Phylogenetics and evolution of nematode-trapping fungi (Orbiliales) estimated from nuclear and protein coding genes

The systematic classification of nematode-trapping fungi is redefined based on phylogenies inferred from sequence analyses of 28S rDNA, 5.8S rDNA and beta-tubulin genes. Molecular data were analyzed with maximum parsimony, maximum likelihood and Bayesian analysis. An emended generic concept of nematode-trapping fungi is provided. Arthrobotrys is characterized by adhesive networks, Dactylellina by adhesive knobs, and Drechslerella by constricting-rings. Phylogenetic placement of taxa characterized by stalked adhesive knobs and non-constricting rings also is confirmed in Dactylellina. Species that produce unstalked adhesive knobs that grow out to form loops are transferred from Gamsylella to Dactylellina, and those that produce unstalked adhesive knobs that grow out to form networks are transferred from Gamsylella to Arthrobotrys. Gamsylella as currently circumscribed cannot be treated as a valid genus. A hypothesis for the evolution of trapping-devices is presented based on multiple gene data and morphological studies. Predatory and nonpredatory fungi appear to have been derived from nonpredatory members of Orbilia. The adhesive knob is considered to be the ancestral type of trapping device from which constricting rings and networks were derived via two pathways. In the first pathway adhesive knobs retained their adhesive material forming simple two-dimension networks, eventually forming complex three-dimension networks. In the second pathway adhesive knobs lost their adhesive materials, with their ends meeting to form nonconstricting rings and they in turn formed constricting rings with three inflated-cells.     

Ecology of nematophagous fungi: vertical distribution in a deciduous woodland

Summary The distribution of nematophagous fungi in soil collected from a deciduous woodland is compared to various biotic and abiotic soil factors. The microfungi are isolated at all depths down to a maximum of 35 cm. Predators forming constricting rings, adhesive branches and adhesive knobs are restricted to the upper litter and humus layers. The net forming predators and endoparasites are isolated at all depths, although they are significantly more abundant in the lower mineral rich soils. A much greater species diversity of nematophagous fungi is recorded in the upper organic zones.Preliminary soil analysis indicates thatCephalosporium balanoides is independent of all soil variables, while predators able to form traps spontaneously are restricted to the organic soils which are rich in nematodes. Non-spontaneous trap forming predators, which are excellent saprophytes, are isolated from the deeper soils which are low in nutrients. The ecological significance of these results is discussed.     

The C. elegans touch response facilitates escape from predacious fungi

Predator-prey interactions are vital determinants in the natural selection of behavioral traits. Gentle touch to the anterior half of the body of Caenorhabditis elegans elicits an escape response in which the animal quickly reverses and suppresses exploratory head movements [1, 2]. Here, we investigate the ecological significance of the touch response in predator-prey interactions between C. elegans and predacious fungi that catch nematodes using constricting hyphal rings. We show that the constricting rings of Drechslerella doedycoides catch early larval stages with a diameter similar to the trap opening. There is a delay between the ring entry and ring closure, which allows the animal to withdraw from the trap before being caught. Mutants that fail to suppress head movements in response to touch are caught more efficiently than the wild-type. This demonstrates that the coordination of motor programs allows C. elegans to smoothly retract from a fungal noose and evade capture. Our results suggest that selective pressures imposed by predacious fungi have shaped the evolution of C. elegans escape behavior.     

The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.     

Induction of trap formation in nematode‐trapping fungi by bacteria‐released ammonia

A total of 11 bacterial strains were assayed for bacteria‐induced trap formation in the nematode‐trapping fungus Arthrobotrys oligospora YMF1·01883 with two‐compartmented Petri dish. These strains were identified on the basis of their 16S rRNA gene sequences. Volatile organic compounds (VOCs) of eight isolates were extracted using solid‐phase micro‐extraction (SPME) and their structures were identified based on gas chromatography‐mass spectrometry (GC‐MS). At the same time, all isolates were used for quantitative measurement of ammonia by the indophenol blue method. The effects of pure commercial compounds on inducement of trap formation in A. oligospora were tested. Taken together, results demonstrated that the predominant bacterial volatile compound inducing trap formation was ammonia. Meanwhile, ammonia also played a role in other nematode‐trapping fungi, including Arthrobotrys guizhouensis YMF1·00014, producing adhesive nets; Dactylellina phymatopaga YMF1·01474, producing adhesive knobs; Dactylellina cionopaga YMF1·01472, producing adhesive columns and Drechslerella brochopaga YMF1·01829, producing constricting rings.     

