昆虫共生菌的次级代谢产物研究进展

微生物与昆虫的共生是一种普遍现象,昆虫种类繁多,与昆虫共生的微生物也多种多样。昆虫共生菌是活性次生代谢产物的重要来源。本文对自2008年以来已报道的177个昆虫共生菌的次级代谢产物进行了统计和分析,结果表明:61.6%的化合物为新天然产物(生物碱类新化合物最多),其中,约75%的新化合物来源于昆虫共生真菌,25%来源于细菌;醌酮类化合物是昆虫共生菌源天然产物的主要结构类型,占23.2%;47.5%的化合物具有显著的抗肿瘤、抗菌、除草和抗氧化等生物活性,且化合物中的主要活性类型是抗菌和抗肿瘤活性,活性范围覆盖面最广的结构类型是生物碱类。以上结果表明昆虫共生菌的次级代谢产物是先导性化合物的重要来源且具有丰富的生物活性类型。本文以天然产物的结构分类为切入点,结合其研究菌株来源、生物活性等进行综述,旨在为充分挖掘昆虫共生菌次级代谢产物提供重要参考。     

昆虫共生放线菌多样性、功能及其活性次生代谢产物研究进展

作为一类具有重要经济价值的微生物资源,已发现的放线菌所产的生物活性物质占到所有微生物生物活性物质的70%,其中包括抗生素、免疫抑制剂和酶抑制剂等。从昆虫体内分离放线菌将会是发现新菌种和新化合物的重要渠道。本文主要阐述了昆虫共生放线菌的多样性及功能,总结了昆虫共生放线菌的活性次生代谢产物的种类及其应用进展情况,最后对昆虫共生放线菌代谢活性物质研究与开发中存在的问题提出了对策。     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termitomyces),相对含量在51.83%以上,菌圃中还鉴定到炭角菌属(1%,Xylaria)。【结论】为今后培菌白蚁-体内外微生物共生关系研究以及微生物的分离培养提供了依据和参考。     

昆虫体外共生菌研究进展

昆虫体外共生菌是指能在体外与宿主发生互利共生关系的微生物。体外共生菌虽然不如肠道微生物那样普遍存在于昆虫中,但也在宿主生长发育过程中扮演着重要的角色。昆虫体外共生菌一般寄生于昆虫体表或体内特异器官(如储菌器),在特定时期转移到植物组织中。体外共生菌产生的挥发物能作为宿主定位寄主植物的信号物质,能为宿主提供生长发育所需的营养物质,还参与了宿主体外免疫。对昆虫体外共生菌的研究,不仅能进一步揭示昆虫与微生物之间的互作关系,丰富昆虫共生菌的研究,还能从共生菌的角度探索害虫引诱剂和昆虫免疫豁免机制。本文对昆虫体外共生菌寄生方式、传播途径、对宿主的影响等研究成果进行了综述,旨在为害虫综合防控提供新思路。     

药用昆虫喙尾琵琶甲肠道抗菌活性放线菌的筛选及鉴定

【目的】探究药用昆虫喙尾琵琶甲Blaps rynchopetera肠道具有抗菌活性的放线菌,为抗菌药物开发提供新的放线菌资源。【方法】结合稀释涂布法和选择培养法从喙尾琵琶甲成虫肠道分离放线菌。以耐甲氧西林金黄色葡萄球菌(methieillin-resistant Staphylococus aureus)、金黄色葡萄球菌、大肠杆菌Escherichia coli、粪肠球菌Enterococcus faecalis、鼠伤寒沙门氏菌Salmonella typhimurium 和铜绿假单胞菌Pseudomonas aeruginosa 6株致病细菌以及黑曲霉Aspergillus niger、青霉菌Penicillium expansum和白色念珠菌Canidia albicans 3株致病真菌为指示菌,通过牛津杯琼脂扩散法测试放线菌次生代谢产物的抗菌活性。随后,采用分子生物学方法进行16S rRNA序列分析鉴定活性显著的18株放线菌并构建系统发育树。【结果】从喙尾琵琶甲成虫肠道中分离到176株共生放线菌,初步活性筛选结果显示其中46株放线菌表现出不同程度的抗菌活性。多株放线菌对致病菌具有广谱的抑菌作用且抑菌圈直径超过阳性对照药。选择抑菌圈直径大于15 mm的18株放线菌进行分子鉴定,结果显示均为链霉属Streptomyces。【结论】喙尾琵琶甲肠道含有丰富的抗菌活性放线菌资源。     

培菌白蚁菌圃微生物降解木质纤维素的研究进展

白蚁及其共生微生物协同降解植物细胞壁的机理一直被世界各国科学家所关注。培菌白蚁作为高等白蚁,相比低等食木白蚁具有更多样化的食性,其利用外共生系统“菌圃”,对多种植物材料进行处理。本文综述了菌圃微生物降解木质纤维素的研究进展,以期为深入研究菌圃中木质纤维素降解过程及其机制,并挖掘利用菌圃降解木质纤维素的能力及仿生模拟菌圃开发新的生物质利用系统提供参考。培 菌白蚁在其巢内利用由植物材料修建的多孔海绵状结构——“菌圃”来培养共生真菌鸡枞菌Termitomyces spp.,形成了独特的木质纤维素食物降解和消化策略,使木质纤维素在培菌白蚁及其共生微生物协同作用下被逐步降解。幼年工蚁取食菌圃上的共生真菌菌丝组成的小白球和老年工蚁觅得食物并排出粪便堆积到菌圃上成为上层菌圃。这一过程中,被幼年工蚁取食的共生真菌释放木质素降解酶对包裹在植物多糖外部的木质素屏障进行解聚。菌圃微生物(包括共生真菌)对解聚的木质素基团进一步降解,将多糖长链或主链剪切成短链,使菌圃基质自下而上被逐步降解。最后下层的老熟菌圃被老年工蚁取食,其中肠的内源酶系及后肠微生物将这些短链进一步剪切和利用。因此,蚁巢菌圃及其微生物是培菌白蚁高效转化利用木质纤维素的基础。化学层面的研究表明,菌圃能够实现对植物次生物质解毒和植 物纤维化学结构解构。对共生真菌相关酶系的研究显示可能其在菌圃的植物纤维化学结构和植物次生物质的降解中发挥了作用,但不同属共生真菌间其效率和具体功能不尽相同。而菌圃中的细菌是否发挥了作用和哪些细菌类群发挥了作用等仍有待进一步的研究。相比于低等食木白蚁利用其后肠共生微生物降解木质纤维素,培菌白蚁利用菌圃降解木质纤维素具有非厌氧和能处理多种类型食物两大优势,仿生模拟菌圃降解木质纤维素的机制对林地表面枯枝落叶的资源化利用具有重要意义。     

小菜蛾肠道成团泛菌PxG45的分离鉴定及其抗真菌活性

【目的】昆虫肠道共生细菌是栖息在昆虫肠道中的微生物,是肠道微生物群落中最主要的成员。它们对昆虫的生长发育、营养吸收以及免疫系统均具有重要影响。本研究通过对小菜蛾Plutella xylostella肠道共生细菌进行分离培养和功能鉴定,以探究肠道共生细菌对小菜蛾适应性的影响及其对真菌的广谱抑制作用。【方法】利用体外平板培养分离纯化小菜蛾3龄幼虫肠道细菌PxG45菌株,结合形态学、16S rDNA基因测序和生理生化测定进行菌种鉴定。利用生物测定探究PxG45对小菜蛾3龄幼虫存活率及球孢白僵菌Beauveria bassiana侵染小菜蛾3龄幼虫对存活率的影响。通过平板对峙法检测PxG45对球孢白僵菌的抑制作用,进一步检测PxG45对植物病原真菌希金斯炭疽菌Colletotrichum higginsianum、刺盘孢炭疽菌C.camelliae、尖孢镰刀菌古巴专化型4号生理小种Fusarium oxysporum f. sp.cubense race 4、茄链格孢菌Alternaria solani、稻瘟病菌Magnaporthe oryzae和丁香假单胞菌杨梅致病变种Pseudomona...     

植菌昆虫及共生真菌共生机制

大约4-6千万年前,3种昆虫类群：白蚁、蚂蚁及食菌甲虫独立进化了培植真菌作为食物的能力,完成了从收集、捕获到主动种植真菌作为食物的生活方式的转变。“耕种”的生活方式最终使得这些昆虫占据重要的生态位。这3类昆虫种植真菌的过程具有明确的人类农业的特点,包括接种、培育、收获以及对培养物的营养依赖。围绕这些环节,昆虫适应不同的功能而进行分工合作,同时通过与一类放线菌共生,利用其产生抗生素来保护菌圃。切叶蚁（attine ant）及其共生真菌、白蚁（termite）及其共生蚁巢伞、食菌甲虫（ambrosia beetles）及其共生真菌是典型的被广泛研究的真菌和昆虫共生体系。而这种培植真菌的能力并不仅仅存在于以上3类昆虫中。植菌卷叶象甲Euops chinensis精心制作叶苞并接种储菌器真菌;蜥蜴甲虫Doubledaya bucculenta以及树蜂Sirex spp.也存在接种共生真菌作物的行为。从本质上讲,昆虫的真菌培植体系与人类的农业体系非常类似,因此对于种植真菌昆虫的系统研究能够为应对全球粮食短缺和农业持续高产提供一些有价值的参考。     

植菌昆虫与其共生真菌协同进化分子机制

昆虫菌业（fungiculture）是一种类似于人类种植业的昆虫种植体系,包括种植、耕作、收获和营养依赖4个过程,可分为高级的社会性昆虫如切叶蚂蚁、白蚁等和低级的非社会性昆虫如食菌小蠹虫、卷叶象甲、蜥蜴甲虫、树蜂等,它们均能种植并取食真菌。近年来随着组学及微生物组技术的发展,植菌昆虫与其共生真菌协同进化的分子机制研究方面取得了重要进展。系统发育分析阐明了植菌昆虫的起源与进化历程,并显示出与共生真菌系统发育的一致性;共生真菌细胞核数量也从双核增加到最多17个核,而染色体倍型也从单倍体增加为二倍体甚至多倍体;组学分析则揭示了植菌昆虫与其共生真菌在精氨酸、碳水化合物、木质素及几丁质合成或降解等方面显示出了高度的协同进化。本文系统综述了植菌昆虫及其共生真菌的系统进化、核进化及基因组进化进展,并探讨这种协同进化机制的生物学意义。     

伯氏致病杆菌IDP16 蛋白抑制大蜡螟的免疫反应

[目的]从伯氏致病杆菌(Xenorhabdus bovienii)胞外组分中分离纯化出能够抑制大蜡螟(Galleria mellonella)免疫反应的一种蛋白,研究其在昆虫病原线虫及其共生菌致病过程中的作用.[方法]采用硫酸铵沉淀和柱层析的方法对活性蛋白进行分离和纯化,通过体内注射并观察血淋巴黑化进行活性蛋白的筛选;采用荧光微球和琼脂糖小球评价活性蛋白对血细胞吞噬、包被作用的影响;采用双向电泳结合质谱分析对活性蛋白进行鉴定,设计引物用PCR的方法克隆其编码基因,利用pET 30a载体进行原核表达,以亲和层析纯化重组蛋白.[结果]纯化得到一个昆虫免疫抑制蛋白,命名为IDP16,该蛋白可显著抑制大蜡螟血淋巴中的多酚氧化酶活性,降低血细胞的吞噬和包被作用.克隆得到其编码基因并进行了原核表达,重组蛋白仍具有免疫抑制活性.[结论]伯氏致病杆菌产生的IDP16蛋白能够抑制昆虫的免疫反应,在共生菌和宿主昆虫互作过程中起着重要的作用.     

