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。     

华山松大小蠹共生真菌对华山松木质部危害的解剖学研究

通过对野外和人工接种条件下,华山松大小蠹（Dendroctonus armandi)共生直菌（Leptographium terebrantis)对寄主华山松木质部危害的解剖学研究。结果表明,华山松大小蠹共生真菌随华山松大小蠹入侵健康寄主华山松木质部组织,真菌菌丝首先在其木质部树脂道内发育,分解木质部树脂道泌脂细胞,堵塞树脂道;通过菌丝在木质部交叉场薄壁细胞内及管胞细胞间和管胞细胞内的扩展,使寄主华山松树脂代谢,抗性物质代谢和水分代谢紊乱,木质部边材组织蓝变。     

松树蜂形态及危害特点的研究

在发生松树蜂的林场利用"五点取样法"进行调查,调查样方内树木种类、胸径大小、生长情况、蛀干害虫种类和在树干上的分布情况;同时,选取虫害木,解剖并统计不同蛀干害虫的羽化孔数量和特点,幼虫钻蛀特征和危害特点;研究松树蜂自然种群的寄主选择规律和垂直分布特点。结果表明,松树蜂在我国仅危害衰弱的樟子松,30-40年生的中龄林受害最严重,平均有虫株率为10.94%±8.67%。松树蜂产卵后的树皮表面有流树脂现象,呈点状或流泪状。同松幽天牛和吉丁危害状不同,松树蜂羽化孔呈正圆形,直径大小约为7 mm,分布在树干0-4.20 m的高度,其中90%的集中分布在0-3 m范围内。松树蜂幼虫坑道横截面为正圆形,无排粪孔,虫粪和蛀屑都呈粉末状,紧实堆满蛀道,颜色和蛀道周围颜色无明显差异。     

松树蜂的产卵行为

【目的】研究松树蜂Sirex noctilio的产卵行为,明确其产卵能力,为评估其繁殖潜力和危害能力提供基础数据。【方法】于黑龙江省大庆市杜尔伯特蒙古族自治县新店林场采集被松树蜂危害的寄主樟子松Pinus sylvestris var.mongolica木段上获得虫源。在室内条件下观察和分析松树蜂产卵的行为过程及规律。解剖松树蜂在樟子松木段上的产卵孔,并观察其结构特征。【结果】松树蜂羽化时即性成熟,能够马上进行交配和产卵。松树蜂雌成虫一次完整的产卵过程主要分为4个动作:树皮钻孔、木质部钻刺、产卵(注入有毒黏液和共生真菌Amylosereum areolatum)和产卵器拔出。产卵时间在360～540 s之间的产卵频数最多,占产卵总频数的41.40%;产卵过程中木质部钻刺用时最长,至少占整个产卵过程用时的90%。松树蜂雌成虫在一个产卵孔处会进行1～4次产卵,产卵时间和产卵次数呈显著正相关,当产卵时间t<360 s时,进行了1次产卵;当360 s≤t<540 s时,进行了2次产卵;当540 s≤t<780 s时,进行了3次产卵;当t≥780 s时,进行了4次或更多次产卵。松树蜂在一个产卵孔处进行1, 2, 3和4次产卵的比例分别为21.66%, 41.40%, 27.39%和9.55%。【结论】松树蜂雌成虫在一个产卵孔处的产卵时间和产卵次数呈正相关。利用产卵时间和产卵次数的关系,在只调查产卵时间的情况下,可以推断产卵次数。松树蜂在一个产卵孔处产卵的次数多,对寄主樟子松危害大。     

蚜虫与其胞内共生细菌的相互作用

蚜虫—巴克纳氏菌之间是一种典型的互利共生关系,两者相互依存,缺少一方,另一方便不能生存。研究表明,共生细菌能为寄主蚜虫提供必需氨基酸和维生索,并对寄主具有一些非营养功能,如促进蚜虫传播循环性病毒等。寄主蚜虫则是为共生菌提供一个合适的生存场所,并对共生菌的生长和繁殖进行调控。现代分子生物学技术从基因水平证明了蚜虫与共生菌的相互依赖性。     

