烟粉虱内共生菌 Rickettsia 在植物体内的分布及转移效率初探

【目的】检测 Q 型烟粉虱 Bemisia tabaci （Gennadius）体内 Rickettsia 的感染情况,研究分析Rickettsia 共生菌经烟粉虱传入豇豆植物后的分布、转移效率等。【方法】以 Q 型烟粉虱为实验材料,利用常规 PCR 及荧光原位杂交技术(FISH),检测了烟粉虱体内 Rickettsia 的感染率,以及 Rickettsia 传入豇豆植物体内后的存留情况。【结果】 Q 型烟粉虱可以通过取食将 Rickettsia 传至豇豆植株内;接虫数量与 Rickettsia传入效率及其在取食部位相邻的下部叶片中检测到的起始时间呈负相关;Rickettsia 经烟粉虱取食传入豇豆叶片后,集中分布在叶片的韧皮部筛管中;基于16S rRNA 的系统发育分析结果表明,Q 型烟粉虱体内的Rickettsia 与经取食传入豇豆叶片的 Rickettsia 高度同源。【结论】 Rickettsia 可以通过烟粉虱的取食传入植物体内,并且可以在相邻叶片之间转移传播,Rickettsia 在由寄主昆虫向植株传播过程中高度保守。     

Wolbachia infections of the whitefly Bemisia tabaci

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed.     

A newly recorded Rickettsia of the Torix group is a recent intruder and an endosymbiont in the whitefly Bemisia tabaci

The bacterium Rickettsia is found widely in phytophagous insects and often exerts profound effects on the phenotype and fitness of its hosts. Here, we decrypt a new, independent, phylogenetically ancient Torix Rickettsia endosymbiont found constantly in a laboratory line of an economically important insect Asia II 7, a putative species of the Bemisia tabaci whitefly complex (Hemiptera: Aleyrodidae), and occasionally in field whitefly populations. This new Rickettsia distributes throughout the body of its whitefly host. Genetically, compared to Rickettsia_bellii_MEAM1 found earlier in whiteflies, the new Rickettsia species has more gene families and pathways, which may be important factors in shaping specific symbiotic relationships. We propose the name ‘Candidatus Rickettsia_Torix_Bemisia_tabaci (RiTBt)’ for this new endosymbiont associated with whiteflies. Comparative genomic analyses indicate that RiTBi may be a relatively recent intruder in whiteflies given its low abundance in the field and relatively larger genome compared to Rickettsia_bellii_MEAM1.     

烟粉虱内共生菌Rickettsia在植物体内的形态及动态变化

[目的]通过研究烟粉虱Bemisia tabaci取食传入植物体内的昆虫内共生菌种类,探明其在不同植物中的分布形态及时空动态.[方法]以B型烟粉虱、棉花、番茄、豇豆为实验材料,利用常规PCR检测烟粉虱取食后传入植物体内的共生菌种类;利用透射电镜(Transmission electron microscope,TEM)检测Rickettsia传入植物后的分布及形态;利用q-PCR技术检测豇豆叶片中Rickettsia含量的动态变化.[结果]B型烟粉虱体内含有原生共生菌P0rtiera、次生共生菌Ricfettsia,Hamiltonella和Hemipteriphilus,但只检测到Rickettsia可经烟粉虱传入棉花、番茄、豇豆植物体内,并可在植物体内存活、转移.在3种植物体内Rickettsia均分布于叶片韧皮部的筛管细胞中.烟粉虱、棉花、番茄组织内的Rickettsia形态基本一致,但豇豆中Rickettsia在形态上较小而钝圆.相同数量的烟粉虱取食,在豇豆体内最先检测到Rickettsia.随着烟粉虱取食时间的增加,豇豆体内的Rickettsia含量先增加后下降;而当无烟粉虱持续取食时,一定时间段内豇豆体内的Rickettsia先下降再小幅度上升,并可以在一定时间内保持不变.基于16S rDNA序列的系统发育分析表明,传入棉花、番茄、豇豆叶片中的Rickettsia与B型烟粉虱体内的Rickettsia高度同源.[结论]Rickettsia可经烟粉虱取食传入植物体内,分布并存活于韧皮部的筛管细胞中,并可在植物不同叶片之间转移;在不同植物宿主中,Rickettsia的形态会发生轻微变化;烟粉虱对Rickettsia的传播效率受到植物种类的影响.     

