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Effects of bromide and iodide on stalk secretion in the biofouling diatom Achnanthes longipes (Bacillariophyceae)
... Quantifying iodine accumulation and growth of microalgae in relation to external iodine concentration is of special relevance for a better understanding on iodine and iodine compounds trophic transfer from phytoplankton in marine ecosystems as well as in marine aquaculture food chains. Several studies looked at the effect of iodide and/or iodate on the growth rate of phytoplankton species (Sugawara and Terada1967;Fuse et al. 1989, Johnson et al. 1995, Imai et al. 2004Zheng et al. 2005;Iwamoto and Shiraiwa 2012). In some cases, a negative effect was observed, in some cases a positive effect was observed, and in some cases no effect was observed of iodide and/or iodate concentration on growth rate. ...
... In some cases, a negative effect was observed, in some cases a positive effect was observed, and in some cases no effect was observed of iodide and/or iodate concentration on growth rate. In many cases, only one concentration of either iodide or iodate was tested (Sugawara and Terada 1967;Johnson et al. 1995;Imai et al. 2004), or only low concentrations (Sugawara and Terada 1967;Imai et al. 2004) or only high concentrations (Zheng et al. 2005;Iwamoto and Shiraiwa 2012) were tested. ...
... At this concentration lipid accumulation may have been a response to stressful conditions, as has been described for Dunaliella in response to nutrient limitation or salt stress (El-Baky et al. 2004;Chen et al. 2011). Two studies reported an increase in extracellular polymeric substances (EPS) of diatoms as a result of I − addition, but it is not clear how this was related to the intracellular carbohydrate metabolism (Johnson et al. 1995;Zheng et al. 2005). No conclusion on any effect of iodine treatments on lipid metabolism could be drawn from the fatty acid profiles of the microalgae investigated in the a b c Fig. 3 Incorporation rate of iodine and production rate of I − in D. salina (a), P. tricornutum (b) and T. lutea (c) plotted against dissolved inorganic iodine concentration in the culture medium. ...
Marine phytoplankton have been found to be involved in the biogeochemical cycle of iodine in the ocean. However, the role of iodine in the metabolism of marine microalgae has hardly been studied and whether iodine is an element required for growth is not known. In this study, three species of marine microalgae were grown in batch cultures at various concentrations of iodide (I−) or iodate (IO3−) in order to evaluate their effect on growth rate and biochemical composition. Changes in inorganic iodine concentrations in the culture medium were also determined. Growth rate was not affected by external inorganic iodine concentration in cultures of Dunaliella salina and Tisochrysis lutea. In cultures of Phaeodactylum tricornutum, growth was inhibited at 2500 µM IO3−. No clear effects of I− or IO3− were observed on biochemical composition, except for the particulate iodine content of the microalgae, which increased with increasing dissolved iodine concentration. Results of dissolved inorganic iodine measurements indicated that IO3− was reduced to I− during the exponential growth phase of microalgae cultures, under both natural and higher IO3− concentrations. Iodine balance calculations showed that the amount of disappeared IO3− was higher than the amount of I− produced in several cultures and that this discrepancy could not be explained by the formation of particulate iodine. I incorporation rates into biomass were low compared to I− production rates. We conclude that although iodine is clearly taken up from the external medium and it is transformed by microalgae, it seems unlikely that it is an element required for growth or that it plays an essential role in microalgal metabolism.
... A. longipes relies on production of highly organized extracellular adhesive biocomposites for cell motility and permanent adhesion to submerged surfaces (Wang et al., 1997). Studies on its adhesion focused on factors that affect the production of stalks both in field and experiments (Johnson et al., 1995;Wang et al., 1997;Lewis et al., 2002). A. longipes may provide a continuous food source for grazing abalone juveniles (Kawamura et al., 1995;Takami et al., 2003). ...
... The results proved that 10 & 25 µM D-600 (α-Isopropyl-α-[(Nmethyl-N-homovertatryl)-γ-aminopropyl]-3,4,5 trimethoxy phenylacetonitrile) inhibited the adhesion and facilitates getting homogenous free inocula of A. longipes. Replacing sulfate in artificial seawater with methionine also resulted in a lack of adhesion (Johnson et al., 1995). ...
... We propose that the origin of the released HOBr and HOI by diatoms is from an extracellular BrPO, residing in the apoplast, the space between the cell membrane and silicified frustule. The process of stalk assembly and adhesion by the temperate benthic diatom A. longipes requires bromide, and the oxidative phenolic crosslinking that occurs is thought to be catalyzed by an extracellular peroxidase, possibly a BrPO (Johnson et al. 1995;Vreeland and Epstein 1996). This species did test positive for the bromination and iodination of PR using the in situ assay (Table 2), thus strongly indicating the presence of an extracellular BrPO. ...
