稻飞虱酵母类胞内共生菌的组织学研究进展

褐飞虱Nilaparvata lugens体内酵母类共生菌在褐飞虱生长发育和繁殖中起着重要作用,并影响着褐飞虱对寄主植物的致害性变异。该文系统总结了国内外已有的研究成果,讨论了褐飞虱共生菌的分类地位、形态特征、在寄主发育中的功能、侵染途径等,提出了开展褐飞虱生物型形成过程中体内共生菌变异的研究建议,以便利用共生菌来监测田间不同生物型褐飞虱的发生情况,最终实现控菌防虫和对不同生物型的准确预测。     

芽孢杆菌的引入对褐飞虱微生物群和生长发育的影响

【目的】明确褐飞虱Nilaparvata lugens体内可培养共生细菌的种类，探索其中芽孢杆菌类共生菌对褐飞虱微生物群和生长发育的影响。【方法】通过离体培养的方法，从褐飞虱两种不同致害性种群(TN1敏感种群和IR56高致害种群)中分离可培养共生细菌。通过16S rDNA测序对获得的可培养共生菌进行鉴定，在此基础上利用原位杂交研究了共生细菌在褐飞虱体内的分布。利用人工饲料添加抗生素和回补共生细菌的方式，研究了减菌和回补芽孢杆菌的处理对褐飞虱生长发育以及共生菌丰度的影响。比较了通过饲喂和显微注射对芽孢杆菌引入的影响，考察了芽孢杆菌的定殖与褐飞虱TN1种群致害性之间的相关性。【结果】利用离体培养法从褐飞虱中获得了15株不同的共生细菌，其中包括2株源于IR56高致害种群的芽孢杆菌类共生细菌BPH-S36和BPH-S33。原位杂交结果表明共生细菌在褐飞虱成虫唾液腺、肠道、脂肪体和雌虫内生殖器中均有分布，而在雄虫内生殖器中鲜有分布。体内共生细菌对褐飞虱的生长发育至关重要，共生细菌的减少导致褐飞虱存活率在第3和6天时显著下降，而回补共生芽孢杆菌BPH-S36或BPH-S33可使其存活率在第6天时显...     

基于高通量测序的褐飞虱肠道微生物多样性分析

【目的】探明褐飞虱Nilaparvata lugens成虫肠道微生物群落结构和多样性。【方法】分离褐飞虱成虫完整肠道并提取总DNA,利用Illumina MiSeq(PE300)技术对其肠道细菌16S rRNA的V3-V4变异区和真菌ITS2序列进行测序,统计肠道微生物的操作分类单元(operational taxonomic unit, OTU)数量,分析其物种组成、丰度及Alpha多样性。并通过qPCR技术验证随机挑选注释到的4种肠道菌的高通量测序数据的有效性。【结果】分别获得褐飞虱成虫肠道细菌16S rRNA和真菌ITS2优质序列32 395和24 986条,根据序列相似性进行聚类分析分别获得235和128个OTUs。其中,细菌共注释到7个门, 15个纲, 26个目, 45个科和73个属;真菌共鉴定到3个门, 9个纲, 12个目, 15个科和18个属。在门分类水平上,细菌以变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)为优势门类;真菌以子囊菌门(Ascomycota)为绝对优势菌门。在属分类水平上,细菌的优势属为不动杆菌属Acinetobacter以及紫单胞菌科(Porphyromonadaceae)未确定属和毛螺菌科(Lachnospiraceae)未确定属,其丰度分别为36.37%, 17.22%和15.01%;真菌的优势属为粪壳菌纲(Sordariomycetes)未确定属,丰度为95.77%。Alpha多样性分析结果显示,褐飞虱肠道细菌(真菌)的观测物种数、Chao1指数、Shannon指数和Simpson 指数分别为235(128), 262.64(165.40), 3.90(0.91)和0.62(0.75)。4种肠道菌的qPCR结果显示高通量测序数据具有较高的有效性。【结论】褐飞虱成虫肠道细菌和真菌群落整体多样性比较丰富。研究结果为从共生微生物角度解析褐飞虱的环境适应性以及开发基于微生物防治的新技术等方面提供了依据。     