Draft genome sequence of the sulfur-oxidizing bacterium "Candidatus Sulfurovum sediminum" AR, which belongs to the Epsilonproteobacteria

Sulfur-oxidizing bacteria are common microorganisms in a variety of sulfide-rich environments. They play important roles in the global sulfur cycle on earth. Here, we present a high-quality draft genome sequence of a sulfur-oxidizing bacterium, "Candidatus Sulfurovum sediminum" strain AR, which belongs to the class Epsilonproteobacteria and dominated an enrichment culture from a marine sediment collected off Svalbard, within the Arctic Circle. Its genome contains genes for sulfur oxidation and carbon fixation. The size of the draft genome is 2.12 Mb, and the G+C content is 39.4%.     

Rickettsia influences thermotolerance in the whitefly Bemisia tabaci B biotype

Abstract The whitefly Bemisia tabaci harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts, including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. In Israel, Rickettsia is found in both the B and Q of B. tabaci biotypes, and while all other secondary symbionts are located in the bacteriomes, Rickettsia can occupy most of the body cavity of the insect. We tested whether Rickettsia influences the biology of B. tabaci and found that exposing a Rickettsia‐containing population to increasing temperatures significantly increases its tolerance to heat shock that reached 40°C, compared to a Rickettsia‐free population. This increase in tolerance to heat shock was not associated with specific induction of heat‐shock protein gene expression; however, it was associated with reduction in Rickettsia numbers as was assessed by quantitative real‐time polymerase chain reaction and fluorescence in situ hybridization analyses. To assess the causes for thermotolerance when Rickettsia is reduced, we tested whether its presence is associated with the induction of genes required for thermotolerance. We found that under normal 25°C rearing temperature, genes associated with response to stress such as cytoskeleton genes are induced in the Rickettsia‐containing population. Thus, the presence of Rickettsia in B. tabaci under normal conditions induces the expression of genes required for thermotolerance that under high temperatures indirectly lead to this tolerance.     

Variation and covariation of life history traits in aphids are related to infection with the facultative bacterial endosymbiont Hamiltonella defensa

Host–symbiont associations play an important role in insects. In aphids, facultative symbionts affect host plant use and increase thermal tolerance and resistance to natural enemies. In spite of these beneficial effects on aphid fitness, the frequency of facultative symbionts in aphids ranges from low to intermediate. Tradeoffs induced by symbionts could prevent the fixation of symbionts in aphid populations. Therefore, we studied the life history traits and correlations between them in 21 clones of the black bean aphid, Aphis fabae, seven of which were infected with the facultative endosymbiont Hamiltonella defensa. We found that clones harbouring H. defensa exhibited significantly higher body mass at maturity and offspring production, and a marginally higher intrinsic rate of increase. However, development time and offspring body size did not differ between symbiont‐free and infected clones. In addition, body mass at maturity was positively correlated with offspring production, offspring body size and intrinsic rate of increase, whereas development time was negatively correlated with body mass at maturity, offspring production and offspring body size. Excluding infected clones had little effect on these correlations; only correlations between body mass at maturity and offspring production, and between development time and offspring body size, became nonsignificant. Therefore, we did not find any evidence for tradeoffs between life history traits induced by symbiont infection. In fact, infected clones had higher overall fitness than symbiont‐free clones under the conditions of our experiment, suggesting that symbionts do not impose costs on aphids harbouring them. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 237–247.     

Diversity of "Candidatus Liberibacter asiaticus," based on the omp gene sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of "Ca. Liberibacter asiaticus" isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species "Ca. Liberibacter asiaticus," even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating "Ca. Liberibacter asiaticus" isolates.     