... Based on the in situ incubation assays, it appears that S. glacialis contains an IPO. A. longipes, T. pseudonona, and T. oceanica tested positive for BrPO in our assay, the latter being a warm-water strain. Our data supports the finding of a bromide-sensitive external peroxidase active in stalk formation of A. longipes (Johnson et al. 1995;Vreeland and Epstein 1996). The finding of activity in T. oceanica is significant in the context of bromoform production in warm water (see below). ...
An in situ incubation assay measuring the bromination and iodination of phenol red was developed to detect the release of reactive bromine and iodine (primarily hypobromous acid (HOBr) and hypoiodous acid (HOI), respectively) from a putative extracellular bromoperoxidase of marine diatoms. Six of 11 species showed significant release compared to controls. Polar species were particularly active, releasing 0.6-180 fmol HOBr cell21 h21 (0.04-1.8 mmol HOBr (mg total chlorophyll)21 h21; at the seawater bromide concentration, 840 mmol L21) and 1.9-271 fmol HOI cell21 h21 (0.02-2.7 mmol HOI (mg total chlorophyll)21 h21, at 100 mmol L21 iodide). Porosira glacialis consistently showed the highest rates of release. Several temperate diatoms, including Achnanthes cf longipes, known to have a bromide-sensitive peroxidase involved in stalk formation, and warm- water species also showed the ability to release reactive bromine and iodine. This release was influenced by light, temperature, bromide (and iodide) concentration, H2O2 concentration, and pH. The rate of HOBr release by polar diatoms was much greater than bromoform emissions measured by others from laboratory cultures and sea- ice algae in the field. This indicates that most of the HOBr released may react with dissolved organic matter (DOM) to form nonvolatile bromine organics. Some fraction of diatom-produced HOBr and HOI may also form volatile Br2 and I2, which could transfer to the polar troposphere. The reaction of diatom-released reactive bromine and iodine with seawater DOM may represent the major mechanism in the formation of oceanic polybromo- and polyiodo-methanes.
... Unialgal cultures of Phaeodactylum tricornutum Bohlin (CCMP 632) were obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton, USA. The diatom was cultured in either f/2-enriched ASPM media (Johnson et al. 1995), f/2-enriched natural seawater (Guillard 1975), or f/2 agar (Lewin et al. 1958) at 181 C with 12:12 light:dark at light intensity of approximately 100 mmol photons Á m À 2 Á s À 1 . In order to maintain axenic cultures, inocula were incubated overnight in media containing 15 mg/mL of penicillin-G and kanamycin. ...
... Effect of phosphate levels. Cultures were grown in f/2-enriched ASPM media because this artificial media enabled us to control the exact P-level in the media (Johnson et al. 1995). Cultures were grown in P-replete (PO 4 3 À concentration 36.3 mM, N/P ratio 24) and P-limited (PO 4 3 À concentration 12.1 mM, N/P ratio 73) conditions. ...
The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media-soluble polymers (CL and cEPS) were enriched in 4-linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3-linked xylosyl residues, whereas HW polymers consisted mainly of 3-linked glucosyl as well as terminal and 2,4-linked glucuronosyl residues. The HB fraction was enriched in terminal and 2-linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3- and 3-linked mannosyl residues. The fusiform morphotype predominated in standard and P-limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O-methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5- to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O-methylated sugars, uronic acids, and sulfate. The increased deoxy- and O-methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross-linking, suggesting that these changes could ameliorate the effects of salinity or P-stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.
... Rost et al. [39] found similar trends in Sierra Nevada streams. The stalk of D. geminata is primarily sulphated polysaccharide [40]; therefore, sulphur limitation may inhibit stalk formation as observed in the stalkforming marine diatom Achnanthes longipes C. Agardh [41]. The presence of D. geminata in Norway was associated with a minimum sulphate concentration of 2.5 mg l −1 . ...
Didymosphenia geminata (Lyngbye) M. Schmidt is a stalked freshwater diatom that is expanding its range globally. In some rivers, D. geminata forms thick and expansive polysaccharide-dominated mats. Like other stalked diatoms, D. geminata cells attach to the substratum with a pad of adhesive extracellular polymeric substance. Research on D. geminata and other diatoms suggests that bacterial biofilm composition may contribute to successful attachment. The aim of this study was to investigate the composition and role of bacterial biofilm communities in D. geminata attachment and survival. Bacterial biofilms were collected at four sites in the main stem of a river (containing D. geminata) and in four tributaries (free of D. geminata). Samples were characterised using automated rRNA intergenic spacer analysis and high-throughput sequencing (HTS). Mat-associated bacteria were isolated and their effect on the early establishment of D. geminata cells assessed using co-culturing experiments. ARISA and HTS data showed differences in bacterial communities between samples with and without D. geminata at two of the four sites. Samples with D. geminata had a higher relative abundance of Sphingobacteria (p < 0.01) and variability in community composition was reduced. Analysis of the 76 bacteria isolated from the mat revealed 12 different strains representing 8 genera. Co-culturing of a Carnobacterium sp. with D. geminata reduced survival (p < 0.001) and attachment (p < 0.001) of D. geminata. Attachment was enhanced by Micrococcus sp. and Pseudomonas sp. (p < 0.001 and p < 0.01, respectively). These data provide evidence that bacteria play a role in the initial attachment and on-going survival of D. geminata, and may partly explain observed distribution patterns.