水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析

利用16S rDNA通用引物扩增了水稻褐飞虱Nilaparvata lugens（Stål）体内共生细菌的序列,经克隆、测序和NCBI数据库比对,发现褐飞虱体内存在杀雄菌属Arsenophonus类共生细菌,系统发育上与粉虱科和木虱科体内的Arsenophonus属亲源关系较近。在褐飞虱体内该共生细菌具有两种长度不同的16S rDNA序列,分别为1 504 bp和547 bp,其中后者为前者中间缺失了957 bp,其余序列相同。通过重新设计两对引物进行扩增,进一步确认不同褐飞虱地理种群及寄主种群均存在两种片段。Arsenophonus特异的 23S rDNA引物的扩增结果表明,Arsenophonus存在于所有检测的褐飞虱种群中,但不存在于水稻寄主中。荧光定量PCR检测发现3个褐飞虱室内寄主种群Arsenophonus属共生细菌含量不同,其中TN1种群明显高于Mudgo种群和ASD7种群。此为水稻褐飞虱体内存在Arsenophonus属共生细菌的首次报道。     

不同地理种群褐飞虱体内共生酵母菌的个体大小及数量差异

褐飞虱腹部脂肪体内普遍存在共生酵母菌,该类共生菌在褐飞虱的生理代谢和营养利用等方面起着重要作用。采用冷冻切片技术结合显微摄像系统观察法测定不同地理种群褐飞虱体内共生酵母菌的个体大小和数量。试验结果表明,不同地理种群褐飞虱雌(雄)成虫体内共生菌的个体大小和数量差异显著,依次为:对照种群>广西种群>浙江种群>福建种群。结合虫体内脂肪和糖元含量的分析得出,褐飞虱体内共生酵母菌的长、宽度和数量与脂肪和糖元含量显著正相关。文章从共生酵母菌的角度解释了不同地理种群褐飞虱致害性变异的内在原因,并推测,迁飞过程所导致的褐飞虱体内脂肪和糖元的消耗影响了该类共生菌的数量和质量,并最终导致褐飞虱对抗性品种水稻的致害性减弱。     

褐飞虱成虫唾液细菌蛋白的鉴定

【目的】通过鉴定褐飞虱Nilaparvata lugens(St?l)水状唾液中的细菌蛋白,了解其唾液中的细菌种类。【方法】利用双层膜夹蔗糖溶液的方法对褐飞虱成虫唾液进行收集,超滤浓缩后进行电泳,然后利用液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)方法鉴定蛋白,与Uniprot的细菌蛋白数据库进行比对从而鉴定褐飞虱成虫唾液中的细菌蛋白。【结果】在褐飞虱成虫唾液中鉴定到22种细菌的35种蛋白,这些蛋白主要参与能量代谢过程、蛋白的折叠和合成以及氨基酸代谢过程。这些细菌分布在变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes)3个门。蛋白种类较多来源于变形菌门。【结论】鉴定到蛋白的这些细菌可能在褐飞虱的生活史中扮演重要角色。     

褐飞虱体内Arsenophonus、Wolbachia、Spiroplasma和Cardinium四种次生共生菌感染分析

【目的】Arsenophonus、Wolbachia、Spiroplasma和Cardinium是广泛分布于昆虫体内的次生共生菌,能对寄主昆虫的生殖和发育生理产生影响,明确不同地理种群褐飞虱体内4种共生菌的感染和系统发育关系可为利用共生菌防治害虫提供技术依据。【方法】利用Arsenophonus特异性23S rRNA基因引物,Wolbachia、Spiroplasma和Cardinium各自的16S rRNA基因引物PCR检测广西10个地市、海南海口及上海、江苏南京、湖南长沙共14个地理种群褐飞虱雌、雄虫体内共生菌感染情况,并对扩增到的基因片段进行测序、构建系统发育树。【结果】不同地区2014年和2015年褐飞虱体内Arsenophonus感染率最高的分别为河池种群(30%)和长沙种群(20%)、PWolbachia感染率最高为河池种群(20%)和南京种群(30%),而Spiroplasma和Cardinium在褐飞虱各种群内均未检测到。序列比对和发育分析表明,褐飞虱体内Arsenophonus与同翅目昆虫白蜡虫Ericerus pela Chavannes体内感染的该菌关系最近;而感染Wolbachia菌的系统发育分析表明,除海南种群有一个个体感染Wolbachia菌A群外,其余种群感染的全部为B群;同时在所有感染两类共生菌的个体中并未发现共感染现象。【结论】不同地理种群褐飞虱感染Arsenophonus和Wolbachia两类共生菌的感染率并不一致;总体上褐飞虱雌虫Wolbachia感染率要高于雄虫,褐飞虱体内感染的两类共生菌与其它昆虫体内的共生菌序列高度一致或类似,且在所有检测的地理种群中均未发现有Spiroplasma和Cardinium感染。     

