昆虫病原线虫资源概况和分类技术进展

昆虫病原线虫是具有重要潜在应用价值的害虫生物防治资源,主要包括斯氏线虫科（Steinernematidae）的斯氏线虫属Steinernema与新斯氏线虫属Neosteinernema线虫和异小杆线虫科（Heterorhabditidae）的异小杆线虫属Heterorhabditis线虫。近10年来,分子生物学方法与传统的形态学方法相结合应用到线虫的鉴定与分类,昆虫病原线虫的分类进入稳定与发展时期,越来越多的新种或品系被发现及应用于生物防治。目前已描述的昆虫病原线虫种类达65种,其中斯氏线虫属52种,新斯氏线虫属1种,异小杆线虫属12种。本文整理列出了迄今报道的昆虫病原线虫种类及其来源,并综述了昆虫病原线虫分类现状以及鉴定与分类方法上的研究进展,重点阐述了分子生物学技术在昆虫病原线虫鉴定与分类的应用状况。     

Species confirmation of fungal isolates by molecular analysis

Traditional taxonomy of hyphomycetes has been based on conidial morphology and development. In order to confirm species level for the detection and identification of the entomopathogenic fungus, we analysed the species-specific fingerprints to investigate molecular characteristics within isolates of six species and to resolve morphologically atypical isolates. The extent of fingerprint profile observed by RAPD was sufficient to confirm the species level of all the isolates. The genetic similarity among morphologically identified isolates of each species was considerably higher, allowing us to conclude that all the isolates are of same species. These results establish a molecular framework for further taxonomic, phylogenetic and comparative biological investigations.     

Marine Nematode Taxonomy in Africa: Promising Prospects Against Scarcity of Information

From the late 19th century, Africa has faced heavy exploitation of its natural resources with increasing land/water pollution, and several described species have already become extinct or close to extinction. This could also be the case for marine nematodes, which are the most abundant and diverse benthic group in marine sediments, and play major roles in ecosystem functioning. Compared to Europe and North America, only a handful of investigations on marine nematodes have been conducted to date in Africa. This is due to the scarcity of experienced taxonomists, absence of identification guides, as well as local appropriate infrastructures. A pivotal project has started recently between nematologists from Africa (Tunisia), India, and Europe (Italy) to promote taxonomic study and biodiversity estimation of marine nematodes in the African continent. To do this, as a first step, collection of permanent slides of marine nematodes (235 nominal species and 14 new to science but not yet described) was recently established at the Faculty of Sciences of Bizerte (Tunisia). Capacity building of next generation of African taxonomists have been carried out at level of both traditional and molecular taxonomy (DNA barcoding and next-generation sequencing [NGS]), but they need to be implemented. Indeed, the integration of these two approaches appears crucial to overcome lack of information on the taxonomy, ecology, and biodiversity of marine nematodes from African coastal waters.     

Parameters affecting plant defense pathway mediated recruitment of entomopathogenic nematodes

Entomopathogenic nematodes are natural enemies and effective biological control agents of subterranean insect herbivores. Interactions between herbivores, plants, and entomopathogenic nematodes are mediated by plant defense pathways. These pathways can induce release of volatiles and recruit entomopathogenic nematodes. Stimulation of these plant defense pathways for induced defense against belowground herbivory may enhance biological control in the field. Knowledge of the factors affecting entomopathogenic nematode behaviour belowground is needed to effectively implement such strategies. To that end, we explore the effect of elicitor, elicitor dose, mechanical damage, and entomopathogenic nematode release distance on recruitment of entomopathogenic nematode infective juveniles to corn seedlings. Increasing doses of methyl jasmonate and methyl salicylate elicitors recruited more entomopathogenic nematodes as did mechanical damage. Recruitment of entomopathogenic nematodes was higher at greater release distances. These results suggest entomopathogenic nematodes are highly tuned to plant status and present a strategy for enhancing biological control using elicitor-stimulated recruitment of entomopathogenic nematodes.     