An ultrastructural study of cell-cell interactions in capture organs of the nematophagous fungus Arthrobotrys oligospora

Abstract A detailed ultrastructural analysis was made of interactions between individual cells within the same adhesive network (trap) of the nematophagous fungus Arthrobotrys oligospora . These interactions were confined to traps which had captured nematodes, and occurred concurrently with the fungus-nematode interactions. The process was initiated by the anchoring of 2 cells of different loops constituting the trap network, by means of the adhesive coating of these cells. Subsequently, penetration tubes were formed. As in nematode-fungus interactions, the walls of these tubes arose from underneath the original trap cell walls. Two response were observed: either only one of the anchored cells formed a penetration tube, which penetrated the opposite cell and subsequently digested its contents; or both cells formed penetration tubes simultaneously, which were directed against each other. In the latter case, no penetration of other cells was observed, and elongated tubes were occasionally formed. The above mechanism differed from 2 other modes of interaction also observed, namely fusion of intact cells (anastomosis) and development of new hyphae inside dead hyphal cells. In the latter case the newly formed cells developed from the cross-wall of the neighbouring intact cell.     

An electron-microscopical analysis of capture and initial stages of penetration of nematodes byArthrobotrys oligospora

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungusArthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.     

Two new species of nematode-trapping fungi: relationships inferred from morphology,rDNA and protein gene sequence analyses

Two new nematode-trapping fungi, Dactylellina sichuanensis and D. varietas from China, which capture nematodes by both adhesive knobs and non-constricting rings, are described and illustrated. D. sichuanensis is characterized by both adhesive knobs and non-constricting rings, solitary conidiophores and 3-(4)-6 septate conidia, as compared with species such as D. appendiculata, D. candida, D. leptospora and D. lysipaga. Although D. sichuanensis shares similar types of trapping devices, the presence of simple conidiophores and spindle-shaped conidia with these species, it can be distinguished by its larger conidia and presence of more than four septa. In D. sichuanensis, a single conidium is born at the tip of conidiophore, while in D. candida, 3–10 conidia are born near the apex of conidiophore in a capitate arrangement. It produces spindle-shaped conidia with 3–6 septa, whereas D. leptospora produces cylindrical-shaped conidia with 5–15 septa. D. appendiculata captures nematodes by adhesive knobs whereas D. sichuanensis captures nematodes by both adhesive knobs and non-constricting rings. They also differ in conidial size (35–82.5 μm in D. sichuanensis as compared with 57–108 μm in D. appendiculata). D. varietas is characterized by conidiophores that are branched at right-angles, and elongate to fusoid conidia, with 7–8 septa (more than 25% of which are curved). D. varietas resembles D. asthenopaga, Dactylella oxyspora and Monacrosporium multiseptatum, but has elongate-fusoid conidia, whereas D. asthenopaga possesses obconical or clavate conidia. D. varietas forms both adhesive knobs and non-constricting rings whereas Dactylella oxyspora does not produce any trapping device. M. multiseptatum differs from D. varietas in having larger conidia with an inflated middle cell. Phylogenetic analyses based on nuclear and protein coding DNA sequences (18 S, and a combined 28 S + 5.8 S + β-tubulin dataset) indicate that these two taxa should be assigned to the family Orbiliaceae.     

坡向尺度差异对大理苍山捕食线虫真菌分布格局的影响

为了解大理苍山地区不同尺度坡向上捕食线虫真菌的分布格局及其驱动因素,利用系统采样法对大理苍山东、西坡及其次级南、北坡的捕食线虫真菌进行调查。按照五点采样法采集苍山东、西坡及其次级南、北坡土壤样品共300份。采用传统方法对捕食线虫真菌进行分离纯化,结合形态特征和分子生物学方法鉴定所得菌株,按照新分类系统进行分类,鉴定为3属12种57株捕食线虫真菌。结果表明：苍山各尺度坡向捕食线虫真菌检出率和多样性指数均是西坡大于东坡,次级南坡大于次级北坡。次级南、北坡向间捕食线虫真菌群落差异大于东、西坡向间;西坡次级南、北坡向间的群落差异最大;东、西坡向的次级南坡向间群落差异也较大;东坡次级北坡与西坡次级北坡间的群落最为相似。产黏性菌网的Arthrobotrys属、产黏性球和黏性分枝的Dactylellina属在东、西坡及其次级南、北坡间均有分布,而产收缩环的Drechslerella属仅在西坡有分布。因此,坡向是影响苍山捕食线虫真菌空间分布格局的驱动因子之一,不同尺度坡向上捕食线虫真菌分布格局存在差异;次级南、北坡间捕食线虫真菌群落差异性比东、西坡间大,小尺度坡向间地理屏障对捕食线虫真菌分布格局的影响大于大尺度坡向间环境异质性的作用;环境筛选和扩散限制均会影响到捕食线虫真菌的群落构建过程。     