三种稻飞虱成虫体内酵母类共生菌的形态差异

试验采用冷冻切片技术结合显微摄像系统观察法研究了酵母类共生菌在3种稻飞虱成虫体内的存在状态、形态特性以及种类和组成.光学显微镜观察证实,3种稻飞虱成虫的头部和胸部均未观察到共生菌,在其腹部脂肪体中有大量酵母类共生菌,且以出芽进行无性繁殖,并伴有菌胞的出现.3种稻飞虱成虫体内共生菌的形态和组成明显不同.其中,褐飞虱体内的共生菌个体较大,以梭形、杆状和卵形为主,分别占共生菌总数的30.7%、53.5%和15.1%;灰飞虱和白背飞虱体内的共生菌个体较小,以卵形为主,分别占共生菌总数的93.4%和94.7%.此外,褐飞虱成虫体内的各类共生菌都显著大于灰飞虱和白背飞虱成虫体内的同种类型个体,且灰飞虱和白背飞虱体内的同种共生菌个体大小之间也存在一定差异.     

Phylogenetic position of yeast-like symbiotes from three rice planthoppers (Homoptera: Delphacidae ) based on 18S rDNA and ITS-5.8S rDNA sequences

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌( yeast-like symbiotes,YLS)的种属地位及与寄主的进化关系,测定了其体内YIS的18SrDNA及ITS-5.8S rDNA的全长序列.基于3种稻飞虱体内YLS的18S rDNA序列比对表明,褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLs的高(褐飞虱YLS和白背飞虱YLS为98.91％,灰飞虱YLS和褐飞虱YLS为95.74％,灰飞虱YLS和白背飞虱YLS为96.02％),而基于ITS-5.8S rDNA序列比对,灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高(白背飞虱YLS和灰飞虱YLS为99.57％,灰飞虱YLS和褐飞虱YIS为91.91％,白背飞虱YLS和褐飞虱YLS为90.46％).基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明,3种稻飞虱体内YLS与其他已知真菌进化关系较远.本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系,从而形成了不同于已知真菌的分类地位.     

三种稻飞虱体内类酵母共生菌18S rDNA和 ITS 5.8S rDNA序列克隆及进化分析

为研究水稻3种主要害虫灰飞虱Laodelphax striatellus、 褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera体内类酵母共生菌（yeast-like symbiotes, YLS）的种属地位及与寄主的进化关系, 测定了其体内YLS的18S rDNA及ITS-5.8S rDNA的全长序列。基于3种稻飞虱体内YLS的18S rDNA序列比对表明, 褐飞虱YLS和白背飞虱YLS的一致性比其与灰飞虱YLS的高（褐飞虱YLS和白背飞虱YLS为98.91%, 灰飞虱YLS和褐飞虱YLS为95.74%, 灰飞虱YLS和白背飞虱YLS为96.02%）, 而基于ITS-5.8S rDNA序列比对, 灰飞虱YLS和白背飞虱YLS的一致性比其与褐飞虱YLS的要高（白背飞虱YLS和灰飞虱YLS为99.57%, 灰飞虱YLS和褐飞虱YLS为91.91%, 白背飞虱YLS和褐飞虱YLS为90.46%）。基于真菌18S rDNA和ITS-5.8S rDNA的系统发育树均表明, 3种稻飞虱体内YLS与其他已知真菌进化关系较远。本研究证实了昆虫真菌类共生菌与寄主形成了长期的进化关系, 从而形成了不同于已知真菌的分类地位。     

互惠共生微生物多样性研究概况

所谓互惠共生微生物(mutualistic symbiotic microbes,MSM)是指能定殖其他生物构建互惠共生体系的微生物,主要包括互惠共生细菌、互惠共生放线菌和互惠共生真菌等。MSM种类繁多、分布广泛、物种多样性丰富,涉及原核生物界和真菌界等。MSM定殖人体、动物、植物、藻类或其他真菌,可构建各自相应的互惠共生体系,进而形成范围更加巨大的共生网络,发挥不可替代的生理生态功能。本文在介绍MSM概念的基础上,重点总结了MSM多样性研究进展,指出了目前研究中尚存在的问题,探讨了今后应该开展的工作,MSM多样性研究成果可望为研发MSM应用技术提供依据和材料。     

Bacterial symbionts in insects or the story of communities affecting communities

Bacterial symbionts are widespread in insects and other animals. Most of them are predominantly vertically transmitted, along with their hosts' genes, and thus extend the heritable genetic variation present in one species. These passengers have a variety of repercussions on the host's phenotypes: besides the cost imposed on the host for maintaining the symbiont population, they can provide fitness advantages to the host or manipulate the host's reproduction. We argue that insect symbioses are ideal model systems for community genetics. First, bacterial symbionts directly or indirectly affect the interactions with other species within a community. Examples include their involvement in modifying the use of host plants by phytophagous insects, in providing resistance to natural enemies, but also in reducing the global genetic diversity or gene flow between populations within some species. Second, one emerging picture in insect symbioses is that many species are simultaneously infected with more than one symbiont, which permits studying the factors that shape bacterial communities; for example, horizontal transmission, interactions between host genotype, symbiont genotype and the environment and interactions among symbionts. One conclusion is that insects' symbiotic complements are dynamic communities that affect and are affected by the communities in which they are embedded.     

