蝗虫微孢子虫的生产及田间应用现状

蝗虫微孢子虫是国际上商品化的微孢子虫杀虫剂,在对蝗虫的生物防治方面具有重要作用。介绍了关于蝗虫微孢子虫的基础理论知识、大规模生产技术及田间应用现状,同时对利用蝗虫微孢子虫进行害虫生物防治提出了一些有益的建议。     

昆虫的微孢子虫病

介绍了昆虫微孢子虫病的病理学、传播途径、寄主特异性、病害管理以及利用昆虫微孢子虫作为生物杀虫剂的应用等方面的研究进展 ,以期为益虫微孢子虫病害的防治和昆虫微孢子虫杀虫剂的研制提参考。     

蝗虫微孢子虫及其在蝗害治理中的作用

扼要阐述了蝗虫微孢子虫的生活史、形态特征及其防治蝗虫的原理和方法。指出,用蝗虫微孢子虫治蝗具有显著的社会、经济及生态效益。     

蝗虫微孢子虫及其在蝗害治疗中的作用

扼要阐述了蝗虫微孢子虫的生活史,形态特征及其防治蝗虫的原理和方法。指出用蝗虫微孢子虫治蝗具有显著的社会,经济及生态效益。     

梨花迁粉蝶微孢子虫对家蚕的侵染力与胚传性

【目的】对梨花迁粉蝶Catopsilia pyranthe分离的微孢子虫进行形态与分子鉴定,探究其对非天然宿主家蚕Bombyx mori的侵染力与胚传性。【方法】从田间采集的梨花迁粉蝶中分离得到梨花迁粉蝶微孢子虫液,测定其孢子的形态、大小、体积、长短轴比,同时对该孢子虫的16S r DNA进行PCR克隆测序与分析。将梨花迁粉蝶微孢子虫Nosema sp.CP与家蚕微孢子虫N.bombycis分别对2龄起蚕、4龄起蚕进行添食感染比对,测定家蚕食下两种微孢子虫的感染率和胚种传染能力。【结果】本研究分离的梨花迁粉蝶微孢子虫形态为长椭圆形,具双核;其16S r DNA序列与已报道的梨花迁粉蝶微孢子虫的序列一致性大于99%,为梨花迁粉蝶微孢子虫。梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕综合感染率分别是68.8%和98.3%;在继代蚁蚕中,感染梨花迁粉蝶微孢子虫和家蚕微孢子虫的雌蛾所产蚕卵次代蚁蚕检出有孢子虫的检出率分别为100%和100%,卵壳的孢子虫的检出率分别为92.9%和100%;梨花迁粉蝶微孢子虫和家蚕微孢子虫对家蚕的胚种传染力分别为9.6%和23.2%。【结论】本研究分离得到的微孢子虫为梨花迁粉蝶微孢子虫,具有微孢子虫Nosema属的典型特征。梨花迁粉蝶微孢子虫能感染危害家蚕,也具有家蚕胚种传染性,但感染率和胚传率均明显低于家蚕微孢子虫,是蚕业生产中必须防控的对象。     

一株家蚕异型微孢子虫感染特征和分类地位

【背景】家蚕微粒子病是一种对蚕业生产危害巨大的蚕病,该病病原是蚕种生产唯一检疫对象,而家蚕病原性微孢子虫种类多、来源复杂,给蚕种生产微粒子病的防控增加了难度。【目的】研究一株从蚕种检疫样品中分离的微孢子虫(命名为GXM15)的致病性和分类地位,鉴定并分析其来源,完善家蚕病原性微孢子虫分类和数据库,为蚕种生产控制家蚕微粒子病提供参考依据。【方法】采用生物试验方法测定GXM15微孢子虫对家蚕的半数感染浓度(IC50)和胚种传染率;显微镜观察GXM15微孢子虫孢子形态,利用透射电子显微镜观察GXM15微孢子虫的超微结构;采用PCR扩增、T克隆和测序获得GXM15微孢子虫的SSU rRNA基因和ITS片段DNA序列,并利用MEGA 5.0和DNAstar软件构建GXM15微孢子虫的系统发育树和遗传距离分析。【结果】GXM15微孢子虫对家蚕的IC50为8.29×104个/mL,是家蚕微孢子虫(Nosema bombycis,Nb)的2.28倍;GXM15微孢子虫对家蚕的胚种传染率为3.6%,明显低于Nb;GXM15微孢子虫形态呈短卵圆形,大小为(2.05±0.20)× (3.25±0.30) μm,GXM15微孢子虫体积是Nb微孢子虫的2.19倍;GXM15微孢子虫超微结构具双核,极丝13圈,极丝倾斜角约45°,符合Nosema属的特征;GXM15微孢子虫SSU rRNA基因在系统发育树中位于Nosema属分支中,遗传距离分析表明GXM15微孢子虫与Nb同属异种,是一株新微孢子虫。【结论】GXM15微孢子虫是一株家蚕病原性微孢子虫,根据GXM15微孢子虫致病性和分类地位研究,可以为蚕种生产防控家蚕微粒子病提供参考依据。     

