The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α-Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50-90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.     

Effect of biofilm age on settlement of Mytilus edulis

Biofilm ageing is commonly assumed to improve mussel settlement on artificial substrata, but the structure and taxonomic composition of biofilms remains unclear. In the present study, multi-species biofilms were characterized at different ages (1, 2, and 3 weeks) and their influence on settlement of the blue mussel, Mytilus edulis, was tested in the field. As biofilms can constitute a consistent food resource for larvae, the lipid quality, defined as the proportion of related essential fatty acids, may be a selection criterion for settlement. Overall mussel settlement increased on biofilms older than 1 week, and the enhanced settlement corresponded to the abundance and composition of the biofilm community, rather than to essential fatty acid levels. However, during a pulse of phytoplankton, the positive influence of biofilm was not detected, suggesting that pelagic cues overwhelmed those associated with biofilms. The influence of biofilms on mussel settlement could be more crucial when planktonic resources are limited.     

Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae

Bacterial biofilms are increasingly seen as important for the successful settlement of marine invertebrate larvae. Here we tested the effects of biofilms on settlement of the sea urchin Heliocidaris erythrogramma. Larvae settled on many surfaces including various algal species, rocks, sand and shells. Settlement was reduced by autoclaving rocks and algae, and by treatment of algae with antibiotics. These results, and molecular and culture-based analyses, suggested that the bacterial community on plants was important for settlement. To test this, approximately 250 strains of bacteria were isolated from coralline algae, and larvae were exposed to single-strain biofilms. Many induced rates of settlement comparable to coralline algae. The genus Pseudoalteromonas dominated these highly inductive strains, with representatives from Vibrio, Shewanella, Photobacterium and Pseudomonas also responsible for a high settlement response. The settlement response to different bacteria was species specific, as low inducers were also dominated by species in the genera Pseudoalteromonas and Vibrio. We also, for the first time, assessed settlement of larvae in response to characterised, monospecific biofilms in the field. Larvae metamorphosed in higher numbers on an inducing biofilm, Pseudoalteromonas luteoviolacea, than on either a low-inducing biofilm, Pseudoalteromonas rubra, or an unfilmed control. We conclude that the bacterial community on the surface of coralline algae is important as a settlement cue for H. erythrogramma larvae. This study is also an example of the emerging integration of molecular microbiology and more traditional marine eukaryote ecology.     

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.     

Antifouling properties of zinc oxide nanorod coatings

In laboratory experiments, the antifouling (AF) properties of zinc oxide (ZnO) nanorod coatings were investigated using the marine bacterium Acinetobacter sp. AZ4C, larvae of the bryozoan Bugula neritina and the microalga Tetraselmis sp. ZnO nanorod coatings were fabricated on microscope glass substrata by a simple hydrothermal technique using two different molar concentrations (5 and 10?mM) of zinc precursors. These coatings were tested for 5?h under artificial sunlight (1060?W?m^?2 or 530?W?m^?2) and in the dark (no irradiation). In the presence of light, both the ZnO nanorod coatings significantly reduced the density of Acinetobacter sp. AZ4C and Tetraselmis sp. in comparison to the control (microscope glass substratum without a ZnO coating). High mortality and low settlement of B. neritina larvae was observed on ZnO nanorod coatings subjected to light irradiation. In darkness, neither mortality nor enhanced settlement of larvae was observed. Larvae of B. neritina were not affected by Zn²⁺ ions. The AF effect of the ZnO nanorod coatings was thus attributed to the reactive oxygen species (ROS) produced by photocatalysis. It was concluded that ZnO nanorod coatings effectively prevented marine micro and macrofouling in static conditions.     

The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals

Microbial biofilms induce larval settlement for some invertebrates, including corals; however, the chemical cues involved have rarely been identified. Here, we demonstrate the role of microbial biofilms in inducing larval settlement with the Caribbean coral Porites astreoides and report the first instance of a chemical cue isolated from a marine biofilm bacterium that induces complete settlement (attachment and metamorphosis) of Caribbean coral larvae. Larvae settled in response to natural biofilms, and the response was eliminated when biofilms were treated with antibiotics. A similar settlement response was elicited by monospecific biofilms of a single bacterial strain, Pseudoalteromonas sp. PS5, isolated from the surface biofilm of a crustose coralline alga. The activity of Pseudoalteromonas sp. PS5 was attributed to the production of a single compound, tetrabromopyrrole (TBP), which has been shown previously to induce metamorphosis without attachment in Pacific acroporid corals. In addition to inducing settlement of brooded larvae (P. astreoides), TBP also induced larval settlement for two broadcast-spawning species, Orbicella (formerly Montastraea) franksi and Acropora palmata, indicating that this compound may have widespread importance among Caribbean coral species.     