... Very few studies (Johnson et al. 1995, Lewis et al. 2002, Kilroy & Bothwell 2011 have been conducted to define the conditions promoting stalk formation in fouling diatoms. However, EPS production has been extensively investigated in several marine benthic and planktonic diatoms (see review by Hoagland et al. 1993). ...
Submerged natural and artificial marine substrata can rapidly become covered by biofouling organisms, of which diatoms are the early algal colonisers. Licmophora flabellata is an estuarine stalk-forming diatom that strongly adheres to substrata via a compound-branching polysaccharide stalk. In the present study, the effects of light, nutrients, temperature and turbulence were examined in laboratory cultures to determine how they affected stalk formation and growth rates. Growth rates in multiwell plates were estimated using in vivo fluorescence in a plate reader. Low light intensities (50, 100 and 150 µmol photons m−2 s−1) produced no or poor growth, whereas growth rates of 0.24-0.42 divisions per day were achieved at 233 µmol photons m−2 s−1. High growth rates coincided with long stationary growth periods (21-36 days) and long branching stalks (800-3500 µm). N, P and Si nutrients had no effect on growth or stalks, whereas high turbulence (from an orbital shaker) reduced stalk length but not growth. The results call for species-specific approaches towards mitigating the impact of fouling diatoms.
... Adhesion usually requires an oxidase mediated polymerisation of quinine (Vreeland et al., 1998;Hellio et al., 2000a). There are some common adhesion mechanisms among micro and macroalgae, such as the involvement of bromide for stalk assembly and adhesion (Johnson et al., 1995;Wustman et al., 1997;Passow and Alldredge, 1995;Waite et al., 1997) or of vanadium for the cross-linking of polyphenols to extracellular carbohydrate fibers (Vreeland and Epstein, 1996). Testing new AF compounds for their inhibitory activities towards various oxidases such as haloperoxidase, bromoperoxidase or similar oxidase-activated mechanisms could the next breakthrough for the development of fast, reliable, targeted and nonexpensive anti-algal assays. ...
Human impacts on coastal waters, through eutrophication and overfishing, associated with the global change phenomenon are leading to a proliferation of both micro- and macroalgae. Algae can settle and develop on a wide range of surfaces including both natural and man-made structures. They are fast colonisers and can out-compete numerous species. The fixation of marine organisms on immersed man-made structures is responsible for major economic costs and the recent recrudescence of algae in the environment might increase the fouling phenomenon observed: the resistance penalty for ships is known to be increased by 11% in presence of a light microalgal slime to 34% in case of colonisation by macroalgae (Schultz, 2007). The paints used nowadays often contain biocides, are non selective against target organisms and are not environmentally friendly. The development of new antifouling paints requires a better understanding of both the mechanisms of fixation and colonisation of the fouler organisms. This chapter focuses on the fixation of algae (both micro and macro), the damages they cause and the method of prevention currently used.
... In marine macroalgae, the enzyme vanadiumbromoperoxidase is essential for production of bromoform that plays an important role in eliminating epiphytic microalgae (Ohsawa et al. 2001). In species of biofouling diatoms, an absolute requirement of the same enzyme for substratum attachment has been reported (Johnson et al. 1995). An elevated concentration of vanadium has also been demonstrated to have an inhibitory effect on the photosynthesis of phytoplankton (Nalewajko et al. 1995). ...
Abundance and frequency of dinoflagellate cysts in 19 surface sediment samples from the northern part of the Swedish west coast has been related to physical and chemical characters of the sediment, hydrography of the overlying water column, and plankton species data from the area. Density of cysts varied between 5000 and 101000 cysts g-1 dw, and the most commonly encountered species were Lingulodinium polyedrum and Protoceratium reticulatum. In all, 46 environmental variables were tested for their relation to dinoflagellate cyst densities, proportion of autotrophic and heterotrophic taxa, and individual species distribution and frequency. The outcomes of multivariate analyses, projection to latent structures (PLS) and canonical correspondence analysis (CCA) were consistent with each other and the actual cyst count. The density of the total cyst assemblage (>90% autotrophic taxa) was primarily related to surface temperature, macronutrients, and inversely to phytoplankton competitors, such as diatoms. The abundance of heterotrophic taxa was governed by the preferences of their prey, i.e. diatom-favourable conditions, and, in most cases, higher proportions of heterotrophic taxa were found at well-mixed sites. Some possible effects of anthropogenic contaminants were also noted. Several taxa showed distinct distribution patterns with respect to the environmental variables. A discrepancy between the species constituting the planktonic and the benthic community was revealed when data from 6 yr of plankton monitoring was compared to the data on distribution of dinoflagellate cysts. In particular, cyst-forming species were only a minor part of the plankton, suggesting that these dinoflagellates spend much of their life in the sediments.
... Achnanthes exhibits an absolute requirement for bromide for stalk production and substratum attachment, while elevated iodide concentrations in the growth medium inhibit insoluble extracellular polymer formation and adhesion (Johnson, 1995;Johnson, et al., 1995). Alterations in bromide and iodide levels resulted in varying extracellular adhesive morphologies (including pads, stalk-pads and none). ...
We have answered fundamental questions regarding the mechanisms of attachment and the nature of the biocomposite adhesives utilized by the marine biofouling diatom Achnanthes longipes and the freshwater Cymbella cistula. During the course of this grant we have: (1) delineated the sequence of events involved in attachment of the organisms to a variety of surfaces; (2) discovered that initial adhesion is mediated by different methods/polymers on hydrophilic surfaces vs. those more hydrophobic and that bacterial 'preconditioning' has variable effects on adhesion; (3) developed methodology for mass culture of fouling diatoms and isolation of adhesive components; (4) characterized the 'proteoglycan' bioadhesives using monosaccharide and methylation analysis, NMR and other analytical techniques; (5) localized specific carbohydrate moieties of the adhesive with lectins and produced monoclonal antibodies against the adhesives and applied them as probes of structure/function of the adhesives; (6) determined that adhesion is disrupted at several levels by dichlorobenzonitrile (DCB) and related specific inhibitors of plant extracellular polysaccharide synthesis; (7) ascertained that DCB and other potential anti-fouling chemicals act intracellularly on an 18 KD membrane associated protein and that DCB doped polyimide surfaces do not inhibit adhesion; (8) discovered that adhesive structures are not assembled in the presence of high concentrations of iodide and that bromide is a limiting requirement for adhesion; and (9) created an expression library to screen for a 50 kD polypeptide from the adhesive and a peroxidase involved in crosslinking the adhesive.
... Nevertheless, the option of studying D. geminata in a mixed algal community colonizing outdoor channels has proved successful, because, as has been pointed out previously (Whitton et al. 2009), the large size of D. geminata makes it an ideal model organism. Direct measurements of diatom stalk length have rarely been employed in previous studies on species-environment relationships, exceptions being studies on A. longipes (Johnson et al. 1995, Lewis et al. 2002. In the present experiments, our ability to accurately measure lengths of individual stalk segments was facilitated by the reaction of methylene blue with anionic polysaccharides providing contrast between the stalks and the non-staining polystyrene (Styrofoam TM ) substratum. ...
Blooms of the freshwater stalked diatom Didymosphenia geminata (Lyngb.) M. Schmidt in A. Schmidt typically occur in oligotrophic, unshaded streams and rivers. Observations that proliferations comprise primarily stalk material composed of extracellular polymeric substances (EPS) led us to ask whether or not the production of excessive EPS is favored under nutrient-limited, high-light conditions. We conducted experiments in outdoor flumes colonized by D. geminata using water from the oligotrophic, D. geminata–affected Waitaki River, South Island, New Zealand, to determine the relationship between D. geminata stalk length, cell division rates, and light intensity under ambient and nutrient-enriched conditions. Stalk lengths were measured in situ, and cell division rates were estimated as the frequency of dividing cells (FDC). FDC responded positively to increasing light intensity and to nutrient additions (N+P and P). Under ambient conditions, stalk length increased as light level increased except at low ambient light levels and temperature. Nutrient enrichment resulted in decreased stalk length and negative correlations with FDC, with this effect most evident under high light. Our results are consistent with the hypothesis that extensive stalk production in D. geminata occurs when cell division rates are nutrient limited and light levels are high. Thus, photosynthetically driven EPS production in the form of stalks, under nutrient-limited conditions, may explain the development of very high biomass in this species in oligotrophic rivers. The responses of FDC and stalk length under nutrient-replete conditions are also consistent with occurrences of D. geminata as a nondominant component of mixed periphyton communities in high-nutrient streams.
... The dominant diatom species in Tarraleah No. 1 Canal is Gomphonema, a raphid diatom with cells 35 µm long and 6 µm wide (Fig. 1a). Gomphonema secretes a mucous stalk which pushes the cells away from the wall (Fig. 1b) towards better light and nutrient conditions [9]. The stalks observed from the fouling at Tarraleah are hundreds of microns long, tens of times the size of the cell itself. ...