鳞翅目昆虫病原微孢子虫研究进展

微孢子虫广泛存在于鳞翅目昆虫中,是一类重要的病原微生物。微孢子虫病一方面影响野外昆虫种群的自然平衡,另一方面对家蚕、柞蚕等经济和资源昆虫造成了严重的危害。微孢子虫分子生物学研究基础相对薄弱,再加上微孢子虫表面坚厚的孢壁,无疑增加了研究难度。随着核酸、蛋白质等生物大分子分离制备方法和高通量测序技术的不断更新发展,基于各种组学(Omics)研究微孢子虫的工作方兴未艾,并且有了一些重要的发现。本文综述了微孢子虫与鳞翅目昆虫寄主的相互作用及寄生于鳞翅目昆虫的病原微孢子虫基因组、转录组和蛋白质组进展情况,以期为微孢子虫的深入研究提供参考。这些昆虫微生物研究将为鳞翅目害虫生物防治提供新的思路,并对家蚕等经济昆虫微粒子病的诊断、防控及治疗产生积极影响。     

家蚕微孢子虫极管蛋白1 (NbPTP1) 在果蝇S2细胞中的表达与糖基化修饰

极管蛋白(Polar tube protein)是极管的主要成分,能特异性定位于微孢子虫极管,在微孢子虫侵染宿主过程中发挥重要作用。文中分析了家蚕微孢子虫极管蛋白1中潜在的O-、N-糖基化修饰位点,克隆了家蚕微孢子虫极管蛋白1全基因序列,并将其插入带有V5和His标签的真核表达载体pMT/Bip/V5-His A中,成功构建了pMT/Bip/V5-His A-NbPTP1重组质粒,经转染果蝇S2细胞后,发现NbPTP1基因能在果蝇细胞中高效表达。此外,Lectin blotting和β-消除反应分析结果表明:果蝇S2细胞内表达的NbPTP1具有O-糖基化修饰特征。以上结果为研究NbPTP1的糖基化修饰特征与其功能之间的关系提供了基础,有助于揭示微孢子虫侵染机制,建立可行有效的微孢子虫病诊断和防治措施。     

两株不同地域的家蚕微孢子虫分离株的细胞侵染力 比较及遗传多样性分析

家蚕微孢子虫(Nosema bombycis)是蚕业生产上一种毁灭性病害—— 微粒子病的病原体。文章将安徽和重庆两地域来源的病原性家蚕微孢子虫分离株进行草地贪夜蛾Sf9细胞感染性检测, 结果显示二者对细胞的侵染能力存在显著差异。为进一步探讨不同家蚕微孢子虫分离株的种群多态性, 进行了孢子虫核糖体DNA序列的测定和系统聚类比较, 结果表明SSU rDNA(Small subunit ribosomal DNA)和ITS(Internal transcribed spacer)在不同地域的种群差异性并不显著。通过检索重庆株全基因组数据及其他地域株rDNA序列, 显示在家蚕微孢子虫rDNA元件的部分SSU rDNA结构复制子中存在MITE-like转座元件的插入, 表明家蚕微孢子虫rDNA结构的多样性。     

微孢子虫的进化起源与系统分类现状

系统回顾了微孢子虫起源进化的研究进展及其系统分类现状。近30年来,一些依据SSU r DNA序列、翻译延伸因子1α序列、直系同源蛋白基因树及真菌蛋白质组生命树等的研究支持微孢子虫起源于原生生物。但同时大量的单基因或多基因系统发育研究又支持微孢子虫起源于真菌。最近一系列独立的系统基因组学研究结果,加上在罗兹菌门中发现了介于真菌和微孢子虫中间类型的近微孢虫菌属(Paramicrosporidium)、线孢虫菌属(Mitosporidium)和噬核菌属(Nucleophaga),进一步支持微孢子虫起源于罗兹菌门的噬核菌属谱系。     

Temporal variations of Microsporidia diversity and discovery of new host–parasite interactions in a lake ecosystem