Adrenoceptor Compounds Prevent the Settlement of Marine Invertebrate Larvae: Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) and Hydroides elegans (Polychaeta)

The effects of the neurotransmitter blockers idazoxan and phentolamine on the larval settlement of three marine invertebrate species belonging to three different phyla were investigated by using in vitro concentration-response bioassays. Since neurotransmitters are known to influence metamorphic transitions in invertebrate larvae, neurotransmitter blockers were tested to evaluate their sublethal effects on larvae. The α-adrenergic antagonists idazoxan and phentolamine inhibited settlement of Balanus amphitrite (Cirripedia), Bugula neritina (Bryozoa) larvae, and larvae of the polychaete Hydroides elegans (Polychaeta) in a concentration- and taxon-dependent manner. At concentrations of 10^?3 M of both agents, larvae of all three species became immobile and subsequently died within 24 h. While cumulative settlement rates were observed after 48 h for B. amphitrite and H. elegans, and after 5 h for B. neritina, > 90% of the larvae that settled did so within 24 h for the first two species and within 1 h for B. neritina. The tendency of the hydrophobic idazoxan and phentolamine to accumulate at solid surfaces most probably contributes to their successful inhibition of larval settlement. This ability makes them particularly attractive as candidates for the development of slow-release carriers in antifouling paints.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

Larval settlement and metamorphosis in the slipper limpet Crepidula onyx (Sowerby) in response to conspecific cues and the cues from biofilm

In the marine environment, aggregated distribution in the genus Crepidula is a very common phenomenon. Works from Pechenik's group suggested that this is the result of gregarious settlement of larvae in response to cues associated with conspecific adults. In this study, we investigated the existence of larval metamorphic cues associated with adults of C. onyx, a slipper limpet introduced to Hong Kong from the U.S. in the 1970s, through a series of laboratory bioassays. The results showed that derived cues in adult C. onyx were waterborne and the waterborne cues were not derived from bacteria associated with the shell and soft body of the adult Crepidula. The natural biofilm also induced the larval metamorphosis of C. onyx. The cues from the biofilm were associated with the surface of the biofilm and were not waterborne. The aggregated distribution in nature of adult C. onyx may result from a selective larval settlement process. On a small scale in the water column near the conspecific adults, larvae of C. onyx initially detect the waterborne conspecific cues, which then lead to positive downward swimming or passive sinking. This activity increases the chances for larvae to make contact with the biofilm and to be exposed into the higher concentration of waterborne conspecific cues. This may eventually lead to the enhanced larval settlement pattern on or near the conspecific adults.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Facilitation and inhibition of larval attachment of the bryozoan Bugula neritina in association with mono-species and multi-species biofilms

In this study, we investigated the effect of mono-species and multi-species biofilms on larval attachment of the bryozoan Bugula neritina. The effect of biofilms was examined through a double-dish choice bioassay in which larvae were given the choice of attaching either to a clean surface of a container or to surfaces covered with biofilms. Larvae attached in response to mono-species biofilms of 5 out of 7 bacterial isolates from a subtidal region, but they avoided surfaces covered by biofilms of 7 out of 8 isolates obtained from an intertidal region. In the follow-up choice experiments with multi-species biofilms developed for 2 days, 7 days, 14 days, 28 days and 30 days, larvae preferentially attached to filmed surfaces over the unfilmed surfaces. When biofilms from 2 different tidal regions (intertidal and subtidal) were offered as choices in the double-dish bioassay, larvae in all cases attached on the subtidal biofilms. Two-day-old subtidal biofilms with low densities of bacteria induced significantly higher (p < 0.05) attachment than did 30- day-old intertidal biofilms, which had high bacterial density. Terminal Restriction Fragment Polymorphism (T-RFLP) analysis revealed that the bacterial communities were substantially different in the subtidal and intertidal regions during all periods of the experiment. Attachment of B. neritina on subtidal biofilms did not depend on the bacterial density but rather was negatively correlated with diatom density, thickness of the exopolysaccharide layer and biofilm age. Our results suggest that the larvae of B. neritina can discriminate between biofilmed and clean surfaces and between biofilms developed under different tidal zones.     