Freshwater biofilms are currently being studied to determine their effect on the capacity of hydroelectric power scheme canals and the structure of their turbulent wall layers. Mean velocity boundary layer profiles and total drag measurements have been conducted in a purpose built recirculating water tunnel on freshwater biofilms grown in a hydroelectric canal. Two different fouled surfaces were compared with a smooth painted surface to determine the effects of the physical characteristics of a biofilm on skin friction drag. A 310% increase in local skin friction coefficient was measured for a biofilm dominated by long filamentous algae streamers, and a 50% increase was measured for a biofilm dominated by a low-form gelatinous diatom.
... Therefore, significant amounts of SO 4 2may be required to support stalk production during bloom conditions. In fact, a previous study shows that in the absence of sulfur, the stalk forming marine diatom, Achnanthes longipes, is unable to produce stalks (Johnson et al., 1995). Although SO 4 2typically occurs in quantities sufficient to supply biological demands, nuisance blooms of D. geminata may present unique circumstances. ...
The freshwater stalked diatom, Didymosphenia geminata, has increased its proliferation in new and native ranges with the potential for significant ecological impacts to streams
around the world. Uncertainty remains regarding the water chemistry in which D. geminata thrives despite recent advances in the understanding of its suitable physical habitat. Stream surveys were conducted in the
central and the northern Sierra Nevada, CA, USA, to determine whether D. geminata distribution patterns were related to water chemistry and bedrock geology. Surveys included measurements of physical, chemical,
and biological parameters from 50 stream reaches in 35 different streams. The water chemistry variable, percent Ca2+ (P<0.001) provided the best fit models (binary logistic regression AUC 0.84) for predicting the presence of D. geminata in Sierra Nevada streams. Other significant water chemistry predictors included the percent SO4
2− and Mg2+ concentrations. The bedrock geology variable, percent meta-sedimentary rock in a watershed (P<0.001), provided the best fit model (binary logistic regression AUC 0.77) for predicting the presence of D. geminata. This study further defines the environmental conditions in which D. geminata thrives, provides insight into D. geminata ecophsyiology, and will help refine current distribution and risk assessment models.
Keywords
Didymosphenia geminata
–Stalked diatoms–Water chemistry–Bedrock geology–Nuisance algal blooms
... As with a number of other fouling diatom species Gomphonema secretes a mucous stalk which pushes the cells away from the wall (Figure 2b). It is believed stalking is used to push crowded cells towards better light and nutrient conditions [12]. The stalks observed from the fouling at Tarraleah are hundreds of microns long, tens of times the size of the cell itself. ...
Gomphonema, a freshwater diatom, is currently being studied to determine its effect on the capacity of hydroelectric canals and the structure of turbulent boundary layers. Gomphonema is the primary fouling organism present in Tarraleah No.1 Canal (operated by Hydro Tasmania) and was found to cause a 10% reduction in flow carrying capacity. Filamentous algae streamers up to 200mm long have also been observed in the canal. A recirculating water tunnel was used to measure the characteristics of the boundary layer flow over a 997mm x 597mm test plate covered with a biofilm grown in the field in an attempt to better understand the mechanisms for the increase in drag. Mean velocity profiles for the fouled test plate have been compared with results for a smooth test plate.
... In marine macroalgae the enzyme vanadium-bromoperoxidase is essential for production of bromoform that plays an important role in eliminating epiphytic microalgae (Ohsawa et al. 2001). In species of biofouling diatoms an absolute requirement of the same enzyme for substratum attachment has been reported (Johnson et al. 1995). An elevated concentration of vanadium has also been demonstrated to have an inhibitory effect on the photosynthesis of phytoplankton (Nalewajko et al. 1995). ...
Thesis (doctoral)--Göteborg University, 2002.
... The physiological requirement for bromine is poorly documented in diatoms, with most reports confined to the red and brown algae (Fries 1966;McLachlan and Craigie 1967;Wolk 1968). However, Johnson et al. (1995) recently demonstrated a nutritional requirement of bromine in the stalked marine diatom Achnanthes. Extracellular polymeric substance (EPS) secretion in this species apparently exhibits an absolute requirement for bromide as both stalk formation and adhesion require this compound. ...
Here we introduce a combined experimental and descriptive approach (termed resurrection ecology) to reconstructing historical perturbations, pointing out how direct tests with sediments and hatched resting eggs complement the traditional descriptive calculation of microfossil fluxes. In the Keweenaw Waterway, a freshwater estuary off Lake Superior, turn-of-the-century copper mining impacted the resident biota. Remain fluxes document that diatom, rhizopod, and Bosmina production all declined during stamp sand discharges but recovered rapidly after World War II, moving above background levels due to developing eutrophication. In addition to biogenic silica, we discovered that bromine flux holds promise as an indicator of diatom production and confirmed that this element is present in several genera. Fluxes of Daphnia resting eggs also increased dramatically since the 1940s, dominated by a hybrid apparently produced from crosses between offshore and interior Waterway species, after channeling promoted greater mixing of water masses. Toxicity studies with sediments and Daphnia clones directly tested recovery of environments after cessation of mining activities. The studies document that increased concentrations and fluxes of copper in the Waterway during mining discharges were toxic to invertebrates. Once stamp sand discharges ceased,the biota recovered rapidly due to a combination of decreased copper cycling and organic complexation. Although sedimentation has returned to near-background conditions and surficial sediments in much of Portage Lake are no longer toxic, eutrophication and faunal exchange with Lake superior make it unlikely that the original zooplankton community composition will return to the Waterway system.
... That A. Zongipes polymers are organized and assembled into networks by phenolic cross-linking components is suggested by several results: (a) the blue autofluorescence of the stalks when excited by UV light >360 nm; (b) Johnson et al. (1995) have characterized a bromide dependence for stalk synthesis in A. longipes and also found that iodide inhibits stalk production; and (c) V. Vreeland (personal communication) found preliminary cytochemical evidence for peroxidase activity within intracellular vesicles in A. longipes cells. Phenolic compounds such as dityrosine, ferulic acid, feruloyl esters, caffeic acid, and sinapic acid are known to fluoresce blue when exposed to 366-nm UV light (Fry, 1995). ...
Extracellular adhesives from the diatoms Achnanthes longipes, Amphora coffeaeformis, Cymbella cistula, and Cymbella mexicana were characterized by monosaccharide and methylation analysis, lectin-fluorescein isothiocyanate localization, and cytochemical staining. Polysaccharide was the major component of adhesives formed during cell motility, synthesis of a basal pad, and/or production of a highly organized shaft. Hot water-insoluble/hot 0.5 M NaHCO3-soluble anionic polysaccharides from A. longipes and A. coffeaeformis adhesives were primarily composed of galactosyl (64-70%) and fucosyl (32-42%) residues. In A. longipes polymers, 2,3-, t-, 3-, and 4-linked/substituted galactosyl, t-, 3-, 4-, and 2-linked fucosyl, and t- and 2-linked glucuronic acid residues predominated. Adhesive polysaccharides from C. cistula were EDTA-soluble, sulfated, consisted of 83% galactosyl (4-, 4,6-, and 3,4-linked/substituted) and 13% xylosyl (t-, 4f/5p-, and 3p-linked/substituted) residues, and contained no uronosyl residues. Ulex europaeus agglutinin uniformly localized [alpha](1,2)-L-fucose units in C. cistula and Achnanthes adhesives formed during motility and in the pads of A. longipes. D-Galactose residues were localized throughout the shafts of C. cistula and capsules of A. coffeaeformis. D-Mannose and/or D-glucose, D-galactose, and [alpha](t)-L-fucose residues were uniformly localized in the outer layers of A. longipes shafts by Cancavalia ensiformis, Abrus precatorius, and Lotus tetragonolobus agglutinin, respectively. A model for diatom cell adhesive structure was developed from chemical characterization, localization, and microscopic observation of extracellular adhesive components formed during the diatom cell-attachment process.
... The pad then tears in half, producing the 'collar' adhered to the cell, additional to the adhesive pad on the substrate, after which the shaft is secreted from the raphe binding the two, thus forming the stalk (Figure 11e,f). In ideal growth conditions, the stalk can grow to a length of 500 mm (Johnson et al. 1995). Once attached via a stalk, a cell may divide several times, forming a large stack (ie colony) of cells adhered to a single adhesive stalk. ...
Diatoms are a major component of microbial slimes that develop on man-made surfaces placed in the marine environment. Toxic antifouling paints, as well as environmentally friendly, fouling-release coatings, tend to be effective against most fouling organisms, yet fail badly to diatom slimes. Biofouling diatoms have been found to tenaciously adhere to and colonise even the most resistant of artificial surfaces. This review covers the basic biology of fouling marine diatoms, their mechanisms of adhesion and the nature of their adhesives, as well as documenting the various approaches that have been utilised to understand the formation and maintenance of diatom biofouling layers.
