Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Holographic microscopy provides new insights into the settlement of zoospores of the green alga Ulva linza on cationic oligopeptide surfaces

Interaction of zoospores of Ulva linza with cationic, arginine-rich oligopeptide self-assembled monolayers (SAMs) is characterized by rapid settlement. Some spores settle (ie permanently attach) in a ‘normal’ manner involving the secretion of a permanent adhesive, retraction of the flagella and cell wall formation, whilst others undergo ‘pseudosettlement’ whereby motile spores are trapped (attached) on the SAM surface without undergoing the normal metamorphosis into a settled spore. Holographic microscopy was used to record videos of swimming zoospores in the vicinity of surfaces with different cationic oligopeptide concentrations to provide time-resolved insights into processes associated with attachment of spores. The data reveal that spore attachment rate increases with increasing cationic peptide content. Accordingly, the decrease in swimming activity in the volume of seawater above the surface accelerated with increasing surface charge. Three-dimensional trajectories of individual swimming spores showed a ‘hit and stick’ motion pattern, exclusively observed for the arginine-rich peptide SAMs, whereby spores were immediately trapped upon contact with the surface.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). I. ZOOSPORES1

Zoospores of 17 species in 14 genera of Laminariales, collected in the northeast Pacific Ocean, were studied by electron microscopy. These zoospores are unique in the brown algae in lacking both an eyespot in the single chloroplast and any associated swelling at the base of the shorter, posterior flagellum. Spores of all species examined possess a distal whiplash portion on the longer, mastigoneme-bearing anterior flagellum. This appendage may sometimes be as long as the mastigoneme-bearing portion of the flagellum, but it is only seldom preserved in the preparations for electron microscopy. A microtubular cytoskeleton is probably responsible for maintaining the shape of the spore. It consists of a short band of about 10 microtubules between the two basal bodies, scattered tubules converging at the anterior of the spore, a band of 7–9 tubules directed anteriorly from the anterior basal body, and a band directed posteriorly from the posterior basal body. These anterior and posterior bands may form one continuous band looping around the periphery of the spore. Variation with possible taxonomic significance was found in the ultrastructure of vesicles which apparently contain adhesive material, and which are extruded through the plasmalemma when the zoospores settle.     

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

Alteration of the Cell Surface during the Vegetative Cell Cycle of the Unicellular Green Alga Chlamydomonas reinhardtii

Alterations of the cell surface during the vegetative cell cycleof the unicellular green alga Chlamydomonas reinhardtii wereinvestigated using polyclonal antibodies against the purifiedand subsequently deglycosylated insoluble cell wall componentand against a 100 kDa polypeptide of the deglycosylated, chaotrope-solublewall fraction, respectively. Both antibodies recognized epitopeswithin the non-glycosylated domains of a ‘150 kDa’chaotrope-soluble glycoprotein (=GP3B) localized in the outerlayers of the C. reinhardtii cell wall. Immunofluorescence studiesindicated that both antibodies reacted with the surface of ‘late’sporangia (harvested 1 h before liberation of the zoospores),but not with the cell surfaces of released zoospores, growingcells and young sporangia, respectively. After pretreatmentwith aqueous LiCl, however, the cell surfaces of zoospores,growing cells and young sporangia became accessible to theseparticular antibodies. Highly purified preparations of the insolublewall fraction revealed strong immunofluorescence with both antibodiesbut not with the corresponding preimmune sera. Based on thesedata, we concluded that the antigenic sites of the insolubleglycoprotein framework of the C. reinhardtii wall are maskedby LiCl-soluble glycoproteins in single cell stages and youngsporangia, but not or to a lesser extent in the case of themother walls of ‘late’ sporangia. The conclusionwas supported by findings that (I) the multilayered structureof the mother-cell wall was disturbed in ‘late’,but not in young sporangia and that (II) the amounts of chaotropesolublecell wall glycoproteins present in the LiCl-extracts from intactsporangia decreased during ripening of the sporangia. (Received January 10, 1996; Accepted May 27, 1996)     

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin

Two hybridoma cell lines were selected after the fusion of the myeloma cell line X-63 Ag8-653 with spleen cells from mice immunized with bovine brain microtubules. These lines, clones 3F3 and 16D3, secrete IgM antibodies both staining a fibrillar network in fibroblasts. Autoradiography of immunoblots of SDS gels showed that the antigenic determinants defined by these antibodies are present on tubulin and also on several other polypeptides in mammalian cells. In contrast, they were found to react only with tubulin in Trypanosoma brucei, parasitic protozoan which are the causative agent of sleeping sickness. By immunofluorescence microscopy, 3F3 bound only to a subpopulation of microtubules associated with the flagellum of these cells when, under the same conditions, 16D3 stained other microtubule populations including sub-pellicular microtubules. These results show that flagellar tubulin differs from tubulin of other locations in the same cell by at least one antigenic determinant which could be involved in microtubule specialization.     