Invasive Robinia pseudoacacia in China is nodulated by Mesorhizobium and Sinorhizobium species that share similar nodulation genes with native American symbionts

The aim of this work is to describe the diversity of potentially symbiotic bacteria associated with the invasive introduced legume Robinia pseudoacacia in China. Thirty-three isolates from 33 separate trees and nodules were characterized using restriction length fragment polymorphism and sequencing of 16S rRNA, nodA, nodC and nifH genes. Their 16S rRNA gene patterns and sequences placed them in three clades: 85% of isolates were related to the Mesorhizobium mediterraneum/temperatum group, whereas the remaining were similar either to Mesorhizobium amorphae or to Sinorhizobium meliloti . However, despite their diverse taxonomic positions, the nodA, nodC and nifH genes' phylogenies indicated that these R. pseudoacacia symbionts share similar symbiosis genes, implying gene transfers and a degree of host specificity. Comparison of R. pseudoacacia symbiotic diversity in native and other invaded areas suggests that most Chinese symbionts may not have arrived with the seed but were local bacteria that acquired specific symbiotic genes from native American rhizobia.     

Diversity of fungus‐growing termites (Macrotermes) and their fungal symbionts (Termitomyces) in the semiarid Tsavo Ecosystem,Kenya

Fungus‐growing termites of the subfamily Macrotermitinae together with their highly specialized fungal symbionts (Termitomyces) are primary decomposers of dead plant matter in many African savanna ecosystems. The termites provide crucial ecosystem services also by modifying soil properties, translocating nutrients, and as important drivers of plant succession. Despite their obvious ecological importance, many basic features in the biology of fungus‐growing termites and especially their fungal symbionts remain poorly known, and no studies have so far focused on possible habitat‐level differences in symbiont diversity across heterogeneous landscapes. We studied the species identities of Macrotermes termites and their Termitomyces symbionts by excavating 143 termite mounds at eight study sites in the semiarid Tsavo Ecosystem of southern Kenya. Reference specimens were identified by sequencing the COI region from termites and the ITS region from symbiotic fungi. The results demonstrate that the regional Macrotermes community in Tsavo includes two sympatric species (M. subhyalinus and M. michaelseni) which cultivate and largely share three species of Termitomyces symbionts. A single species of fungus is always found in each termite mound, but even closely adjacent colonies of the same termite species often house evolutionarily divergent fungi. The species identities of both partners vary markedly between sites, suggesting hitherto unknown differences in their ecological requirements. It is apparent that both habitat heterogeneity and disturbance history can influence the regional distribution patterns of both partners in symbiosis.     

Diversity and specificity of Rhizobium leguminosarum biovar viciae on wild and cultivated legumes

The symbiotic partnerships between legumes and their root-nodule bacteria (rhizobia) vary widely in their degree of specificity, but the underlying reasons are not understood. To assess the potential for host-range evolution, we have investigated microheterogeneity among the shared symbionts of a group of related legume species. Host specificity and genetic diversity were characterized for a soil population of Rhizobium leguminosarum biovar viciae (Rlv) sampled using six wild Vicia and Lathyrus species and the crop plants pea (Pisum sativum) and broad bean (Vicia faba). Genetic variation among 625 isolates was assessed by restriction fragment length polymorphism (RFLP) of loci on the chromosome (ribosomal gene spacer) and symbiosis plasmid (nodD region). Broad bean strongly favoured a particular symbiotic genotype that formed a distinct phylogenetic subgroup of Rlv nodulation genotypes but was associated with a range of chromosomal backgrounds. Host range tests of 80 isolates demonstrated that only 34% of isolates were able to nodulate V. faba. By contrast, 89% were able to nodulate all the local wild hosts tested, so high genetic diversity of the rhizobial population cannot be ascribed directly to the diversity of host species at the site. Overall the picture is of a population of symbionts that is diversified by plasmid transfer and shared fairly indiscriminately by local wild legume hosts. The crop species are less promiscuous in their interaction with symbionts than the wild legumes.     