This cooperative Augmentation Award for Science and Engineering Research Training has provided training for two doctoral students in several important areas of marine microalgal biofouling, including marine diatom mass culturing and adhesive isolation, biochemistry of adhesives, and laboratory experiments on the effects of substrate preconditioning on diatom adhesion. Students have gained significant experience through exchange between cooperating labs. This grant augmented research described in "parent" award #N000T4-94-1-0273. The overall goal of this research has been to provide a better understanding of the diatom portion of the biofouling process, to facilitate the development of more effective methods to control marine biofouling. We have identified bromide as an absolute requirement for the formation of the principal attachment organ for a common biofouling diatom, which provides a unique and powerful tool for initiating synchronous stalk production on a large scale and facilitating further extracellular adhesive biosynthesis, secretion and physiology research. Several monoclonal antibodies directed against the adhesive of Achnanthes were produced, allowing for effective isolation, localization and analysis of adhesive components. Detailed chemical characterization of antibody epitopes in adhesives from Achnanthes provides requisite information to determine the nature of the interactions responsible for assembly and function of the adhesives.
Many experiments utilize static immersion tests to evaluate the performance of ship hull coatings. These provide valuable data; however, they do not accurately represent the conditions both the hull and fouling organisms encounter while a ship is underway. This study investigated the effect of static and dynamic immersion on the adhesion and settlement of diatoms to one antifouling coating (BRA 640), four fouling-release coatings (Intersleek(®) 700, Intersleek(®) 900, Hempasil X3, and Dow Corning 3140) and one standard surface (Intergard(®) 240 Epoxy). Differences in community composition were observed between the static and dynamic treatments. Achnanthes longipes was present on all coatings under static immersion, but was not present under dynamic immersion. This was also found for diatoms in the genera Bacillaria and Gyrosigma. Melosira moniformis was the only diatom present under dynamic conditions, but not static conditions. Several common fouling diatom genera were present on panels regardless of treatment: Amphora, Cocconeis, Entomoneis Cylindrotheca, Licmophora, Navicula, Nitzschia, Plagiotropis, and Synedra. Biofilm adhesion, diatom abundance and diatom diversity were found to be significantly different between static and dynamic treatments; however, the difference was dependent on coating and sampling date. Several coatings (Epoxy, DC 3140 and IS 700) had significantly higher biofilm adhesion on dynamically treated panels on at least one of the four sampling dates, while all coatings had significantly higher diatom abundance on at least one sampling date. Diversity was significantly greater on static panels than dynamic panels for Epoxy, IS 700 and HX3 at least once during the sampling period. The results demonstrate how hydrodynamic stress will significantly influence the microfouling community. Dynamic immersion testing is required to fully understand how antifouling surfaces will respond to biofilm formation when subjected to the stresses experienced by a ship underway.
The attachment of diatoms to surfaces is an important and poorly understood step in the development of biofouling communities. Experiments were performed in vitro on a common fouling diatom (Achnanthes longipes) to determine the influence of the base material and bacterial conditioning on diatom attachment. The first series of experiments compared attachment of A. longipes to four different base materials, and the influence of a bacterial film on attachment to these materials. A. longipes preferentially attached to polystyrene, a hydrophobic surface, but was inhibited by the presence of a bacterial biofilm. On other surfaces, bacteria either facilitated or had no effect on algal attachment. The second series of experiments found no difference in the attachment of A. longipes to a surface covered with bacterial exopolymer compared to a surface with a film of living bacteria. Attachment of A. longipes was found to vary depending on the conditions under which the bacterial film developed and the species of bacteria within the film. These results help to illustrate the complexity of the relationship between surfaces and attaching organisms and show that bacteria may either facilitate, have no effect or inhibit attachment by diatoms. The mechanisms underlying these patterns require further investigation.
Achnanthes longipes Ag. is a marine stalk-forming diatom that grows in dense biofilms. The effects of cell density, temperature, and light on growth and stalk production were examined in the laboratory to determine how they affected the ability of this diatom to form a biofilm. Stalk production abruptly increased when A. longipes was cultured at a density of 5.4 × 103 cells·mL−†, with a lag before stalk production occurring in cultures initiated at lower densities. Growth occurred at all temperatures from 8 to 32° C, with maximum growth at 26° C. Growth rate was light saturated at 60 μmol photons·m−2·s−†. Stalk production was determined as the proportion of cells producing stalks and stalk length in response to various temperatures and light intensities at high (5000 cells·mL−†) and low (500 cells·mL−†) densities. More cells formed stalks at high density, with no difference in stalk length. The proportion of cells producing stalks was maximal at 20° C, with little change at 17–32° C. Stalk length was at a maximum between 14 and 26° C. Stalk production showed little change in response to varying light intensity. The results of an earlier investigation on the effects of bromide concentration on stalk formation were expressed as the proportion of cells forming stalks and the lengths of the stalks. Both measures of stalk production varied with bromide concentration, with maximum values at 30 mM bromide. The increased stalk production at higher densities may be a means of elevating cells above the substrate to avoid competition in the dense biofilm.
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Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high-resolution visualization of surfaces using cryo-field emission SEM (cryo-FESEM) and preservation for TEM observation of thin sections by high-pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge-frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo-FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe-associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo-FESEM revealed a sheet-like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra- and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron-opaque layer organized as a sheath at the stalk periphery.