Use of Self-Assembled Monolayers of Different Wettabilities To Study Surface Selection and Primary Adhesion Processes of Green Algal (Enteromorpha) Zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH₃) or hydroxyl (OH) groups or mixtures of CH₃- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and “searching” above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to “sense” that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Moments of weaknesses – exploiting vulnerabilities between germination and encystment in the Phytomyxea

Attempts at management of diseases caused by protozoan plant parasitic Phytomyxea have often been ineffective. The dormant life stage is characterised by long-lived highly robust resting spores that are largely impervious to chemical treatment and environmental stress. This review explores some life stage weaknesses and highlights possible control measures associated with resting spore germination and zoospore taxis. With phytomyxid pathogens of agricultural importance, zoospore release from resting spores is stimulated by plant root exudates. On germination, the zoospores are attracted to host roots by chemoattractant components of root exudates. Both the relatively metabolically inactive resting spore and motile zoospore need to sense the chemical environment to determine the suitability of these germination stimulants or attractants respectively, before they can initiate an appropriate response. Blocking such sensing could inhibit resting spore germination or zoospore taxis. Conversely, the short life span and the vulnerability of zoospores to the environment require them to infect their host within a few hours after release. Identifying a mechanism or conditions that could synchronise resting spore germination in the absence of host plants could lead to diminished pathogen populations in the field.     

Characterization of the structural proteins of the murine coronavirus strain A59 using monoclonal antibodies

Monoclonal antibodies reacting with the A59 strain of mouse hepatitis virus (MHV-A59) were characterized and those specific to the E2 major envelope glycoprotein were studied in detail. Antibodies were tested for their ability to neutralize viral infectivity (N+ characteristic) and prevent viral-induced cell-to-cell fusion (F+ characteristic). All four possible combinations of activities reflecting E2 functions were found, i.e., N+F+, N-F-, N+F-, and N-F+. In addition, competitive binding studies with these monoclonal antibodies revealed two nonoverlapping antigenic regions. The first region, designated A, was recognized by antibodies which included each of the four functional types. Region B was identified by a single monoclonal antibody with N-F- activities. The existence of antibodies which only neutralize virus or only block viral-induced fusion implies that the structures on E2 which serve as targets for neutralization and which induce fusion are not identical. The critical determinants for neutralization and fusion must be closely related topographically on E2 since both N+F- and N-F+ antibodies recognize the same antigenic region.     

Engineered antifouling microtopographies - effect of feature size, geometry, and roughness on settlement of zoospores of the green alga Ulva

The effect of feature size, geometry, and roughness on the settlement of zoospores of the ship fouling alga Ulva was evaluated using engineered microtopographies in polydimethylsiloxane elastomer. The topographies studied were designed at a feature spacing of 2 microm and all significantly reduced spore settlement compared to a smooth surface. An indirect correlation between spore settlement and a newly described engineered roughness index (ERI) was identified. ERI is a dimensionless ratio based on Wenzel's roughness factor, depressed surface fraction, and the degree of freedom of spore movement. Uniform surfaces of either 2 mum diameter circular pillars (ERI=5.0) or 2 microm wide ridges (ERI=6.1) reduced settlement by 36% and 31%, respectively. A novel multi-feature topography consisting of 2 mum diameter circular pillars and 10 microm equilateral triangles (ERI=8.7) reduced spore settlement by 58%. The largest reduction in spore settlement, 77%, was obtained with the Sharklet AF topography (ERI=9.5).     

