The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

The functional morphology and attachment mechanism of pandarid adhesion pads (Crustacea: Copepoda: Pandaridae)

The attachment mechanism of pandarid adhesion pads is described from observations of their externally ridged structure and internal construction in three species; Pandarus bicolor Leach, 1816, Dinemoura latifolia (Steenstrup and Lutken, 1861) and Echthrogaleus coleoptratus (Guerin-Meneville, 1837). The host's external skin morphology was also examined, since parasite attachment mechanism and host surface can be considered as components of a single system.

The results emphasise the importance of the physical nature of the pad's surface. This is inferred from the compliance of the cuticle and subsurface structure, and the presence of cuticular ridging. The pads probably prevent pandarids from being dislodged by hydrodynamic drag, by increasing overall adhesion. It is proposed that this is achieved in different ways, by two types of adhesion pad identified here, distinguishable by their external structure and location. Type I pads are suggested to remove interfacial water and increase surface contact by one of two contrasting methods. The ridges may act as tyre treads, by channelling water from the contact surface. Alternatively, the channels between ridges may be hydrophobic and behave as dewetting structures, preventing water from entering in the same way that troughs between surface nodules function to produce superhydrophobicity on lotus leaves. Type I adhesion pads are also suggested to aid attachment by hindering the process of peeling, by which they are thought to be removed by hydrodynamic drag. Type II pads are more likely to function as one-way frictional attachments. Both types of pad appear to be attached passively, since they lack muscles inserting into them. The adhesive mechanism of each, which functions most effectively on hard surfaces, may explain why pads are absent or reduced on pandarids which parasitise the softer, unscaled surfaces of hosts.

Pandarids predominantly parasitise the skin and fins of fast-swimming sharks. This may be because the scales are characteristically smaller in these species and are more easily encircled by the primary attachment appendages, the maxillipeds.

This is thought to be the first published report to reveal frictional attachment structures from the Crustacea, which have convergently evolved in many terrestrial Arthropoda.     

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.     

Fungal spore attachment to substrata

A critical factor in the success of fungal growth is spore adhesion to host surfaces. Generating spores capable of rapid and firm bonding to their hosts is not only important for keeping spores from prematurely detaching from the host surface but can also serve as a trigger for spore germination and the development of infection structures. In this paper fungal spore adhesion mechanisms are reviewed as well as factors influencing spore adhesion, germination, and differentiation. This review ends with a brief discussion on the future of fungal adhesion research.     

Ultrastructural architecture and mechanical properties of attachment pads in Tettigonia viridissima (Orthoptera Tettigoniidae)

Natural releasable attachment systems of insect legs, where attachment-detachment performances are often very fast, seem to be optimized to get a maximum of real contact to the substratum. Tarsi of Tettigonia viridissima bear flexible attachment pads with unusual ultrastructural architecture of the cuticle. The indentation of the attachment pads was measured under different loads using a force-tester. Since the mechanical properties are influenced by material structure, the freeze-substitution experiments were undertaken to investigate the influence of loads on material structure. Both profile changes of the surface and the orientation of cuticle microfibrils were visualized by means of scanning electron microscopy followed by fracturing of the frozen material. The results show that the flexible pad material deforms replicating the substrate profile down to the micrometer roughness. The pad material showed both elastic and viscous behavior under loads. Elastic deformation of the pad occurred under normal force applied for 4-6 s (elastic modulus 27.2 +/- 11.6 kPa). Two viscous relaxation processes were found, of time constants tau1 = 1.88+/-0.616 s and tau2 =41.2 +/- 9.95 s. Low stiffness of material studied here aids in surface replication and increase of area of real contact between the pad and the underlying substrate.     

Chemical composition of the attachment pad secretion of the locust Locusta migratoria

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C₁₆ and C₂₀ in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.     

Direct Probing of the Surface Ultrastructure and Molecular Interactions of Dormant and Germinating Spores of Phanerochaete chrysosporium

Atomic force microscopy (AFM) has been used to probe, under physiological conditions, the surface ultrastructure and molecular interactions of spores of the filamentous fungus Phanerochaete chrysosporium. High-resolution images revealed that the surface of dormant spores was uniformly covered with rodlets having a periodicity of 10 +/- 1 nm, which is in agreement with earlier freeze-etching measurements. In contrast, germinating spores had a very smooth surface partially covered with rough granular structures. Force-distance curve measurements demonstrated that the changes in spore surface ultrastructure during germination are correlated with profound modifications of molecular interactions: while dormant spores showed no adhesion with the AFM probe, germinating spores exhibited strong adhesion forces, of 9 +/- 2 nN magnitude. These forces are attributed to polysaccharide binding and suggested to be responsible for spore aggregation. This study represents the first direct characterization of the surface ultrastructure and molecular interactions of living fungal spores at the nanometer scale and offers new prospects for mapping microbial cell surface properties under native conditions.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.     