田间系统调查表明山东省农区烟粉虱优势种为Q隐种

【目的】为了揭示山东省农区烟粉虱 Bemisia tabaci 隐种的分布情况,对2013年7-8月份期间在该省不同农区采集的烟粉虱隐种组成进行了系统调查研究。【方法】在山东省15市22个地点作物上采集了69份烟粉虱样品,利用mtCOI基因PCR-RFLP方法进行了分子鉴定,并分析了烟粉虱Q隐种在不同寄主作物及地理区域内的分布状况。【结果】22个采集地点中,17个地点的采集样品全部为烟粉虱Q隐种;其他5个地点（潍坊、菏泽、泰安、淄博和临沂）的烟粉虱Q隐种所占比例均大于90%,B隐种比例较低（<10%）。烟粉虱Q隐种比例在5种寄主（茄子、辣椒、番茄、黄瓜和棉花）种群间以及在山东省东部丘陵、中部山地和西部平原地区间均没有显著差异。【结论】2013年田间调查发现烟粉虱Q隐种在山东各地已广泛取代B隐种成为该地区优势种群。     

Ultrastructure of the endosymbionts of the whitefly,Bemisia tabaci andTrialeurodes vaporariorum

Summary The ultrastructure of the mycetocytes and mycetome micro-organisms of the sweetpotato whitefly,Bemisia tabaci Genn. andTrialeurodes vaporariorum West are described. InB. tabaci, two morphologically distinct types of micro-organisms were observed in mycetocytes. The predominant type lacked a distinct cell wall, was pleomorphic in shape with a surrounding vacuole. The second type was a coccoid organism, with inner and outer cell membranes. The coccoid organism was often found in groups of varying number within vacuoles, and in many cases appeared to be undergoing degradation. InT. vaporariorum mycetocytes, pleomorphic and coccoid organisms were found, although the coccoid micro-organism inT. vaporariorum, had a thicker cell wall than the coccoid micro-organism inB. tabaci.Abbreviations C coccoid micro-organism - P pleomorphic micro-organism     

Genome sequence for "Candidatus Mycoplasma haemominutum," a low-pathogenicity hemoplasma species

We present the genome sequence of "Candidatus Mycoplasma haemominutum" strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.     

Localized migration and dispersal by the sweet potato whitefly,Bemisia tabaci

Laboratory populations of the sweet potato whitefly, Bemisia tabaci, have been shown to consist of both migratory and trivial flying morphs. The behavior of these forms as part of the process of short-range migration was examined under field conditions. Insects were marked in a field of melons using fluorescent dust during two consecutive growing seasons. During the first growing season, passive traps used to collect living whiteflies, were placed along 16 equally spaced transects radiating from the field to a distance of up to 1.0 km. Wind out of the north-east consistently carried migrating whiteflies to traps placed along transects in the south-western quadrant because cold air drainages dictate wind direction during early morning hours in the desert South-west. For this reason, during the second season traps were laid out over fallow ground in a rectangular grid extending 2.7 km to the south-west of the marked field. If dispersal was entirely passive, patterns could be described using a diffusion model. Statistical examination of the data, howèver, demonstrated that the distribution on all days was patchy. Geostatistical techniques were used to describe the observed patchiness. Traps in the immediate vicinity of the marked field caught more whiteflies than the daily median. Large numbers were also collected from near the periphery of the grid. White-flies were far less prevalent in the grid's center. As a result, the distribution of captured whiteflies can be described as bimodal. These patterns confirm behavior observed in the laboratory, i.e., a portion of the population are trivial fliers that do not engage in migration and are consequently captured in traps near the field, and a portion initially respond to cues associated with skylight, ignoring cues provided by the ground, and fly for a period of time before landing in distant traps. During both years movement out of the field had an exaggerated directional component on 13 of 14 days.     

Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci

Abstract Endosymbionts are important components of arthropod biology. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex composed of ≥ 28 putative species. In addition to the primary endosymbiont Portiera aleyrodidarum, six secondary endosymbionts (S‐endosymbionts), Hamiltonella, Rickettsia, Wolbachia, Cardinium, Arsenophonus and Fritschea, have been identified in B. tabaci thus far. Here, we tested five of the six S‐endosymbiont lineages (excluding Fritschea) from 340 whitely individuals representing six putative species from China. Hamiltonella was detected only in the two exotic invaders, Middle East‐Asia Minor 1 (MEAM1) and Mediterranean (MED). Rickettsia was absent in Asia II 1 and MED, scarce in Asia II 3 (13%), but abundant in Asia II 7 (63.2%), China 1 (84.7%) and MEAM1 (100%). Wolbachia, Cardinium and Arsenophonus were absent in the invasive MEAM1 and MED but mostly abundant in the native putative species. Furthermore, phylogenetic analyses revealed that some S‐endosymbionts have several clades and different B. tabaci putative species can harbor different clades of a given S‐endosymbiont, demonstrating further the complexity of S‐endosymbionts in B. tabaci. All together, our results demonstrate the variation and diversity of S‐endosymbionts in different putative species of B. tabaci, especially between invasive and native whiteflies.     

Factors Limiting the Spread of the Protective Symbiont Hamiltonella defensa in Aphis craccivora Aphids

Many insects are associated with heritable symbionts that mediate ecological interactions, including host protection against natural enemies. The cowpea aphid, Aphis craccivora, is a polyphagous pest that harbors Hamiltonella defensa, which defends against parasitic wasps. Despite this protective benefit, this symbiont occurs only at intermediate frequencies in field populations. To identify factors constraining H. defensa invasion in Ap. craccivora, we estimated symbiont transmission rates, performed fitness assays, and measured infection dynamics in population cages to evaluate effects of infection. Similar to results with the pea aphid, Acyrthosiphon pisum, we found no consistent costs to infection using component fitness assays, but we did identify clear costs to infection in population cages when no enemies were present. Maternal transmission rates of H. defensa in Ap. craccivora were high (ca. 99%) but not perfect. Transmission failures and infection costs likely limit the spread of protective H. defensa in Ap. craccivora. We also characterized several parameters of H. defensa infection potentially relevant to the protective phenotype. We confirmed the presence of H. defensa in aphid hemolymph, where it potentially interacts with endoparasites, and performed real-time quantitative PCR (qPCR) to estimate symbiont and phage abundance during aphid development. We also examined strain variation of H. defensa and its bacteriophage at multiple loci, and despite our lines being collected in different regions of North America, they were infected with a nearly identical strains of H. defensa and APSE4 phage. The limited strain diversity observed for these defensive elements may result in relatively static protection profile for this defensive symbiosis.     

Isolation, pure culture, and characterization of "Candidatus Arsenophonus arthropodicus," an intracellular secondary endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as "Candidatus Arsenophonus arthropodicus." Phylogenetic analyses indicate that "Candidatus Arsenophonus arthropodicus" is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of "Candidatus Arsenophonus arthropodicus" in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.     

Complete genome sequence of Candidatus Ruthia magnifica

The hydrothermal vent clam Calyptogena magnifica (Bivalvia: Mollusca) is a member of the Vesicomyidae. Species within this family form symbioses with chemosynthetic Gammaproteobacteria. They exist in environments such as hydrothermal vents and cold seeps and have a rudimentary gut and feeding groove, indicating a large dependence on their endosymbionts for nutrition. The C. magnifica symbiont, Candidatus Ruthia magnifica, was the first intracellular sulfur-oxidizing endosymbiont to have its genome sequenced (Newton et al. 2007). Here we expand upon the original report and provide additional details complying with the emerging MIGS/MIMS standards. The complete genome exposed the genetic blueprint of the metabolic capabilities of the symbiont. Genes which were predicted to encode the proteins required for all the metabolic pathways typical of free-living chemoautotrophs were detected in the symbiont genome. These include major pathways including carbon fixation, sulfur oxidation, nitrogen assimilation, as well as amino acid and cofactor/vitamin biosynthesis. This genome sequence is invaluable in the study of these enigmatic associations and provides insights into the origin and evolution of autotrophic endosymbiosis.     