Impact of ultraviolet radiation on physiology and development of zoospores of the brown alga Alaria esculenta from Spitsbergen

Zoospores of the brown alga Alaria esculenta from Spitsbergen were exposed in the laboratory to photosynthetically active radiation [(P), 400–700 nm], P + UVA radiation (PA, 320–700 nm) and PA + UVB radiation (PAB, 280–700 nm). Germination rates were determined, and the germination process was documented by light microscopy. In parallel, the presence of UV-absorbing phlorotannins was studied. Photoinhibition and recovery of photosynthesis were monitored as well as DNA damage and repair. After 8 h of exposure to PAB, germination was inhibited. A 16-h exposure to PA and PAB resulted in a dramatic reduction of germination rates. Phlorotannin-containing physodes were observed in the peripheral cytoplasm and physode-like bodies were found outside the spore, still attached to the zoospore as well as freely floating in the medium; this suggested exocytosis. The absorbance of spore suspensions below 300 nm was higher after 20 h exposure to P, PA and PAB compared with the dark treatment. About 50% of the radiation was absorbed by the zoospores, and the rest was absorbed by the medium, especially after PA and PAB exposure. In this way, harmful UV radiation is absorbed before reaching the cells and this is probably the major reason for the relatively low DNA damage after ≤8 h exposure to PAB. Under these conditions, DNA damage was efficiently repaired and there was a notable recovery of photosynthesis. However, after 16 h exposure to PA and PAB, the protective and repair mechanisms are surcharged and the zoospores degenerate. The results cast light on the potential impact of enhanced UVB radiation on a dominant component of the seaweed community on Spitsbergen because of stratospheric ozone depletion. The acclimation potential of zoospores of this species to UV radiation as demonstrated here is regarded as conferring a competitive advantage in recruitment over similar species in the upper sublittoral zone.     

Species-specific engineered antifouling topographies: correlations between the settlement of algal zoospores and barnacle cyprids

Novel, non-toxic antifouling technologies are focused on the manipulation of surface topography to deter settlement of the dispersal stages of fouling organisms. This study investigated the effect of the aspect ratio (feature height/feature width) of topographical features engineered in polydimethylsiloxane, on the settlement of cyprids of Balanus amphitrite and zoospores of Ulva linza. The correlation of relative aspect ratios to antifouling efficacy was proven to be significant. An increase in aspect ratio resulted in an increase of fouling deterrence for both zoospores and cyprids. The spore density of Ulva was reduced 42% with each unit increase in aspect ratio of the Ulva-specific Sharklet AF topography. Similarly, the number of settled cyprids was reduced 45% with each unit increase in aspect ratio. The newly described barnacle-specific Sharklet AF topography (40 microm feature height, aspect ratio of 2) reduced cyprid settled by 97%. Techniques have been developed to superimpose the smaller Ulva-specific topographies onto the barnacle-specific surfaces into a hierarchical structure to repel both organisms simultaneously. The results for spore settlement on first-generation hierarchical surfaces provide insight for the efficacious design of such structures when targeting multiple settling species.     

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.     

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.     

PRIMARY ADHESION OF ENTEROMORPHA (CHLOROPHYTA,ULVALES) PROPAGULES: QUANTITATIVE SETTLEMENT STUDIES AND VIDEO MICROSCOPY1

Quantitative methods and associated kinetic analyses have been used for the first time to study detailed aspects of the settlement and adhesion of various types of Enteromorpha popagule. Time course experiments showed that quadri and biflagellate zoospores and zygotes adhered rapidly, but a proportion within any one population appeared to be incompetent at adhering to the substratum. Kinetic (Scatchard) analysis of adhesion experiments performed at a range of zoospore concentrations revealed density-dependent effects not previously reported, with positive cooperativity at low spore densities and negative cooperativity at high spore densities. High-resolution video microscopy was used for the first time to reveal details of the various stages in the settlement and adhesion of zoospores and zygotes. Novel observations were made of an initial, temporary phase of attachment via the apical papilla, followed by a permanent phase of commitment, characterized by discharge of adhesive-containing cytoplasmic vesicles, as the cell contracted against the surface, and adsorption of flagella. The phase of commitment was followed by exploitation of the surface through amoeboid-like movements at the interface. Gregarious settlement behavior was frequently observed leading to the formation of rafts of cells. The possible mechanisms and significance of density-dependent spore adhesion are discussed.     

Oxidation of Manganese and Formation of Mn(3)O(4) (Hausmannite) by Spore Coats of a Marine Bacillus sp

Isolated spore coats of a marine Bacillus species were incubated in 25 mM MnCl(2) at pH 7.5. Manganese precipitates, formed on the coat surfaces, were analyzed by transmission electron microscopy, electron diffraction, and energy-dispersive X-ray spectroscopy. Initially, an amorphous manganese oxide was observed on the coats which recrystallized to hausmannite after prolonged incubation in the MnCl(2) solution. The spore coats catalyze the oxidation of Mn(II) and have no structural influence on the final mineral phase precipitated.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth