher on orange jessamine and, in all instances, females had greater longevity than males. A fixed number of instars (five) was obtained on the three hosts. Rangpur lime and orange jessamine provided better D. citri development when compared with mandarin. Duration of the egg and nymphal stages varied from 2.6 to 7.7 and from 9.4 to 35.8 days, respectively, at temperatures from 18 to 32°C. Egg viability was higher than 81.6% at the temperature range studied (18–32°C); nymphal viability was higher than 70% at the 18–30°C range, differing from viability at 32°C, which was dramatically reduced. The D. citri lower temperature development threshold (TT) and thermal constant ( K ) values for the egg, nymphal and biological cycle (egg–adult) stages were 12.0°C and 52.6 Degree-Day (DD); 13.9°C and 156.9 DD; and 13.5°C and 210.9 DD respectively.