网隙裂粉韧革菌不同生长时期转录组分析

网隙裂粉韧革菌Amylostereum areolatum是松树蜂Sirex noctilio携带并传播的共生真菌,与松树蜂之间存在严格的互利共生关系,其正常生长发育是松树蜂完成生活史的关键因子之一。为研究该共生菌生长发育的相关机制,我们对共生菌菌丝最大生长速率前期（7d）和后期（12d）样本进行转录组测序,在转录水平上分析差异表达基因在共生菌生长发育中的功能。结果显示,两个不同生长时期共有差异基因2 425个,其中在共生菌最大生长速率前期样本中上调的基因有946个,下调的有1 479个。Nr注释和GO功能富集分析结果表明,共生菌最大生长速率前期的差异表达基因主要与碳水化合物代谢、蛋白质合成以及水解酶活性相关。Pathway富集分析表明,差异表达基因显著富集在糖酵解/糖异生代谢通路上,并且这些基因在共生菌最大生长速率前期的样本中显著上调,表明其可能在网隙裂粉韧革菌的生长发育中发挥重要作用。通过分析两个不同生长时期共生菌基因的表达情况,挖掘参与共生菌生长发育的关键基因,旨在为探索松树蜂与其共生菌的互利共生机制提供理论基础。     

危害松树的小蠹虫与其伴生菌的相互关系

危害健康松属植物的小蠹虫经常与一些特殊的真菌相联系。在小蠹虫危害松属植物的过程中,这些真菌被小蠹虫的一些特殊结构或者体表携带到松属植物上。小蠹虫与其伴生菌的联系表明小蠹虫和其伴生菌之间是一种互惠互利的关系。伴生菌随小蠹虫扩散而被带到新的寄主树木。而伴生菌或作为小蠹虫的食物来源,但更重要的是,有些伴生菌能够通过其菌丝渗透寄主组织,释放毒素,致死寄主树木,以帮助小蠹虫降低寄主抗性。许多研究致力于探索小蠹虫/伴生菌联合体与寄主树木之间关系的特征和确定小蠹虫与其伴生菌相互关系在生态学上的意义。然而,不同小蠹虫和其伴生菌所组成的共生体系,不同小蠹虫的种群数量,和不同环境条件下同种小蠹虫与其伴生菌相互作用方式的差异使我们在研究小蠹虫和其伴生菌这个共生体系时,对它们各自在成功聚集寄主树木过程中所发挥的重要作用的概括变得非常困难。     

昆虫内共生菌在昆虫防御中的作用

共生菌在昆虫体内的生长、繁殖过程中起着十分重要的作用,如营养功能、解毒作用、调控生殖及与寄主适应性等等。近些年,一些学者提出了共生菌在寄主抵御天敌等逆境的胁迫时也具有重要的作用。其在寄主防御中的作用可分为3类:(1)昆虫体内共生菌能产生具有抗真菌作用的代谢物,能够抵御病原真菌的入侵,提高寄主的适应性。(2)昆虫抵御寄生蜂的作用与其体内的共生细菌密切相关。(3)共生菌也能产生有毒物质保护寄主免于被捕食。昆虫共生菌在寄主防御中的作用机制除了产生抗菌物质和聚酮类等有毒物质外,还可能通过与水平传播的寄生物(horizontal transmission parasites,HTPs)竞争寄主资源来直接保护寄主。对共生菌的作用的深入研究将有可能进一步明确其功能,并加以利用。     

褐飞虱体内酵母类共生菌的形态观察

试验采用冷冻切片技术结合显微摄像系统观察研究了褐飞虱雌成虫体内酵母类共生菌的形态特性和在寄主虫体内的存在状态.光学显微镜观察证实,褐飞虱雌成虫的头部和胸部均未观察到共生菌,在其腹部脂肪体中却发现大量酵母类共生菌的存在,且有大量的菌胞出现.菌胞已经成为褐飞虱特有的细胞器,其发育可划分为发育初期、共生菌增殖适应期、对数增殖期(高峰期)、释放期和衰竭期5个阶段.按照形态划分,褐飞虱体内的酵母类共生菌有4大类型,即长梭形、杆状、卵形和球状.其中,长梭形和杆状个体最多、占绝大多数,卵形和球状个体很少见.此外,还发现具假膈梭形和不规则共生菌的存在.同时观察到该类共生菌通过多边芽殖进行无性繁殖,且以顶端芽殖(包括两端芽殖)为主.     