感染羽茅的香柱菌属内生真菌对丛枝菌根真菌孢子萌发的影响

内生真菌与丛枝菌根(AM)真菌是构成草原生态系统的重要组成部分.内生真菌会抑制其宿主植物的AM真菌侵染率.本研究以感染2种香柱菌属内生真菌[Epichloё gansuensis(Eg)和E. sibirica(Es)]的天然禾草羽茅为供试材料,进行体外纯培养的内生真菌培养滤液、感染内生真菌的羽茅叶片(包括鲜叶和枯叶)浸提液,以及根系分泌物对摩西球囊霉(Gm)和幼套球囊霉(Ge)2种AM真菌孢子萌发影响试验.结果表明: 香柱菌属内生真菌的培养滤液会显著抑制2种AM真菌孢子的萌发,而感染香柱菌属内生真菌的羽茅根系分泌物只对Ge孢子萌发有显著抑制作用,且上述抑制作用与内生真菌种类无关;鲜叶浸提液对Gm和Ge的孢子萌发率均无显著影响,而枯叶浸提液对Ge的孢子萌发有显著抑制作用.在自然生态系统中,香柱菌属内生真菌通常存在于宿主植物体内,可能通过影响宿主植物的根系分泌物来影响AM真菌孢子的萌发.     

Diversity and partial characterization of putative virulence determinants in Pasteuria penetrans, the hyperparasitic bacterium of root-knot nematodes (Meloidogyne spp.)

Antigens recognized by monoclonal antibodies (Mabs) raised to the surface of the obligate nematode hyperparasite Pasteuria penetrans were characterized. Using the attachment of spores of the bacterium to host nematodes to determine the biological variability present on the spore surface greatly underestimated the amount of surface heterogeneity present compared with estimates from immunological techniques. This heterogeneity differed not only between different individual spores from the same population but also between different spore populations. None of the Mabs completely inhibited any spore population from attaching to the nematode cuticle, suggesting that the mechanism of attachment may be more complex than previously supposed. Chemical degradation of one particular epitope recognized by monoclonal antibody PP1/117, and designated ep117, occurred after treatment with NaOH, periodate or Proteinase K, suggesting that an O-linked glycoprotein may be involved. Fibronectin, which had been found to bind to Pasteuria spores through hydrophobic interactions, also prohibited the Mab from recognizing ep117. However, SDS-PAGE of spore extracts followed by immunoblotting showed that none of the Mabs could detect this epitope and so ep117 may be conformational in nature. Thus, the conformation of any particular epitope recognized by a Mab may be important in determining to which nematode a particular spore will attach. The distribution of a particular epitope within a population of spores will in turn therefore determine its virulence on a particular nematode.     

A new oomycete species parasitic in nematodes, Chlamydomyzium dictyuchoides sp. nov.: Developmental biology and phylogenetic studies

The genus Chlamydomyzium is a little studied holocarpic oomycete parasite of nematodes of uncertain phylogenetic and taxonomic position. A new holocarpic species, Chlamydomyzium dictyuchoides, is described which has usually refractile cytoplasm and a dictyuchoid pattern of spore release. This new species infects bacteriotrophic rhabditid nematodes and was isolated from diverse geographical locations. Infection was initiated by zoospore encystment on the host surface and direct penetration of the cuticle. A sparsely branched, constricted, refractile thallus was formed which eventually occupied almost the entire host body cavity, often accompanied by complete dissolution of the host cuticle. Walled primary cysts formed throughout the thallus and each cyst released a single zoospore via an individual exit papillum, leaving a characteristic dictyuchoid wall net behind. At later stages of infection some thalli formed thick-walled stellate resting spores in uniseriate rows. Resting spore formation appeared to be parthenogenetic and was not accompanied by the formation of antheridial compartments. These spores had ooplast-like vacuoles and thick multi-layered walls, both of which suggest they were oospores. The maximum likelihood tree of sequences of the small ribosomal subunit (SSU) gene placed this new isolate in a clade before the main saprolegnialean and peronosporalean lines diverge. A second undescribed Chlamydomyzium sp., which has direct spore release forms a paraphyletic clade, close to C. dictyuchoides and Sapromyces. The fine structure of other documented Chlamydomyzium species was compared, including an undescribed (but sequenced) isolate, SL02, from Japan, Chlamydomyzium anomalum and Chlamydomyzium oviparasiticum. Chlamydomyzium as currently constituted is a paraphyletic genus that is part of a group of phylogenetically problematic early diverging clades that lie close to both the Leptomitales and Rhipidiales.     