Phylogenetics of the millipede genus Brachycybe Wood, 1864 (Diplopoda: Platydesmida: Andrognathidae): patterns of deep evolutionary history and recent speciation

The genus Brachycybe Wood is a little known group of millipedes comprising eight nominal species distributed throughout North America, Japan, South Korea, Taiwan, and China. The group's species are relatively morphologically homogenous and have been described primarily on the basis of differences in somatic morphology largely ignoring the often-used characters in millipede taxonomy and systematics - male genitalia (the gonopods). The objectives of this study were to survey male gonopods with the aim of evaluating inter-specific variation, assess existing species boundaries and phylogeny using molecular characters, examine the historical biogeography of the genus, and estimate the timing of lineage divergence using a molecular clock. We surveyed two mitochondrial genes (cytochrome c oxidase I and cytochrome b) and one nuclear protein-coding gene (glutamyl and prolyl-tRNA synthetase) each comprising 952, 746, and 555 aligned base pairs respectively. Phylogenetic inference coupled with an analysis of species delimitation using a generalized mixed Yule coalescent model recovered eight species, two of which were considered to be cryptic. Molecular dating analyses coupled with ancestral range reconstructions indicate that the group is quite ancient (age>50 million years) and its origins are likely traced back geographically to the mountains of California's Sierra Nevada and Coastal Ranges. The genus Brachycybe appears to have expanded its range at least twice out of present day California into eastern North America and at least once into Asia. This study highlights the need for integrative approaches to describe biodiversity and furthers the evidence for cryptic diversity even in groups where genitalia are generally thought to be rapidly evolving, diagnostic features.     

昆虫病原线虫对非生物胁迫的响应机制

昆虫病原线虫是农林害虫生物防治中重要的生防因子之一。它对非生物胁迫的耐受能力决定着线虫在田间的个体生存及控制害虫效果。线虫对环境胁迫的响应是一个整体性的复杂过程, 体现在群体遗传、发育阶段、生理生化和抗逆相关基因的表达调控等不同层次、不同水平上。本文综述了昆虫病原线虫抗逆相关领域的主要研究进展, 重点介绍线虫响应非生物胁迫的生理生化机制和相关抗性基因的分离鉴定, 并对该研究领域发展趋势进行了讨论和展望, 期望为我国研究线虫抗逆机理提供一些新的信息。     

Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.     

座壳孢属真菌的分类研究

对昆虫病原真菌座壳孢的功能、资源调查、经典分类发展历程及其传统分类存在的问题、以及应用现代分子生物学方法研究座壳孢的系统发育等进行了回顾和总结,旨在为澄清该属真菌的种类及其系统发育关系提供科学依据。     

Free-living nematodes research: State of the art,prospects, and future directions. A bibliometric analysis approach

Nematodes are among the most successful metazoans inhabiting the Earth and they are pivotal components as in terrestrial as in aquatic (both in marine and freshwater) environments providing important ecosystem services. The aim of this study was to understand major research trends and topics on free-living nematodes inhabiting soil, marine and freshwater environments and to highlight possible differences among them. To achieve this objective, a bibliometric analysis was performed using Scopus database. The indexed global scientific literature on free-living nematodes from 1912 to 2021 was explored using VOSviewer software, allowing a comprehensive overview of the topic. The analyses of co-authorship (among researchers and countries), the co-occurrence of keywords and the analysis of citation of journals were performed. Overall, free-living soil nematodes found a wider audience in high ranked journals especially when compared with freshwater nematodes. Marine nematodes stand in between them and many aspects of biodiversity research in marine ecosystems are covered by high-medium ranked journals (i.e. taxonomy, systematic, phylogeny, morphological and genetic diversity). Although, the estimation of the taxonomic diversity of the phylum Nematoda enumerated a high number of documents, an increasing attention emerged for the investigation of pollution effects (i.e. nematodes as bioindicators of environmental status) and the use of nematodes as model organisms for addressing scientific questions in line with the Eco-Evo-Devo (Ecological Evolutionary Developmental biology) approach. These fundamental themes were indirectly confirmed by the co-authorship analysis, which revealed that taking integrative approaches between taxonomy (both morphological and molecular), ecological and evolutionary aspects attracted a higher number of citations.     

PCR-Ribotyping of Xenorhabdus and Photorhabdus Isolates from the Caribbean Region in Relation to the Taxonomy and Geographic Distribution of Their Nematode Hosts

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.     

