A New Antifouling Bioassay Monitoring Brown Algal Spore Swimming Behaviour in the Presence of Echinoderm Extracts

Epibiosis, the colonization of biogenic surfaces by epibiotic organisms such as bacteria, filamentous algae, and sessile invertebrates, poses a major threat to the fitness and survival of macroorganisms which could potentially be fouled. Fouling of artificial submerged structures can also cause severe economic problems, making the need for refined bioassays to determine the efficacy of potential antifouling compounds even more important. The aim of this study was to use the distinct swimming behaviour of zoospores of the fouling brown alga Hincksia irregularis to develop a new laboratory antifouling bioassay to test the effect of marine natural products. Spores were exposed to different concentrations of aqueous and organic extracts from body walls of sympatric echinoderms (Asteroidea: Luidia clathrata, Astropecten articulatus; Ophiuroidea: Astrocyclus caecilia). Computer-assisted motion analysis was used to distinguish between the straight and fast swimming movements of undisturbed spores (controls) and the helical and erratic swimming patterns of chemically irritated spores, using the quantitative parameters rate of direction change (RCD) and swimming speed (SPD). The ratio RCD/SPD of spore swimming paths at extract treatments compared to controls can be used to quantify the detrimental effect of echinoderm extracts. Echinoderm extracts had significant effects on spore swimming behaviour at concentrations three orders of magnitude lower than that present naturally in the echinoderm body walls (mg extract/dry weight echinoderm body wall). Comparative studies on spore settlement and germination under similar treatment conditions show that changes in spore swimming behaviour reflect decreased fitness and survivourship of algal spores. It is suggested that this bioassay can be used to screen potential antifouling extracts and compounds at very low concentrations, making this assay particularly suitable for detection of concentration dependent effects and for bioassay-guided fractionation of extracts to identify active compounds.     

Bioassay-guided fractionation of antifouling compounds using computer-assisted motion analysis of brown algal spore swimming

Antifouling extracts from the sea stars Astropecten articulatus and Luidia clathrata and from the brittle star Astrocyclus caecilia were fractionated by solid phase extraction and high performance liquid chromatography. Bioactive fractions were identified with the use of computer-assisted motion analysis-based bioassays utilising previously described Hincksia irregularis spore swimming behaviour parameters. Quantified parameters of spore movement were rate of change of direction (RCD) and speed (SPEE). The methods used initially required only 10 microg equivalent amounts of total crude extract and each resultant resolving step (normalised to 1 mg ml(-1) of crude, unfractionated extract) required far less material. Statistical analyses of RCD and ratios of RCD:SPEE values in experiments comparing swimming in the presence of extract fractions to controls revealed that both parameters were useful individually and in combination for efficiently following compound bioactivity throughout the fractionation procedure. This technique was also able to detect synergistic or additive interactions between compounds.     

Individual and coupled effects of echinoderm extracts and surface hydrophobicity on spore settlement and germination in the brown alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30 min were > 400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were > 300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30 min and the settled spores allowed to subsequently germinate for 24 h. Spore germling numbers were consistently > 25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24 h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30 min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24 h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.     

Effects of Macroalgal Chemical Extracts on Spore Behavior of the Antarctic Epiphyte Elachista antarctica Phaeophyceae

Most macroalgal species along the Western Antarctic Peninsula (WAP) are defended against predation, many using chemical defenses. These subtidal communities are also mostly devoid of free living filamentous algae. However, one endo/epiphyte, Elachista antarctica, is found growing exclusively out of the palatable rhodophyte Palmaria decipiens. To understand this unusual and exclusive epiphytization, we tested whether macroalgal secondary metabolites such as those responsible for deterring sympatric grazers, affect the behaviors of the epiphyte's spores. Settlement, germination, and swimming behaviors of the epiphyte's motile spores were quantified in the presence of fractionated lipophilic and hydrophilic extracts of host P. decipiens and other rhodophytes from the shallow subtidal. Host P. decipiens was the only alga tested that did not inhibit spore settlement or germination. We also examined whether extracts from these chemically rich algae affect spore swimming behaviors and found spores to be chemotactically attracted to seawater soluble extract fractions of host P. decipiens. These results indicate that chemosensory behaviors of the epiphyte's spores to metabolites associated with these chemically defended macrophytes can explain this exclusive epiphyte–host interaction.     

Exo-β-1,3-Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

Yeast exo-β-1,3-glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 10³ to 80 × 10³ spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 10³ to 80 × 10³, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5-day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β-glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea , respectively, could be considered.     

Regulation of trehalose metabolism by Streptomyces griseus spores.