Microsporidia are a large group of obligate intracellular eukaryotic parasites related to Fungi. Recent studies suggest that their diversity has been greatly underestimated and little is known about their hosts other than metazoans, and thus about their impact on the communities at the base of the food web. In this work, we therefore studied the diversity of Microsporidia over one year and identified potential new hosts in small-sized fractions (<150 μm) in a lake ecosystem using a metabarcoding approach coupled with co-occurrence networks and tyramide signal amplification-fluorescent in situ hybridization. Our analysis shows a great Microsporidia diversity (1 472 OTUs), with an important part of this diversity being unknown. Temporal variations of this diversity have been observed, which might follow temporal variations of their potential hosts such as protists and microzooplankton. New hosts among them were identified as well as associations with phytoplankton. Indeed, repeated infections were observed in Kellicottia (rotifers) with a prevalence of 38% (infected individuals). Microsporidia inside a Stentor (ciliate) were also observed. Finally, potential infections of the diatom Asterionella were identified (prevalence <0.1%). The microsporidian host spectrum could be therefore even more important than previously described, and their role in the functioning of lake ecosystems is undoubtedly largely unknown.     

Microsporidian Diversity in Soil,Sand, and Compost of the Pacific Northwest

Microsporidia are intracellular parasites considered to be ubiquitous in the environment. Yet the true extent of their diversity in soils, sand, and compost remains unclear. We examined microsporidian diversity found in the common urban environments of soil, sand, and compost. We retrieved 22 novel microsporidian sequences and only four from described species. Their distribution was generally restricted to a single site and sample type. Surprisingly, one novel microsporidian showed a wide distribution, and high prevalence, as it was detected in five different compost samples and in soil samples collected over 200 km apart. These results suggest that the majority of Microsporidia appear to have a narrow distribution. Our phylogenetic analysis indicated that the Microsporidia detected in this study include representatives from four of the five major microsporidian groups. Furthermore, the addition of our new sequences calls into question the cohesiveness of microsporidian clade II. These results highlight the importance of increasing our knowledge of microsporidian diversity to better understand the phylogenetic relationships and evolutionary history of this important group of emerging parasites.     

A phylogenetic framework to investigate the microsporidian communities through metabarcoding and its application to lake ecosystems

Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of ‘universal’ primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high-throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on < 150 μm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within seven clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio-temporal variations of their potential hosts such as microeukaryotes and metazooplankton.     

Diseases of Mites

An overview is given of studies on diseases of mites. Knowledge of diseases of mites is still fragmentary but in recent years more attention has been paid to acaropathogens, often because of the economic importance of many mite species. Most research on mite pathogens concerns studies on fungal pathogens of eriophyoids and spider mites especially. These fungi often play an important role in the regulation of natural mite populations and are sometimes able to decimate populations of phytophagous mites. Studies are being conducted to develop some of these fungi as commercial acaricides.Virus diseases are known in only a few mites, namely, the citrus red mite and the European red mite. In both cases, non-occluded viruses play an important role in the regulation of mite populations in citrus and peach orchards, respectively, but application of these viruses as biological control agents does not seem feasible. A putative iridovirus has been observed in association with Varroa mites in moribund honeybee colonies. The virus is probably also pathogenic for honeybees and may be transmitted to them through this parasitic mite.Few bacteria have been reported as pathogens of the Acari but in recent years research has been concentrated on intracellular organisms such as Wolbachia that may cause distorted sex ratios in offspring and incompatibility between populations. The role of these organisms in natural populations of spider mites is in particular discussed. The effect of Bacillus thuringiensis on mites is also treated in this review, although its mode of action in arthropods is mainly due to the presence of toxins and it is, therefore, not considered to be a pathogen in the true sense of the word.Microsporidia have been observed in several mite species especially in oribatid mites, although other groups of mites may also be affected. In recent years, Microsporidia infections in Phytoseiidae have received considerable attention, as they are often found in mass rearings of beneficial arthropods. They affect the efficacy of these predators as biological control agent of insect and mite pests. Microsporidia do not seem to have potential for biological control of mites.     