Brown algae, such as kelps and fucoids, occur over large areas of the subtidal and intertidal rocky shores, including tropical reef habitats, producing high biomass and determining the structure of the ecosystem (i.e. kelp forests). Brown algae live firmly attached to the substratum and are often exposed to high gradients of turbulence. Therefore, they experience drag and lift forces of currents and waves with velocities that may exceed 10 m/s. As other sessile marine organisms, to disperse in certain phases of their life histories and to survive in such a stressful environment, they had to evolve strategies to adhere strongly and durably underwater from the microscopic reproductive cell stages to the large thalli of giant kelps. Brown algal filamentous species such as Ectocarpus were also identified as major ship fouling organisms in the 1960s, when the use of anti-fouling paints containing organo-metallic compounds–which was successful against fouling green algae–enabled Ectocarpus to bloom (Baker and Evans 1973). Unfortunately, studies on adhesion mechanisms of marine brown algal adhesives have consisted mostly of analogy and hypothesis (Vreeland et al. 1998). Better characterization of these adhesives will eventually lead to new strategies to prevent biofouling or to produce or design synthetic water-resistant adhesives of commercial value. The properties of the adhesives in terms of spreading and curing underwater are crucial to successful colonization of substrata. However, much of the evidence on the composition of algal adhesives is circumstantial and based on methodologies such as histochemistry (Fletcher and Baier 1984; Gonzales and Goff 1989; Fletcher and Callow 1992; Callow and Callow 2002). These studies indicate that macroalgal adhesion involves carbohydrate and glycoprotein-containing mucilage (Fletcher and Callow 1992) that ‘cure’ with time after discharge, thereby increasing the strength of attachment to the substratum. In contrast with the progress in the chemical characterization of diatom adhesives (Chamberlain 1976; Stossel 1993; Lind et al. 1997; Wustman et al. 1997; Wetherbee et al. 1998; Higgins et al. 2002), the composition and physicochemical nature of adhesives used by different macroalgae remain unknown. Therefore, it is difficult to infer by which processes they
A programme of research was initiated to provide information on the efficacy of the antifouling agents Irgarol 1051™ and Sea-Nine 211™ to zygotes of the fucoid alga Fucus serratus Linnaeus. The development of methodologies that incorporated the use of image capture and analysis, which minimised the problems of subjectivity and bias that can be typical, are also described. The generation of macro-instructions allowed rapid generation and interpretation of accurate quantitative data. Emphasis was placed on the use of methodologies that did not rely on the subjective determination of spore germination. Consequently, affects due to the biocides were determined that, otherwise, would not have been realised through normal analytical routines.Zygotes were exposed, in static non-renewal systems, for 3 days, to a range of concentrations of biocide dissolved in seawater. Data for seven zygote parameters were recorded daily and values of percentage germination were calculated. From these data, NOEC values of 8 μg l−1 were calculated for both Sea-Nine 211 and Irgarol 1051. An EC50 of 19.4 μg l−1 was also determined for Sea-Nine 211. It is, therefore, suggested that F. serratus zygote germination and early development is unaffected by published environmental concentrations of these biocides. Additionally, it has been demonstrated that the image capture and analysis methodologies developed provide an invaluable tool for use in future bioassays.
The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.
We have shown that a phenolic polymer (PP) extracted from Fucus serratus can be cross-linked using a vanadium-dependent bromoperoxidase (BPO). The methanol extracted PP was adsorbed to a quartz crystal sensor and the cross-linking was initiated by the addition of BPO, KBr, and H2O2. The decreased dissipation upon addition of the cross-linking agents, as measured with the quartz crystal microbalance with dissipation monitoring (QCM-D) method, was interpreted as intramolecular cross-links were formed between different phloroglucinol units in the PP. With surface plasmon resonance, it was shown that no desorption occurred from the sensor surface during the cross-linking. UV/vis spectroscopy verified the results achieved with QCM-D that all components, i.e., BPO, KBr, and H2O2, were necessary in order to achieve intramolecular oxidative cross-linking of the polymer.
Diatoms produce diverse three-dimensional structures that, due to their exponential rate of growth, may be of use in the manufacture of components for nanotechnology as an alternative to present linear lithographic techniques. Vapor replacement of the silicon permits the conversion of diatom silica valves and other structures to metal/ceramics, with no loss of structure. The literature on diatom nanotechnology is reviewed, along with suggestions on how diatomists might enhance this emerging technology. There is a need for a systematics based catalog of parts (via genomics technologies), improved diatom culture techniques, better understanding of the mechanisms of diatom morphogenesis and motility, and genetic manipulation, mutagenesis, and selection, as via the chemostat-like compustat. Given the self-motility of raphid diatoms, they could form the basis for industrially useful nanobots.
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