Regulation of hyphal growth and sporulation of the insect pathogenic fungus Entomophthora thripidum in vitro

Entomophthora thripidum is an obligate biotrophic insect pathogenic fungus that grows as protoplasts within the hemocoel of thrips. Prior to penetration through the insect cuticle and spore formation at the insect surface the protoplasts switch to hyphal growth. In vitro, the differentiation to hyphal growth was a prerequisite for the subsequent formation of infectious spores and was detected 10-20 days after inoculation. E. thripidum secreted a factor that autoinduced the differentiation to hyphal growth. The discovery of this activity inducing hyphal growth made possible the reliable production of spores, the infection of host insects and the consecutive re-isolation of the fungus from the infected insects.     

Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera)

The morphology, ultrastrucure, effective elastic modulus, and adhesive properties of two different smooth-type attachment pads were studied in two orthopteran species. Tettigonia viridissima (Ensifera) and Locusta migratoria (Caelifera) have a similar structural organization of their attachment pads. They both possess a flexible exocuticle, where the cuticular fibrils are fused into relatively large rods oriented at an angle to the surface. The compliant material of the pad contributes to the contact formation with the substrate. However, the pad material structure was found to be different in these two species. L. migratoria pads bear a thick sub-superficial layer, as well as a higher density of rods. The indentation experiments showed a higher effective elastic modulus and a lower work of adhesion for L. migratoria pads. When the indentations were made at different depths, a higher effective elastic modulus was revealed at lower indentation depths in both species. This effect is explained by the higher stiffness of the superficial pad layer. The obtained results demonstrate a clear correlation between density of the fibres, thickness of the superficial layer, compliance of the pad, and its adhesive properties. Such material structures and properties may be dependent on the preferred environment of each species.     

Rapid preflexes in smooth adhesive pads of insects prevent sudden detachment

Many insects possess adhesive organs that can produce extreme attachment forces of more than 100 times body weight but they can rapidly release adhesion to allow locomotion. During walking, weaver ants (Oecophylla smaragdina) use only a fraction of their maximally available contact area, even upside-down on a smooth surface. To test whether the reduced contact area makes the ants more susceptible to sudden and unexpected detachment forces, for example, by rain or wind gusts, we investigated the reaction of untethered ants to rapid horizontal displacements of the substrate. High-speed video recordings revealed that the pad''s contact area could more than double within the first millisecond after the perturbation. This contact area expansion is much faster than any neuromuscular reflex and therefore represents a passive ‘preflex’, resulting from the mechanical properties and geometrical arrangement of the (pre-)tarsus. This preflex reaction protects ants effectively against unexpected detachment, and allows them to use less contact area during locomotion. Contact area expanded most strongly when the substrate displacement generated a pull along the axis of the tarsus, showing that the ants'' preflex is direction-dependent. The preflex may be based on the ability of Hymenopteran adhesive pads to unfold when pulled towards the body. We tested Indian stick insects (Carausius morosus), which have smooth pads that lack this motility. Similar to the ants, they showed a rapid and direction-dependent expansion of the contact area mainly in the lateral direction. We propose that the preflex reaction in stick insects is based on the reorientation of internal cuticle fibrils in a constant-volume system, whereas the ants'' pad cuticle is probably not a hydrostat, and pad extension is achieved by the arcus, an endoscelerite of the arolium.     

Proof for the Production of Cutinase by Fusarium solani f. pisi during Penetration into Its Host, Pisum sativum

Rabbit antibody to cutinase-I, isolated from Fusarium solani f. pisi, was conjugated to ferritin. With this ferritin-conjugated antibody it was shown that germinating spores of this fungus excreted cutinase during the penetration of the host pisum sativum. This result constitutes the most specific and strongest evidence for an enzymic penetration of a plant cuticle by a pathogen during infection.     

Functionally Different Pads on the Same Foot Allow Control of Attachment: Stick Insects Have Load-Sensitive “Heel” Pads for Friction and Shear-Sensitive “Toe” Pads for Adhesion

Stick insects (Carausius morosus) have two distinct types of attachment pad per leg, tarsal “heel” pads (euplantulae) and a pre-tarsal “toe” pad (arolium). Here we show that these two pad types are specialised for fundamentally different functions. When standing upright, stick insects rested on their proximal euplantulae, while arolia were the only pads in surface contact when hanging upside down. Single-pad force measurements showed that the adhesion of euplantulae was extremely small, but friction forces strongly increased with normal load and coefficients of friction were 1. The pre-tarsal arolium, in contrast, generated adhesion that strongly increased with pulling forces, allowing adhesion to be activated and deactivated by shear forces, which can be produced actively, or passively as a result of the insects'' sprawled posture. The shear-sensitivity of the arolium was present even when corrected for contact area, and was independent of normal preloads covering nearly an order of magnitude. Attachment of both heel and toe pads is thus activated partly by the forces that arise passively in the situations in which they are used by the insects, ensuring safe attachment. Our results suggest that stick insect euplantulae are specialised “friction pads” that produce traction when pressed against the substrate, while arolia are “true” adhesive pads that stick to the substrate when activated by pulling forces.     