How carnivorous fungi use three-celled constricting rings to trap nematodes

Predacious fungi form specialized hyphae structures to trap nematodes and other microscopic animals. Among the six kinds of trapping devices, the constricting ring is the only one that actively captures nematodes. When a nematode enters the aperture of the ring, which is formed by three cells, the cells rapidly triple their volume, close the aperture and hold the nematode in place. Hyphae then penetrate and consume the nematode. This paper reviews the data and hypotheses on conserving the evolution of constricting rings and their cytological and molecular mechanisms.     

The distribution of nematophagous fungi in the maritime Antarctic

Endoparasitic and predatory nematophagous fungi are widely distributed throughout the maritime Antarctic, being recorded along the Antarctic Peninsula as far south as 68° S. Fungi were recorded from 71% of the sites examined with Cephalosporium balanoides and Dactylaria gracilis being the commonest recorded endoparasite and predator, respectively.Endoparasites with adhesive and nematode-attracting conidia were shown to be more abundant and to have a competitive advantage in the Antarctic ecosystem over those parasites requiring their conidia to be ingested before infection could occur. Predators able to form traps spontaneously on germination were shown to be far more abundant than those species with a more saprophytic mode of existence, with constricting rings being the most commonly isolated trapping mechanism. Species capturing nematodes by three-dimensional networks were restricted to bird-associated sites indicating that they are able to grow saprophytically in such organically enriched material.Nematophagous basidiomycetes and phycomycetes were absent except for a single Myzocytium sp. isolated from heated soil.     

A QUANTITATIVE STUDY OF TISSUE DYNAMICS DURING CLOSURE IN THE TRAPS OF VENUS'S FLYTRAP DIONAEA MUSCIPULA ELLIS

The mechanisms by which Venus's Flytrap (Dionaea muscipula Ellis) close are not clearly understood, and several conflicting models have been proposed. We have measured the dynamics of five trap tissues from three trap regions during full closure of young, fully developed, previously unclosed traps. Closure was divided into three distinct stages: 1) Capture–occurred immediately after stimulation of the trigger hairs and involved the rapid inward flexure of the trap margin and tynes. This motion interlocked the tynes, effectively capturing the prey. This was the only rapid movement of the trap; 2) Appression–completed by 30 min poststimulation, was characterized by contact of the margins; and 3) Sealing–completed by 1 hr poststimulation, was characterized by a sealed “digestive” sac formed around the potential prey, also by tight appression and recurved bending of the trap–margins. Major tissue dynamics that facilitated changes in trap morphology (hence, closure) occurred in different regions of the trap during different periods of time. The first regions where activity occurred were the A and C regions (Fig. 1), after approximately 15 min poststimulation; tissues in the C regions were most active followed by those in the B region of the trap (30 min to 1 hr poststimulation). Thus, shape changes during each stage of closure were the result of temporally separated changes in trap tissue volume. The complete sequence of events was elicited by a single 5–sec period of trigger hair stimulation. Our study showed that changes in the curvature of the trap during closure involved the expansion of opposing tissue groups (i.e., on opposite sides of trap medullary tissues). The pressure from contact of opposing trap lobes during the Appression stage may play an important role in regulating further trap closure and morphology.     

Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa

Many filamentous fungi are capable of undergoing conspecific hyphal fusion with a genetically different individual to form a heterokaryon. However, the viability of such heterokaryons is dependent upon vegetative (heterokaryon) incompatibility (het) loci. If two individuals undergo hyphal anastomosis, but differ in allelic specificity at one or more het loci, the fusion cell is usually compartmentalized and self-destructs. Many of the microscopic features associated with vegetative incompatibility resemble apoptosis in metazoans and plants. To test the hypothesis whether vegetative incompatibility results in nuclear degradation, a characteristic of apoptosis, the cytology of hyphal fusions between incompatible Neurospora crassa strains that differed at three het loci, mat, het-c and het-6, and the cytology of transformants containing incompatible het-c alleles were examined using fluorescent DNA stains and terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL). Hyphal fusion cells between het incompatible strains and hyphal segments in het-c incompatible transformants were compartmentalized by septal plugging and contained heavily degraded nuclear DNA. Hyphal fusion cells in compatible self-pairings and hyphal cells in het-c compatible transformants were not compartmentalized and rarely showed TUNEL-positive nuclei. Cell death events also were observed in senescent, older hyphae. Morphological features of hyphal compartmentation and death during vegetative incompatibility and the extent to which it is genetically controlled can best be described as a form of programmed cell death.     