Spores of Streptomyces griseus contain trehalose and trehalase, but trehalose is not readily hydrolyzed until spore germination is initiated. Trehalase in crude extracts of spores, germinated spores, and mycelia of S. griseus had a pH optimum of approximately 6.2, had a Km value for trehalose of approximately 11 mM, and was most active in buffers having ionic strengths of 50 to 200 mM. Inhibitors or activators or trehalase activity were not detected in extracts of spores or mycelia. Several lines of evidence indicated that trehalose and trehalase are both located in the spore cytoplasm. Spores retained their trehalose and most of their trehalase activity following brief exposure to dilute acid. Protoplasts formed by enzymatic removal of the spore walls in buffer containing high concentrations of solutes also retained their trehalose and trehalase activity. Protoplasts formed in buffer containing lower levels of solutes contained low levels of trehalose. The mechanism by which trehalose metabolism is regulated in S. griseus spores is unresolved. A low level of hydration of the cytoplasm of the dormant spores and an increased level of hydration during germination may account for the apparent inactivity of trehalase in dormant spores and the rapid hydrolysis of trehalose upon initiation of germination.     

Isolation of antifouling active pyroglutamic acid,triethyl citrate and di-<Emphasis Type="Italic">n</Emphasis>-octylphthalate from the brown seaweed <Emphasis Type="Italic">Ishige okamurae</Emphasis>

Three antifouling active compounds of L-pyroglutamic acid (PGA), triethyl citrate (TEC) and di-n-octylphthalate (DNOP) were isolated from the brown seaweed Ishige okamurae. Approximately 2.8 mg PGA, 1.7 mg TEC, and 2.0 mg DNOP were isolated from 600 g of I. okamurae powder. The concentrations of PGA, TEC, and DNOP required to cause foot repulsion in 50% of mussels (RD₅₀) were 9, 26, and 0.08 mM, respectively. The PGA, TEC, and DNOP concentrations required to inhibit 50% attachment of algal spores (ID₅₀) were 24, 50 and 0.1 mM, respectively. These compounds showed stable antifouling activities against mussel and algal spore attachment.     

A bioassay for insect deterrent compounds found in plant and animal tissues

A general field bioassay for detecting biologically active compounds in plants and insects has been developed and tested for efficacy and sensitivity. Methanolic extracts, in sucrose solution, of 20 plant and six caterpillar species were offered to the ponerine ant Paraponera clavata and the feeding preferences observed. The bioassay resulted in the detection of nine plant and three caterpillar species with ant-deterrent extracts, and 11 plant and three caterpillar species with neutral or attractant extracts. All of the plants showing ant-deterrent characteristics which had been chemically investigated in our laboratory, or for which chemical literature was available, contained secondary metabolites of known deterrence. Both naturally occurring and artificial differences in chemical concentrations could be detected using the bioassay. The method provides a means of screening plants and insects for compounds that are insect anti-feedants or that can modify insect behaviour.     

Antifouling activity of bioactive substances extracted from Holothuria scabra

Methanol extract of Holothuria scabrawas tested for antifouling activity using `mollusc foot adherence bioassay'. It was found that the secondary metabolites of H. scabraeffectively prevented foot adherence of P. vulgataat various concentrations. Based on the present findings it could be inferred that the bioassay guided purification and fractionation may give forth potent novel antifouling compounds.     

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.     

No evidence for a Ganoderma spore dispersal mutualism in an obligate spore-feeding beetle Zearagytodes maculifer

The role of spore dispersal mutualism remains equivocal in many fungus-insect assemblages. We tested experimentally whether an obligate spore-feeding beetle Zearagytodes maculifer has a mutualistic relationship with its host bracket fungus Ganoderma cf. applanatum via spore dispersal. We asked three specific questions: (1) whether or not Ganoderma spore germination rate is increased via beetle digestive activity and (2) is dependent on temperature and sporocarp identity. Spore germination rates were examined in 2×3×2 factorial experiments (spores consumed by beetles or not×temperature 20, 25, and 30°C×two independent pairs of sporocarp-beetle populations) replicated five times in an array of 60 experimental cultures. Analysis showed that consumption by beetles significantly reduced germination rate of Ganoderma spores. The effect of temperature was modulated by the effect of individual sporocarp, and was overridden by beetle feeding. Microscopic analysis revealed that spores from beetle faecal pellets exhibited extensive damage to their thin outer walls (pellicles) and thick inner walls, as well as significant loss of cytoplasm, while control spores were intact. The overall evidence argued against our spore dispersal mutualism hypothesis, suggesting that Z. maculifer can potentially exert a negative, if vanishingly small, fitness effect on its host fungus G. cf. applanatum.     

QUALITATIVE AND QUANTITATIVE STUDIES OF THE SWIMMING BEHAVIOR OF HINCKSIA IRREGULARIS SPORES (PHAEOPHYCEAE)

The swimming behavior of spores of the brown alga Hincksia irregularis was analyzed using computer-assisted motion analysis. We distinguished five main swimming patterns: straight paths, search circles, orientation, gyration, and wobbling. We suggest different functional values for the individual swimming patterns. Straight paths, search circles, and orientation are different but all may be important in small-scale movements in the benthic boundary layer. As such, they could enable a spore to find a suitable microenvironment for germination and growth. Gyration occurs during the initial reversible phase of adhesion that can lead to settlement. Wobbling is typical for irritated or mechanically damaged spores and does not seem to be a typical pattern associated to settlement. The dominant swimming patterns changed with spore age (10 ± 5 to 60 ± 5 min of spore age), with young spores mainly swimming in straight paths and search circles and older spores in orientation and gyration. This change in swimming patterns can be quantified by speed (decrease over time) and rate of change of direction (increase over time). Based on these results, we suggest that computer-assisted motion analysis of the swimming behavior of H. irregularis spores can be used to develop bioassays with both ecological and technological relevance.     

Immunological Homology Between Crystal and Spore Protein of Bacillus thuringiensis

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.     

Immunohistochemical and immunocytochemical detection of SchS34 antigen in <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> spores and spore impacted mouse lungs

The purpose of this study was to evaluate the distribution of a 34 kD antigen isolated from S. chartarum sensu lato in spores and in the mouse lung 48 h after intra-tracheal instillation of spores by immuno-histochemistry. This antigen was localized in spore walls, primarily in the outer and inner wall layers and on the external wall surfaces with modest labelling observed in cytoplasm. Immuno-histochemistry revealed that in spore impacted mouse lung, antigen was again observed in spore walls, along the outside surface of the outer wall and in the intercellular space surrounding spores. In lung granulomas the labelled antigen formed a diffusate, some 2–3× the size of the long axis of spores, with highest concentrations nearest to spores. Collectively, these observations indicated that this protein not only displayed a high degree of specificity with respect to its location in spores and wall fragments, but also that it slowly diffuses into surrounding lungs.     

Isolation and characterization of ribosomes from Bacillus subtilis spores

Bishop, Helen L. (Syracuse University, Syracuse, N.Y.), and Roy H. Doi. Isolation and characterization of ribosomes from Bacillus subtilis spores. J. Bacteriol. 91:695-701. 1966.-The isolation of ribosomes from Bacillus subtilis spores was accomplished by freezing the spores in liquid nitrogen and grinding the spore pellet with an equal weight of levigated alumina. The ribosomes, which were adsorbed to the alumina, were freed by the addition of vegetative-cell ribosomes or bulk ribonucleic acid (RNA) to the crude alumina-ground extract. The spore ribosomes had sedimentation properties and RNA and protein compositions similar to those of vegetative-cell ribosomes. The difficulty encountered in obtaining spore ribosomes by ordinary extraction methods may be the result of nuclease and protease activities which were demonstrated in spore extracts.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Effect of inhibitors of trypsin-like proteolytic enzymes Bacillus cereus T spore germination.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.     

Within-thallus variation in polyphenolic content and antifouling activity in Sargassum vulgare

The brown seaweed Sargassum vulgare C. Agardh abounds along the southeast coast of Rio de Janeiro State, Brazil, creating unique habitats, and its polyphenols play an important ecological role as antifouling agents. In order to understand more precisely this defensive strategy of S. vulgare, we collected this macroalga at Ilha de Itacuruçá in May and October 2009. Thalli were separated according to tissue type: pneumatocysts, receptacles, leaflets, and axis. Phenolic extracts from each specific part of the plant were obtained and the associated antifouling activity was tested in order to assess whether a tissue specialisation/antifouling activity pattern exists. Preliminary separation process was carried out on a phenolic extract to separate the polyphenols from the remaining compounds. Such fractionation allowed us to study the involvement of strictly phenolic compounds in antifouling activity. Within-thallus variation in both phenolic content and antifouling activity were highlighted, and seasonal variation in both those characters was also observed. For both experiments, pneumatocyst and leaflet phenolic extracts were the most active against the attachment of Perna perna, but no correlation was observed between phenolic concentration and antifouling activity. On the contrary, the extract containing only phenolic compounds was twice less active than the extract from which 80 % of the polyphenols were removed. These results led us to hypothesise that polar compounds other than polyphenols are involved in the antifouling activity.     

扁绒泡菌孢子形成过程超微结构

诱导扁绒泡菌显型原质团形成子实体并观察在形成过程中孢囊的超微结构,结果表明,全部原质团参入形成孢子及孢丝;孢子形成初期原质团聚缩使原生质密度加大,脂滴密度也增加;液泡联合形成液泡网体分割原质团,孢子及孢丝一同形成;相邻孢子初始形成的孢子壁可见吻合的突起和凹陷,这是孢子成熟后的表面纹饰部分;孢子壁随孢囊发育逐渐达到适宜位置,孢子壁由透明内层及电子密度较大的外层组成;随后可见外有疣突,内含脂滴的圆形孢子。