Evolutionary basis and ecological role of toxic microbial secondary metabolites

This presentation develops a theory of the evolutionary origin and ecological implications of toxic microbial secondary metabolites. The theory is based on a model system that outlines cause—effect associations between pertinent biotypes in the aflatoxin contamination of developing maize kernels. The model suggests that the aflatoxin-producing fungi are natural digestive tract inhabitants of a number of insect species that feed on developing kernels. During feeding, the insect larvae introduce fungal propagules and provide infection sites on damaged kernels. The fungal association with insects exhibits extraordinary variability, ranging from symbiotic to pathogenic. Elaboration of aflatoxin by the fungus facilitates the pathogenic process in host insects. The theory contends that genetic information for secondary microbial metabolites evolved during ecosystem disequilibria. During periods of ecological stability, mechanisms evolved for repression of toxic secondary metabolite biosynthesis. The theory broadly suggests that contemporary agricultural activities presents the requisite milieu for production or toxic microbial secondary metabolites.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

A meta-analysis of the effects of galling insects on host plant secondary metabolites

The idea that galling insects actively manipulate host plant chemistry has been previously documented but has not been quantified across a range of galler and host plant taxa. We present the first quantitative review of the relationship between insect galling and levels of secondary metabolites in host plants. Using meta-analytic techniques, we examined this relationship across 40 galler and host plant species combinations. We found that galling insects are associated with significantly higher levels of tannins and phenolics; however, no difference was found for volatiles. Hymenoptera, Diptera and Hemiptera were associated with higher levels of secondary metabolites; however, only Hymenoptera was significant. The climatic zone of the study area did not explain significant differences in gall-induced secondary metabolites. Overall the results show that the ability of galling insects to manipulate host plant secondary chemistry is widespread across insect and plant taxa. The evolutionary success of galling insects may be in part due to this unique ability.     

Secondary metabolites as chemical signals for cellular differentiation.

Several microbial secondary metabolites function as essential chemical signals for induction of cellular differentiation in the producing organisms. The role of A-factor and its analogues such as essential autoregulators in actinomycetes is discussed and a review is given of fungal metabolites with hormonal activities. Divergent secondary metabolites with the capability to induce cellular differentiation in other organisms are also discussed as to their possible involvement in a symbiotic relationship in the ecosystem.     

Genetic diversity of termite-egg mimicking fungi “termite balls” within the nests of termites

Antagonistic or mutualistic interactions between insects and fungi are well-known, and the mutualistic interactions of fungus-growing ants, fungus-growing termites, and fungus-gardening beetles with their respective fungal mutualists are model examples of coevolution. However, our understanding of coevolutionary interactions between insects and fungi has been based on a few model systems. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect–fungus associations. This novel termite–fungus interaction is a good model system to compare with the relatively well-studied systems of fungus-growing ants and termites because termite egg-mimicking fungi are protected in the nests of social insects, as are fungi cultivated by fungus-growing ants and termites. Recently, among systems of fungus-growing ants and termites, much attention has been focused on common factors including monoculture system for the ultimate evolutionary stability of mutualism. We examined the genetic diversity of termite egg-mimicking fungi within host termite nests. RFLP analysis demonstrated that termite nests were often infected by multiple strains of termite egg-mimicking fungi, in contrast to single-strain monocultures in fungus combs of fungus-growing ants and termites. Additionally, phylogenetic analyses indicated the existence of a free-living stage of the termite egg-mimicking fungus as well as frequent long-distance gene flow by spores and subsequent horizontal transmission. Comparisons of these results with previous studies of fungus-growing ants and termites suggest that the level of genetic diversity of fungal symbionts within social insect nests may be important in shaping the outcome of the coevolutionary interaction, despite the fact that the mechanism for achieving genetic diversity varies with the evolutionary histories of the component species.     