微孢子虫系统进化研究的变迁与展望

微孢子虫(Microsporidia)是一类专性细胞内寄生的单细胞真核生物,在科研、医疗、农业、商业等领域具有重要影响。由于其不具有某些典型的真核生物细胞结构,如线粒体、过氧化物酶体、高尔基体、鞭毛,曾将其归属于古真核生物谱系,认为其进化历程先于这些细胞器的起源,该假说也得到了一些生物化学和分子生物学研究证据的支持。然而,在最近十年里,通过更深入的研究,尤其是基于分子序列的系统进化分析,表明微孢子虫和真菌具有一定亲缘关系,并认为其结构的简约性恰好体现了微孢子虫营寄生生活的高度退化现象。目前对微孢子虫的系统进化仍存在各种不同意见,对其进化研究历史进行探讨有着重要意义。本文将按照时间顺序回顾微孢子虫进化分类研究过程中的各种研究成果,并讨论为什么微孢子虫独特的细胞和基因组特性会导致众多的学者在其进化分类问题上争执这么久。     

Unique physiology of host–parasite interactions in microsporidia infections

Microsporidia are intracellular parasites of all major animal lineages and have a described diversity of over 1200 species and an actual diversity that is estimated to be much higher. They are important pathogens of mammals, and are now one of the most common infections among immunocompromised humans. Although related to fungi, microsporidia are atypical in genomic biology, cell structure and infection mechanism. Host cell infection involves the rapid expulsion of a polar tube from a dormant spore to pierce the host cell membrane and allow the direct transfer of the spore contents into the host cell cytoplasm. This intimate relationship between parasite and host is unique. It allows the microsporidia to be highly exploitative of the host cell environment and cause such diverse effects as the induction of hypertrophied cells to harbour prolific spore development, host sex ratio distortion and host cell organelle and microtubule reorganization. Genome sequencing has revealed that microsporidia have achieved this high level of parasite sophistication with radically reduced proteomes and with many typical eukaryotic pathways pared-down to what appear to be minimal functional units. These traits make microsporidia intriguing model systems for understanding the extremes of reductive parasite evolution and host cell manipulation.     

Microsporidia: a journey through radical taxonomical revisions

Microsporidia are obligate intracellular parasites of medical and commercial importance, characterized by a severe reduction, or even absence, of cellular components typical of eukaryotes such as mitochondria, Golgi apparatus and flagella. This simplistic cellular organization has made it difficult to infer the evolutionary relationship of Microsporidia to other eukaryotes, because they lack many characters historically used to make such comparisons. Eventually, it was suggested that this simplicity might be due to Microsporidia representing a very early eukaryotic lineage that evolved prior to the origin of many typically eukaryotic features, in particular the mitochondrion. This hypothesis was supported by the first biochemical and molecular studies of the group. In the last decade, however, contrasting evidence has emerged, mostly from molecular sequences, that show Microsporidia are related to fungi, and it is now widely acknowledged that features previously recognized as primitive are instead highly derived adaptations to their obligate parasitic lifestyle. The various sharply differing views on microsporidian evolution resulted in several radical reappraisals of their taxonomy. Here we will chronologically review the causes and consequences for these taxonomic revisions, with a special emphasis on why the unique cellular and genomic features of Microsporidia lured scientists towards the wrong direction for so long.     

Evidence from small-subunit ribosomal RNA sequences for a fungal origin of Microsporidia

The phylum Microsporidia comprises a species-rich group of minute, single-celled, and intra-cellular parasites. Lacking normal mitochondria and with unique cytology, microsporidians have sometimes been thought to be a lineage of ancient eukaryotes. Although phylogenetic analyses using small-subunit ribosomal RNA (SSU-rRNA) genes almost invariably place the Microsporidia among the earliest branches on the eukaryotic tree, many other molecules suggest instead a relationship with fungi. Using maximum likelihood methods and a diverse SSU-rRNA data set, we have re-evaluated the phylogenetic affiliations of Microsporidia. We demonstrate that tree topologies used to estimate likelihood model parameters can materially affect phylogenetic searches. We present a procedure for reducing this bias: "tree-based site partitioning," in which a comprehensive set of alternative topologies is used to estimate sequence data partitions based on inferred evolutionary rates. This hypothesis-driven approach appears to be capable of utilizing phylogenetic information that is not available to standard likelihood implementations (e.g., approximation to a gamma distribution); we have employed it in maximum likelihood and Bayesian analysis. Applying our method to a phylogenetically diverse SSU-rRNA data set revealed that the early diverging ("deep") placement of Microsporidia typically found in SSU-rRNA trees is no better than a fungal placement, and that the likeliest placement of Microsporidia among non-long-branch eukaryotic taxa is actually within fungi. These results illustrate the importance of hypothesis testing in parameter estimation, provide a way to address certain problems in difficult data sets, and support a fungal origin for the Microsporidia.     

Pervasiveness of parasites in pollinators

Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees) in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris) and a third of wasps (Vespula vulgaris), as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.