烟盲蝽对不同虫态烟粉虱的捕食作用及捕食偏好性

杂食性昆虫烟盲蝽Nesidiocoris tenuis是烟粉虱Bemisia tabaci的重要天敌之一。为探索烟盲蝽对不同虫态烟粉虱的捕食作用及捕食偏好性,在室内进行了非选择性和选择性试验。结果表明,在非选择性试验中,烟盲蝽雌成虫、雄成虫以及5龄若虫可以捕食烟粉虱成虫和各龄若虫,但极少捕食卵。其中,烟盲蝽雌成虫、雄成虫及5龄若虫对烟粉虱1龄若虫的捕食量最大,分别为134.2头、101.3头和88.1头,其次是2~3龄若虫,捕食量分别为94.1头、64.2头和53.4头。而在选择性试验中烟盲蝽雄虫更偏好捕食烟粉虱的2~3龄若虫,其次是1龄若虫、4龄若虫和成虫。该结果对了解烟盲蝽控制烟粉虱的作用以及如何对烟盲蝽和其他天敌组合应用提供了参考信息。     

Inheritance of pyriproxyfen resistance in the whitefly, Bemisia tabaci (Q biotype)

The inheritance of resistance to pyriproxyfen, an insect growth regulator (a juvenoid, with ovicidal and larvicidal activities), was studied in the whitefly Bemisia tabaci (Gennadius). Two parental strains, both belonging to Q biotype, were assayed with pyriproxyfen; a susceptible strain (ALM-1) originating from Spain and a pyriproxyfen-resistant one (Pyri-R) from Israel. The resistance ratio between the two parental strains was approximately 7,000-fold. Concentration-mortality lines for F(1) heterozygous females from reciprocal crosses (SS female symbol X R male symbol and RR female symbol X S male symbol ) were derived by statistical modelling and proved intermediate to those of the parents. The pooled degree of dominance from both reciprocal crosses was +0.26, indicating that resistance was incompletely or partially dominant. Mortality curves for F(2) males produced by virgin F(1) heterozygous females displayed a broad plateau at 50% mortality, indicating that resistance to pyriproxyfen in B. tabaci is conferred primarily by a mutant allele at a single locus. The role of arrhenotoky in influencing the mode of inheritance of resistance, and its selection in field populations, is discussed.     

Transovarial transmission of Rickettsia spp. and organ-specific infection of the whitefly Bemisia tabaci

The whitefly Bemisia tabaci is a cosmopolitan insect pest that harbors Portiera aleyrodidarum, the primary obligatory symbiotic bacterium, and several facultative secondary symbionts. Secondary symbionts in B. tabaci are generally associated with the bacteriome, ensuring their vertical transmission; however, Rickettsia is an exception and occupies most of the body cavity, except the bacteriome. The mode of Rickettsia transfer between generations and its subcellular localization in insect organs have not been investigated. Using electron and fluorescence microscopy, we show that Rickettsia infects the digestive, salivary, and reproductive organs of the insect; however, it was not observed in the bacteriome. Rickettsia invades the oocytes during early developmental stages and resides in follicular cells and cytoplasm; it is mostly excluded when the egg matures; however, some bacterial cells remain in the egg, ensuring their transfer to subsequent generations. Rickettsia was localized to testicles and the spermatheca, suggesting a horizontal transfer between males and females during mating. The bacterium was further observed at large amounts in midgut cells, concentrating in vacuole-like structures, and was located in the hemolymph, specifically at exceptionally large amounts around bacteriocytes and in fat bodies. Organs further infected by Rickettsia included the primary salivary glands and stylets, sites of possible secretion of the bacterium outside the whitefly body. The close association between Rickettsia and the B. tabaci digestive system might be important for digestive purposes. The vertical transmission of Rickettsia to subsequent generations occurs via the oocyte and not, like other secondary symbionts, the bacteriome.     

海氏桨角蚜小蜂对两种生物型烟粉虱的功能反应研究

本文报道海氏桨角蚜小蜂Erelmocerus hayati在室内对不同寄主密度下的B型和Q型烟粉虱Bemisiatabaei寄生和取食情况,并比较了该蜂对两种生物型烟粉虱功能反应的差异。结果表明,海氏桨角蚜小蜂对两种生物型烟粉虱的寄生率和致死率可分别达到60％和70％以上,平均每日最大致死量可达40头烟粉虱若虫。该蜂对两种生物型烟粉虱的功能反应曲线符合Holling Ⅱ型,随寄主密度的增加对烟粉虱若虫的寄生和致死数量均增加直至达到每日最大致死数量,而寄生率和致死率则随寄主密度的增加而显著下降。该蜂对两种生物型烟粉虱的功能反应没有显著的差异。