Phylogenetic analysis of mutualistic filamentous bacteria associated with fungus-growing ants

The attine ant-microbe system is a quadripartite symbiosis, involving a complex set of mutualistic and parasitic associations. The symbiosis includes the fungus-growing ants (tribe Attini), the basidiomycetous fungi the ants cultivate for food, specialized microfungal parasites (in the genus Escovopsis) of the cultivar, and ant-associated mu tualistic filamentous bacteria that secrete antibiotics specifically targeted to suppress the growth of Escovopsis. In this study, we conduct the first phylogenetic analysis of the filamentous mutualistic bacteria (actinomycetes) associated with fungus-growing ants. The filamentous bacteria present on 3 genera of fungus-growing ants (Acromyrmex, Trachy myrmex, and Apterostigma) were isolated from 126 colonies. The isolated actinomycetes were grouped into 3 distinct morphological types. Each morphological type was specific to the ant genus from which it was isolated, suggesting some degree of host specificity. The phylogenetic position of the 3 morphotypes was estimated using 16S rDNA for representative strains. The 8 isolates of actinomycetes sequenced are in the family Pseudonocardiaceae (Actino mycetales) and belong to the genus Pseudonocardia. Transmission electron microscopy examination of the actino mycete associated with the cuticle of Acromyrmex sp. revealed bacterial cells with an outer electron-dense membrane, consistent with actinomycetes in the genus Pseudonocardia. Ant-associated Pseudonocardia isolates did not form a monophyletic group, suggesting multiple acquisitions of actinomycetes by fungus-growing ants over their evolutionary history.     

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、共生微生物之间的互作以及在害虫防控中的应用等方面的研究进展进行综述和展望。随着分子微生态学、宏基因组测序等先进研究方法的不断开发应用,对农业昆虫害虫共生微生物研究的广度和深度都有了突破。发现共生微生物主要通过以下方式影响宿主昆虫：(1)合成营养物质或产生消化酶促进宿主生长发育、拓展宿主生态位;(2)产生保护性代谢物直接保护宿主抵御胁迫,或通过调控寄主植物的防御反应间接地保护宿主;(3)产生活性物质调控宿主的生殖、交配、聚集和运动等行为。昆虫共生微生物的种类和数量在一定时空范围内维持动态变化并对宿主表型产生重要影响,是宿主、环境、共生微生物互作因素之间收益权衡的结果。因此建议进一步开展以下研究：影响共生体形成和维持的分子机制;在更多时空维度上研究共生微生物、宿主、寄主、天敌和环境之间的复杂相互作用;通过定向调控共生体设计绿色高效的害虫防治策略。     

Insect endosymbionts: manipulators of insect herbivore trophic interactions?

Throughout their evolutionary history, insects have formed multiple relationships with bacteria. Although many of these bacteria are pathogenic, with deleterious effects on the fitness of infected insects, there are also numerous examples of symbiotic bacteria that are harmless or even beneficial to their insect host. Symbiotic bacteria that form obligate or facultative associations with insects and that are located intracellularly in the host insect are known as endosymbionts. Endosymbiosis can be a strong driving force for evolution when the acquisition and maintenance of a microorganism by the insect host results in the formation of novel structures or changes in physiology and metabolism. The complex evolutionary dynamics of vertically transmitted symbiotic bacteria have led to distinctive symbiont genome characteristics that have profound effects on the phenotype of the host insect. Symbiotic bacteria are key players in insect–plant interactions influencing many aspects of insect ecology and playing a key role in shaping the diversification of many insect groups. In this review, we discuss the role of endosymbionts in manipulating insect herbivore trophic interactions focussing on their impact on plant utilisation patterns and parasitoid biology.     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术。该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化。核糖体工程效果显著,迄今为止,已从百余种放线菌菌株中发掘了10余种新结构分子和提升近30种活性次级代谢产物的生产效价。鉴于此,文中从核糖体工程的发展角度出发,对该技术的建立与优化,及其作用机制的阐明进行了系统的归纳与总结;同时也全面分析探讨了该技术在放线菌次级代谢产物开发中的推广应用,以期为核糖体工程的发展完善及放线菌次级代谢产物的综合开发提供借鉴与参考。