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。     

Putative origin of clonal lineages of Amylostereum areolatum, the fungal symbiont associated with Sirex noctilio, retrieved from Pinus sylvestris, in eastern Canada

The Eurasian Sirex noctilio-Amylostereum areolatum complex was discovered and has become established close to the North American Great Lakes in the 2000s. This invasive forest insect pest represents a very high risk to native and exotic pines in North America. We investigated the geographical origin of clonal lineages of the fungal symbiont A. areolatum in the recently pest-colonized eastern Canadian region by analyzing mitochondrial and nuclear sequence variations and comparing the genetic diversity of a worldwide collection of fungal symbionts among six countries where the Sirex complex is native and four countries from which the insect-fungal complex has been introduced. In total, 102 isolates were analyzed. While 12 multilocus genotypes (MLGs) are observed in the areas where S. noctilio is native, only two MLGs are retrieved from areas where S. noctilio is not native, indicating the wide spread of clonal lineages in the introduced fungal symbiont of S. noctilio. MLG2 comprises 26% of the Canadian isolates and is also observed in Chile and South Africa, where the insect-fungal complex has also been introduced. MLG3 comprises 74% of the Canadian isolates and is also observed in the USA, but nowhere else in our worldwide collection. Thus, at least one of the Canadian clonal lineages shares a common origin with A.?areolatum isolates from the Southern Hemisphere. The source of the second clonal lineage is still unknown, but phylogenetic analyses show that MLG3 is isolated. More extended sampling is necessary to determine the origin of this fungal clonal lineage and investigate its probable symbiotic association with native North American Sirex.     

A comparative study of the phytotoxic effects of siricid woodwasps on conifers

The effects of siricid mucus and the symbiotic fungus injected artificially, or by oviposition of caged females on young conifer trees were investigated in a comparative study of the phytotoxicity caused by seven species of siricid woodwasps. Dilute solutions of mucus were bioassayed using detached shoots. Rapid physiological changes in the radial growth of the stem, quantity of starch in the leaves, leaf pressure and colour of the foliage of living trees were induced by only one species, Sirex noctilio. Solutions of S. noctilio and S. cyaneus mucus caused premature senescence of needles on detached shoots. The other species, Sirex juvencus, Urocerus gigas, U. augur, U. sah and Xeris spectrum , produced no phytotoxic symptoms in living trees and less rapid senescence of needles on detached shoots. These differences are discussed in relation to the ecology of siricids in the northern hemisphere and the successful establishment of S. noctilio in pine plantations of Australasia.     

DNA sequence and RFLP data reflect geographical spread and relationships of Amylostereum areolatum and its insect vectors

The white rot fungus, Amylostereum areolatum (Basidiomycetes), is best known for its symbiotic relationship with various siricid wood wasp species. In this study, the relationship between isolates of A. areolatum associated with two wood wasp species, Sirex noctilio and S. juvencus, are considered to identify possible intraspecific groups. Isolates from the northern (native) and southern (exotic) hemispheres are included to determine patterns of geographical spread and origin of introductions into the southern hemisphere. The phylogenetic relationships of these isolates to authentic isolates of A. chailletii, A. laevigatum and A. ferreum were also investigated. Sequence and restriction fragment length polymorphism (RFLP) analyses of the variable nuc-IGS-rDNA region provided markers to distinguish intraspecific groups within A. areolatum. Isolates of A. areolatum associated with S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These sequences occurred in one of five combinations in each isolate. Some of these sequences were unique to isolates of A. areolatum from either wasp species, while others were present in both groups. This shows the ancient and specialized evolutionary relationship that exists between these insects and fungi. Isolates from the southern hemisphere all share the same sequence group. This supports previous hypotheses that S. noctilio has spread between countries and continents of this region. At the interspecific level, the IGS-rDNA sequence analysis showed that A. ferreum and A. laevigatum are closely related to each other, and they in turn are related to A. chailletii. Amylostereum areolatum was the most distinctly defined species in the genus. This can be attributed to the obligate relationship between A. areolatum and its insect vectors. Polymerase chain reaction-RFLP analysis was also shown to be an effective tool to distinguish between the different species of Amylostereum.     

Behavioural response of the woodwasp Sirex noctilio to volatile emissions of its fungal symbiont

The wood‐boring wasp, Sirex noctilio, is a global invasive pest that infects and kills pine trees by inoculating spores of a symbiotic fungus (Amylostereum areolatum) at oviposition. Wasp larvae depend on fungal growth to feed, while the fungus relies on female wasps to initially condition the pine tree by inoculating a phytotoxic venom and for dispersal. Wasp larvae use the fungus as an external gut for the digestion of lignocellulosic compounds resulting in a strong correlation between fungal growth inside the wood and wasp fitness. This study explores the hypothesis that female wasps will use fungal volatiles as a synomone in the process of locating suitable oviposition areas (i.e. trees). Using a Y‐tube olfactometer, adult female wasp behaviour was assessed towards fungal and pine tree volatiles (i.e. positive control). Our results are the first to demonstrate attraction of female S. noctilio towards volatiles of their fungal symbiont. Furthermore, the positive response towards these volatiles seems to be stronger than the response to a known attractant (i.e. pine volatiles). These results could be an important contribution to improving baits for monitoring and control purposes. Further work is needed, mainly oriented towards the identification of the volatiles that trigger the observed attraction response and their possible synergistic effects with tree volatiles.     