昆虫共生微生物及其功能研究进展

昆虫体内共生微生物能够占到昆虫生物量的1％～10％,主要包括细菌、真菌、古菌和病毒.昆虫与共生微生物共进化形成共生体,共生微生物在昆虫生物学性状、多样性形成、生态适应性与抗逆性等多方面发挥着重要的作用.昆虫中的农作物害虫严重影响农业生产.本文对2000年以来农业害虫共生微生物的多样性、研究方法和功能机制、共生微生物之间...     

昆虫共生细菌研究进展

昆虫体内定殖着大量微生物,经过漫长协同进化,昆虫与这些微生物构建了共生体系,这些昆虫共生微生物参与整个生态过程,对于生态系统中物质转化与交换、能量流动与利用、信息传递与调控等均发挥着重要作用。昆虫共生细菌具有丰富的物种多样性;昆虫与其共生细菌之间通过化学机制、生理机制、生态学机制和遗传学机制构建复杂的共生体系;昆虫为细菌提供稳定的生境并共享特定的代谢途径,共生细菌则协助宿主营养代谢,提供食物中缺乏的养分,促进昆虫生长和繁殖;通过分泌抗菌肽、毒素等,细菌能增强昆虫对寄生物的防御能力和抗病性,并通过调节昆虫对非生物因子的抗逆性和耐药性,扩大昆虫的生态位。昆虫共生细菌在农林牧渔业可持续安全生产与医药研发等领域具有应用潜力和广阔的发展前景。     

Diversity in symbiont consortia in the pea aphid complex is associated with large phenotypic variation in the insect host

Virtually all eukaryotes host microbial symbionts that influence their phenotype in many ways. In a host population, individuals may differ in their symbiotic complement in terms of symbiont species and strains. Hence, the combined expression of symbiont and host genotypes may generate a range of phenotypic diversity on which selection can operate and influence host population ecology and evolution. Here, we used the pea aphid to examine how the infection with various symbiotic complements contributes to phenotypic diversity of this insect species. The pea aphid hosts an obligate symbiont (Buchnera aphidicola) and several secondary symbionts among which is Hamiltonella defensa. This secondary symbiont confers a protection against parasitoids but can also reduce the host’s longevity and fecundity. These phenotypic effects of H. defensa infection have been described for a small fraction of the pea aphid complex which encompasses multiple plant-specialized biotypes. In this study, we examined phenotypic differences in four pea aphid biotypes where H. defensa occurs at high frequency and sometimes associated with other secondary symbionts. For each biotype, we measured the fecundity, lifespan and level of parasitoid protection in several aphid lineages differing in their symbiotic complement. Our results showed little variation in longevity and fecundity among lineages but strong differences in their protection level. These differences in protective levels largely resulted from the strain type of H. defensa and the symbiotic consortium in the host. This study highlights the important role of symbiotic complement in the emergence of phenotypic divergence among host populations of the same species.     

Mycangia of Ambrosia Beetles Host Communities of Bacteria

The research field of animal and plant symbioses is advancing from studying interactions between two species to whole communities of associates. High-throughput sequencing of microbial communities supports multiplexed sampling for statistically robust tests of hypotheses about symbiotic associations. We focus on ambrosia beetles, the increasingly damaging insects primarily associated with fungal symbionts, which have also been reported to support bacteria. To analyze the diversity, composition, and specificity of the beetles' prokaryotic associates, we combine global sampling, insect anatomy, 454 sequencing of bacterial rDNA, and multivariate statistics to analyze prokaryotic communities in ambrosia beetle mycangia, organs mostly known for transporting symbiotic fungi. We analyze six beetle species that represent three types of mycangia and include several globally distributed species, some with major economic importance (Dendroctonus frontalis, Xyleborus affinis, Xyleborus bispinatus-ferrugineus, Xyleborus glabratus, Xylosandrus crassiusculus, and Xylosandrus germanus). Ninety-six beetle mycangia yielded 1,546 bacterial phylotypes. Several phylotypes appear to form the core microbiome of the mycangium. Three Mycoplasma (originally thought restricted to vertebrates), two Burkholderiales, and two Pseudomonadales are repeatedly present worldwide in multiple beetle species. However, no bacterial phylotypes were universally present, suggesting that ambrosia beetles are not obligately dependent on bacterial symbionts. The composition of bacterial communities is structured by the host beetle species more than by the locality of origin, which suggests that more bacteria are vertically transmitted than acquired from the environment. The invasive X. glabratus and the globally distributed X. crassiusculus have unique sets of bacteria, different from species native to North America. We conclude that the mycangium hosts in multiple vertically transmitted bacteria such as Mycoplasma, most of which are likely facultative commensals or parasites.