An Analysis of Hydrozoan Gastrulation by Unipolar Ingression

Unipolar ingression is a common form of gastrulation in the phylum Cnidaria. Here, this process is examined in the hydrozoan Phialidium. Ultrastructural investigation confirmed that, during this process, oral cells elongate, constricting at their apical ends and/or expanding at their basal ends as they ingress. Marking studies show that most entodermal cells originate from the oral area, but a few also come from lateral, aboral-lateral, and aboral areas. Based on orientation of mitotic spindles during gastrulation, a few cells may also enter the entodermal region by delamination. Cell sorting experiments showed that, by postgastrulation, cells from aboral areas of the preingression embryo developed lower adhesive capacities than those from oral regions. Differences in adhesive capacity may allow maintenance of distinct cell layers. Postgastrulae are shorter, thinner, and more streamlined than preingression embryos. Total embryo volume and cell volume decrease, the blastocoel disappears, and 34.4% of the cell mass becomes entoderm. Activities of the ectodermal layer play an important role in gastrulation: during gastrulation, aboral cells become more columnar and oral cells less columnar. Also, cells in oral and oral-lateral regions shift toward the midline, causing oral portions of the embryo to elongate. A model of unipolar ingression is proposed.     

Stability and fine structure of eukaryotic DNA rings in formamide

Folded rings formed from Drosophila and Necturus DNA fragments were examined by electron microscopy in increasing concentrations of formamide, in an effort to identify regions of non-homology within the closure region. Unusual closure regions of this type were not found, in spite of an extensive search. If such regions exist, they must be too short to be detectable (<50 nucleotides), or longer than 1000 nucleotides. In this latter case, they could not be contained within the overlap region of the ring. A study of the thermal (formamide) stability of these rings in relation to the observed closure lengths suggests that extensive (>2 to 3%) mismatching is not possible. At higher formamide concentrations, some rings will partly denature, yet remain circular because the closure region remains intact.     

Occurrence, characterization and development of two different types of microbodies in the nematophagous fungus Arthrobotrys oligospora

Abstract The occurrence of microbodies in different cells of the nematophagous fungus Arthrobotrys oligospora has been investigated. In the predacious phase this organism forms complex 3-dimensional network traps. Mature trap cells generally were crowded with "special" microbodies which possessed an electron dense matrix and were surrounded by a membrane of approx. 9 nm. These organelles developed during the early stages of trap formation and were derived from specialized regions of the endoplasmic reticulum. Cytochemical staining experiments revealed that the electron-dense microbodies contained catalase and d -amino acid oxidase and thus must be considered peroxisomal in nature. Electron-dense bodies were absent in normal vegetative cells of the fungus. These cells contained "normal" microbodies which developed from each other by the separation of small organelles from mature ones. As in yeasts, the metabolic function of these latter organelles was dependent upon environmental conditions.     

Ammonium ions and glutamine inhibit sporulation of Coprinus cinereus basidia assayed in vitro

Basidia of Coprinus cinereus (Schaeff.:Fr) S. F. Gray are committed to their developmental pathway, continuing through meiosis and sporulation even when excised from their parental fruit body. A technique is described which permits this in vitro differentiation to be used as a rapid, small-scale bioassay for chemicals which interfere with these morphogenetic processes. Of a range of compounds tested, only ammonium and glutamine, and some structural analogues, were able to inhibit basidium differentiation. Growth was not inhibited; instead the differentiation inhibitors caused vegetative hyphal tips to grow out from regions of the basidial apparatus expected to be in active growth during sporulation. Depending on the stage reached at the time of exposure to the inhibitors, vegetative hyphal tips emerged from the four apical sites for sterigmata, from the tips of sterigmata, from partially formed or abnormal spores, and from the basal regions of the basidium from which paraphyses would be expected to arise. The experiments show that ammonium ions and glutamine halt meiocyte differentiation. Reports of similar effects in other organisms, animals and plants as well as fungi, may imply that sporulation events are generally sensitive to ammonium inhibition.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.