Natural enemies associated with Sirex noctilio (Hymenoptera: Siricidae) and S. nigricornis in Ontario, Canada

Sirex noctilio F. is an exotic woodwasp now found in eastern North America where it shares natural enemies with native woodwasps of Pinus spp. To study the extent to which native hymenopteran parasitoids and parasitic nematodes could affect woodwasp populations, 60 Pinus trees with symptoms of S. noctilio attack were felled in 2007 and 2008 in Ontario, Canada. Each tree bole was cut into 1-m sections that were placed in individual rearing tubes; emergence was monitored from May to November of the year of felling. Female S. noctilio were dissected to assess parasitism by the nematode Deladenus siricidicola Bedding. Two species of Siricidae emerged from these trees; S. noctilio, which accounted for most of the specimens collected, and S. nigricornis F. Of the three species of parasitoid that emerged, Ibalia leucospoides (Hochenwarth) was the most abundant, accounting for an overall hypothetical Siricidae parasitism rate of almost 20%. This parasitoid emerged over a similar time period as S. noctilio-between early July and early September. Except in trees >15 m in height, parasitism by I. leucospoides generally appeared uniform throughout the bole. Parasitism rates did not vary between the 2 yr, but did between sites in 1 yr. Parasitic nematodes were found in the haemocoel of about one third of S. noctilio females dissected but were never found sterilizing the eggs; none were found in S. noctilio emerging from P. resinosa. These findings suggest that I. leucospoides is currently the primary invertebrate natural enemy of S. noctilio in Ontario.     

STUDIES ON THE BIOLOGY OF RHYSSA PERSUASORIA (L.) (HYMENOPTERA: ICHNEUMONIDAE) INCORPORATING AN X-RAY TECHNIQUE

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Development of the immature stages of Rhyssa persuasoria (L.) on the larvae, prepupae and pupae of Sirex noctilio F. was followed in situ in Pinus radiata D. Don. by an X-ray technique.
R. persuasoria females were allowed to oviposit in wood in which the location of immature stages of S. noctilio had been determined radiographically. By this means it was found that they could not locate their hosts with certainty. When a host was located the female parasite paralysed it by stinging and laid an egg upon it. The host was consumed within five weeks and the parasite passed through four larval instars before pupation.     

Within-tree distributions of the Sirex noctilio Fabricius (Hymenoptera: Siricidae)--parasitoid complex and development of an optimal sampling scheme

Little is known about the introduced European woodwasp, Sirex noctilio F. (Hymenoptera: Siricidae), and its hymenopteran parasitoid complex in North America. To assess within-tree and landscape-level densities and distributions of these insects, and develop a more cost effective approach for sampling and monitoring S. noctilio, 18 infested pines (12 P. resinosa Aiton and six P. sylvestris L.) were felled from six stands within three regions in central New York and cut into 0.5-m bolts. Bolts were carefully split to recover all siricids (S. noctilio and native siricids) and parasitoids. In total, 2,558 siricids were recovered; 1,972 siricid larvae and 586 teneral adult S. noctilio. Parasitism of siricids, a majority of which were S. noctilio based on larval rearing results, was 16.4% with Ibalia leucospoides ensiger Norton causing 10.7% of the documented mortality. Numbers of siricids and parasitoids declined 33-86% from the northern to the southern sampling regions, peak insect densities occurred in sections of the bole 15-19 cm in diameter and numbers of insects were generally higher in P. sylvestris than P. resinosa according to the highest ranked zero-inflated poisson and probit regression models. Bark thickness was not correlated with siricid or parasitoid species densities. We also describe sampling plans where as few as two 0.5-m samples from infested trees provided reliable within-tree insect densities.     