Disappearance of the Cuticle and Wax in Outermost Layer of Callus Cultures and Decrease of Protective Ability against Microorganisms

In electron microscopic observation, neither wax nor cuticle was observed on the outermost layers of callus tissues. Chemical estimation of wax in the callus surface was attempted by thin-layer chromatography of solvent extracts of callus tissues in comparison with those of barley and rice leaves. Hydrocarbons and free alcohols were detected in lyophilized callus tissues, but no wax esters or ketones were detected. Germination test indicated that germination of spores of Aspergillus oryzae was less favored on hydrophobic membranes than that of spores of Alternaria sp. and Botrytis cinerea.

From these results, we inferred that the lack of cuticle and wax in the outermost layer of callus tissues facilitated spore germination and penetration, and A. oryzae, a saprophytic fungus, could also readily penetrate into callus tissues.     

Adhesive Contact in Animal: Morphology, Mechanism and Bio-Inspired Application

Many animals possess adhesive pads on their feet,which are able to attach to various substrates while controlling adhesive forces during locomotion.This review article studies the morphology of adhesive devices in animals,and the physical mechanisms of wet adhesion and dry adhesion.The adhesive pads are either ‘smooth' or densely covered with special adhesive setae.Smooth pads adhere by wet adhesion,which is facilitated by fluid secreted from the pads,whereas hairy pads can adhere by dry adhesion or wet adhesion.Contact area,distance between pad and substrate,viscosity and surface tension of the liquid filling the gap between pad and substrate are the most important factors which determine the wet adhesion.Dry adhesion was found only in hairy pads,which occurs in geckos and spiders.It was demonstrated that van der Waals interaction is the dominant adhesive force in geckos' adhesion.The bio-inspired applications derived from adhesive pads are also reviewed.     

DEVELOPMENT OF THE CUTICULAR LAYERS IN ANGIOSPERM LEAVES

Schieferstein , R. H., and W. E. Loomis . (Iowa State U., Ames.) Development of the cuticular layer in angiosperm leaves. Amer. Jour. Bot. 46(9): 625–635. Illus. 1959.—The cuticularized layers of leaves and other plant surfaces consist of a primary cuticle, formed by the oxidation of oils on exposed cell walls, plus various surface and subsurface wax deposits. The primary cuticle appears to form rapidly on the walls of any living cell which is exposed to air. Surface wax is present on the mature leaves of about half of the 50 or 60 species studied. In general, wax is extruded at random through the newly formed cuticle of young leaves and accumulated in various reticulate to semicrystalline patterns. No wax pores through the cuticle or primary wall can be observed in electron-micrographs of dewaxed mature leaves. Wax accumulations on older leaves are generally subcuticular and may involve the entire epidermal wall. These deposits appear to be of considerably greater ecological significance than those on the surface. Isolated cuticular membranes from Hedera helix increased slightly in permeability to water with age of the leaf, but permeability to 2,4-D decreased 50 times. Evidence based on the patterns of cellulose in primary walls, of surface wax on growing leaves, of the appearance of the cuticle at the margins of growing epidermal cells, of the forms of the cuticle plates digested from growing and older leaves, and of the marginal location of new wax deposits on growing maize leaves is presented to support the thesis that the enlargement of the outer surface of the epidermal cells of leaves occurs at the margins of the surface. Earlier formed cuticle and wax are thus undisturbed during growth. These observations, coupled with evidence for apical growth in fibers, root hairs, etc. suggest that the primary walls of angiosperm cells are formed in specific, localized growth regions, rather than by plastic extension and apposition.     

昆虫足的附着机制

昆虫卓越的爬行和附着能力来源于其精细的功能性黏附系统。根据形态结构的不同,昆虫的黏附系统可分为光滑型黏附垫和刚毛型黏附垫两种类型,二者在分泌液的支持下均能附着于几乎所有的光滑或粗糙的物体表面,而且这两种类型的黏附垫与界面的附着的形成均主要依赖于范德华力。本文综述了昆虫足的附着机制,包括光滑型和刚毛型两种黏附垫的结构和其形成附着的机理,以及黏附垫分泌液的功能、组成成分和释放机制,阐明了昆虫如何巧妙地解决稳定附着和快速脱附这一矛盾的问题,讨论了诸如界面的理化性质和环境湿度等环境因素对昆虫附着的影响,以期帮助人们深入地理解昆虫足的附着机制,并为其在仿生学等方面的应用提供理论依据。