大数据时代土壤微生物地理学研究综述

土壤蕴含极为丰富的微生物多样性,它们在物质分解、元素生物地球化学循环、植物生产力和生物健康中扮演着关键角色。理解土壤微生物的生物地理分布格局、形成机制与群落构建规则,有助于预测在全球变化背景下土壤微生物组的功能演变及其对陆地生态系统的调控影响。自21世纪以来,土壤微生物生物地理学在各种大型国际微生物计划的推动下逐步形成了分子生物学技术耦合大数据分析的模式,实现了多种尺度上的关联研究。阐述了土壤微生物在分布格局和群落构建规则方面的研究进展,重点介绍了分子生物学技术和大数据分析在土壤微生物生物地理研究中的应用,对土壤微生物生物地理学未来在微生物分类分辨率、模型验证与构建和功能基因地理学的发展方向进行了展望。     

MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

Dispersal is fundamental to biogeography and the evolution of biodiversity on oceanic islands

Vicariance biogeography emerged several decades ago from the fusion of cladistics and plate tectonics, and quickly came to dominate historical biogeography. The field has since been largely constrained by the notion that only processes of vicariance and not dispersal offer testable patterns and refutable hypotheses, dispersal being a random process essentially adding only noise to a vicariant system. A consequence of this thinking seems to have been a focus on the biogeography of continents and continental islands, considering the biogeography of oceanic islands less worthy of scientific attention because, being dependent on stochastic dispersal, it was uninteresting. However, the importance of dispersal is increasingly being recognized, and here we stress its fundamental role in the generation of biodiversity on oceanic islands that have been created in situ , never connected to larger land masses. Historical dispersal patterns resulting in modern distributions, once considered unknowable, are now being revealed in many plant and animal taxa, in large part through the analysis of polymorphic molecular markers. We emphasize the profound evolutionary insights that oceanic island biodiversity has provided, and the fact that, although small in area, oceanic islands harbour disproportionately high biodiversity and numbers of endemic taxa. We further stress the importance of continuing research on mechanisms generating oceanic island biodiversity, especially detection of general, non-random patterns of dispersal, and hence the need to acknowledge oceanic dispersal as significant and worthy of research.     

七种我国昆虫病原线虫共生菌的分离与鉴定

昆虫病原线虫共生菌是一类具有广阔发展前景的生物防治资源,由于这类菌的分类鉴定工作起步晚,存在分类混乱和不系统的现象,在本土资源十分丰富的我国尤为突出.本文对本实验室分离的7个共生菌菌株进行了分类描述,建立了以生理形态,生化特征为分类基础,结合16 SrDNA序列分析的有效鉴定方法.     

七种我国昆虫病原线虫共生菌的分离与鉴定

昆虫病原线虫共生菌是一类具有广阔发展前景的生物防治资源, 由于这类菌的分类鉴定工作起步晚, 存在分类混乱和不系统的现象, 在本土资源十分丰富的我国尤为突出。本文对本实验室分离的7个共生菌菌株进行了分类描述, 建立了以生理形态, 生化特征为分类基础, 结合16S rDNA序列分析的有效鉴定方法。     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Shift from morphological to recent advanced molecular approaches for the identification of nematodes

Nematodes are the most diverse but most minor studied microorganisms found in soil, water, animals, or plants. Either beneficial or pathogenic, they significantly affect human and animal health, plant production and ultimately affect the environmental equilibrium. Knowledge of their taxonomy and biology are the main issues to answer the different challenges associated with these microorganisms. The classical morphology-based nematode taxonomy and biodiversity studies have proved insufficient to identify closely related taxa and have challenged most biologists. Several molecular approaches have been used to supplement morphological methods and solve these problems with markable success. The molecular techniques range from enzyme analysis, protein-based information to DNA sequence analysis. For several decades, efforts have been made to integrate molecular approaches with digital 3D image-capturing technology to improve the identification accuracy of such a taxonomically challenging group and communicate morphological data. This review presents various molecular techniques and provides examples of recent advances in these methods to identify free-living and plant-parasitic nematodes.     

First record of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in Costa Rica

A survey of entomopathogenic nematodes was conducted in the north Pacific (Guanacaste Conservation Area) and southeast Caribbean (Gandoca-Manzanillo Natural Refuge) regions of Costa Rica. Out of a total of 41 soil samples, 5 were positive for entomopathogenic nematodes (20.5%), with 3 (12.3%) containing Steinernema and 2 (8.2%) Heterorhabditis isolates. Morphological and molecular studies were undertaken to characterize these isolates. The Heterorhabditis isolates were identified as Heterorhabditis indica and the three Steinernema isolates were identified as two new undescribed species. H. indica was recovered from a coastal dry forest. Steinernema n. sp. 1 was isolated from a rainforest valley, between volcanoes. Steinernema sp. n. 2 was isolated from sand dunes in the Caribbean Coast (Punta Uva) near the rainforest strip along the coast. Although limited to two geographic regions, this study suggests entomopathogenic nematodes may be diverse and perhaps widely distributed in Costa Rica. A more intensive survey, covering all geographic regions is currently undergoing.