Reproductive strategy of a woodwasp with no fungal symbionts, Xeris spectrum (Hymenoptera: Siricidae)

Experiments were conducted to elucidate the reproductive strategy of the siricid woodwasp, Xeris spectrum, which carries no substantial symbiotic fungi in its body, in a comparison with the life cycles of two fungus-carrying siricid woodwasp species, Sirex nitobei and Urocerus japonicus, by considering ecological traits such as seasonal patterns of occurrence, spatial distribution of emergence on a tree, and oviposition activities. Part of the X. spectrum populations emerged in spring, during May and June, while others emerged in summer, during August and September, simultaneously with other siricid fungus-carrying woodwasps. The vertical distribution pattern of X. spectrum emergence holes on the trunk closely coincided with the emergence hole pattern of S. nitobei. X. spectrum laid few eggs on fresh logs, old logs, or on logs inoculated with potato dextrose agar, whereas on logs inoculated with Amylostereum chailletii or A. areolatum, X. spectrum females oviposited no less than 30%, on average, of their potential eggs. Moreover, the oviposition sites on these logs were concentrated near the Amylostereum inoculation positions. These results indicate that X. spectrum has evolved a life history that utilizes fungal symbionts of other woodwasp species without itself possessing any symbiotic fungus. Moreover, X. spectrum has evolved a dual reproductive system in that (1) some adults emerge in summer during the same emergence period as the fungus-carrying woodwasps and thereby oviposit on host trees already inoculated with fungi, and (2) other adults emerge the next spring and oviposit on trees that were inoculated with fungi a year earlier. Received: 16 January 1997 / Accepted: 14 July 1997     

Chemical information from the fungus Amylostereum areolatum and host-foraging behaviour in the parasitoid Ibalia leucospoides

Abstract.  Parasitoids locate hosts using reliable and predictable cues such as smells derived from host plants or from the hosts themselves. For host species that live with mutualistic organisms, such as several wood boring insects, cues derived from the symbionts are likely to be exploited by specific parasitoids. Through a set of bioassays, the behaviour of the parasitoid Ibalia leucospoides Hochenwarth (Hymenoptera: Ibaliidae) is studied in response to the fungus Amylostereum areolatum Boidin (Basidiomycotina: Corticiaceae), a symbiont of its host, the wood wasp Sirex noctilio Fabricius (Hymenoptera: Siricidae). The results show that parasitoids are attracted to the fungus when growing naturally within pine logs, and also when growing on an artificial medium. Fungal volatiles also elicit increased parasitoid activity and may provide information on relative densities of hosts available for parasitization. It is speculated that the the chemical information derived from the host fungal symbiont comprises reliable and detectable host-locating cues used by parasitoids to search for concealed hosts.     

松树蜂毒腺转录组分析

【目的】构建入侵种松树蜂Sirex noctilio毒腺转录组数据库,筛选并分析松树蜂毒腺基因数据。【方法】采用新一代高通量测序平台Illumina HiSeq^TM 4000对松树蜂雌成虫毒腺进行转录组测序、数据组装和生物信息学分析。【结果】共获得12.7 Gb松树蜂雌成虫毒腺有效转录组数据,并组装到37 098条unigenes,平均长度968 bp,N50长度为2 364 bp。将所得的unigenes数据使用BlastX与各大数据库比对,共注释到13 515条unigenes,并且在NR数据库中注释的unigenes最多,共11 108条(占总数的29.94%),其中相似基因占比最高的物种为丽蝇蛹集金小蜂Nasonia vitripennis,达815条(占总数的7.29%)。在GO数据库中注释到5 726条unigenes,根据功能被分为生物学进程、细胞组分和分子功能3大类63个亚类。KEGG代谢通路分析表明,7 602条unigenes注释到357个代谢通路。根据基因注释信息进一步筛选到43条嗅觉相关基因,包括嗅觉受体(odorant receptor, Or)基因25条、化学感受蛋白(chemosensory protein, CSP)基因10条、离子型受体(ionotropic receptor, IR)基因5条和气味结合蛋白(odorant binding protein, OBP)基因3条。此外,还筛选出11条漆酶基因,包括漆酶1(laccase1, LAC1)基因5条、漆酶2(laccase2, LAC2)基因4条、漆酶4(laccase4, LAC4)基因1条和漆酶9(laccase9, LAC9)基因1条,且其中1条LAC2基因在所有被注释的基因中表达量最高(FPKM值=21 126)。【结论】本研究获得的松树蜂毒腺转录组数据为松树蜂毒液组分的鉴定和生物学功能的研究奠定了一定的理论基础。