Antifouling and antibacterial compounds from a marine fungus <Emphasis Type="Italic">Cladosporium</Emphasis> sp. F14

To investigate the antifouling secondary metabolites from marine-derived fungi, we used bioassay-guided column chromatography techniques, such as HPLC, to separate and purify compounds from Cladosporium sp. F14. Extensive spectral analyses including 1D NMR spectra and MS were employed for structure elucidation of the compounds. Antilarval activity of the compounds was evaluated in settlement inhibition assays with laboratory-reared Balanus amphitrite and Bugula neritina larvae, while antibacterial activity was assessed with disc diffusion bioassay on growth inhibition of six marine bacterial species. In total, nine compounds were obtained. Among them, 3-phenyl-2-propenoic acid, cyclo-(Phe-Pro) and cyclo-(Val-Pro) had various antibacterial activities against three fouling bacteria, furthermore, 3-phenyl-2-propenoic acid and bis(2-ethylhexyl)phthalate effectively inhibited larval settlement of B. neritina and B. amphitrite larvae, respectively, indicating that the two compounds are potential natural antifouling agents.     

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta)

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by ‘selection’ to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.     

Localization of sensory mechanisms utilized by coral planulae to detect settlement cues

Coral planulae are induced to settle and metamorphose by contact with either crustose coralline algae or marine bacterial biofilms. Larvae of two coral species, Pocillopora damicornis and Montipora capitata, which respond to different metamorphic cues, were utilized to investigate the sensory mechanisms used to detect metamorphic cues. Because the aboral pole of the coral planula is the point of attachment to the substratum, we predicted that it is also the point of detection for cues. To determine where sensory cells for cues are localized along the body, individual larvae were transversely cut into oral and aboral portions at various levels along the oral–aboral axis, and exposed to settlement‐inducing substrata. Aboral ends of M. capitata metamorphosed, while oral ends continued to swim. However, in larvae of P. damicornis, ¾ oral ends (i.e., lacking the aboral pole) were also able to metamorphose, indicating that the cells that detect cues may be distributed along the sides of the body. These cells do not correspond to FMRFamide‐immunoreactive cells that are present throughout the body. Cesium ions induced both aboral and oral ends of larvae of both species to settle, suggesting that oral ends have not lost their capacity to metamorphose, despite lacking sensory cells to detect natural cues. To determine whether sensory cells in larvae of P. damicornis are restricted to one side of the body, swimming behavior over substrata was observed in larvae labeled with diI, a red fluorescent lipophilic membrane stain. The larvae were found to rotate around the oral–aboral axis, with their surface against the substratum, not favoring a particular side for detecting cues. While clarifying the regions of the larval body important for settlement and metamorphosis in coral planulae, we conclude that significant differences between coral species may be due to differences in the distribution of sensory structures in relation to different planular sizes.     

The interplay of substrate nature and biofilm formation in regulating Balanus amphitrite Darwin, 1854 larval settlement

The settlement of marine larvae is influenced by a wide range of physical and biological factors. It is still poorly known how the nature of substrate and the biofilm can interact in regulating settlement patterns of invertebrate larvae. Here we use laboratory experiments focused on settlement behaviour of the barnacle Balanus amphitrite. The aim of this work is to understand whether: (i) the nature of substratum can affect biofilm formation and its structure, (ii) the nature of substratum can affect B. amphitrite larval settlement, (iii) the age of the biofilms and the nature of substrate can interact in influencing larval settlement.Four kinds of substrata (marble, quartz, glass, and cembonit) were biofilmed under laboratory conditions for 5, 10 and 20 days at the temperature of 28 °C. Settlement response was investigated with 5-day-old cyprids. Biofilms were quantitatively and qualitatively analysed by scanning electron microscopy. The settlement of B. amphitrite larvae significantly differed among substrata; also, the patterns of development of biofilm assemblages changed with substrate. In addition, the larval attractiveness of different substrates tends to disappear with biofilm age.     

Chemical induction of settlement and metamorphosis of Capitella capitata Sp. I (Polychaeta) larvae by juvenile hormone-active compounds

Summary

The influence of juvenile hormone (JH)-active chemicals on the settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. I of the Capitella complex has been investigated. These studies demonstrate that JH-active chemicals are able to induce settlement and metamorphosis of Capitella larvae, and that these effects may possibly be mediated by protein kinase C induction. Evidence for the presence of JH-active compounds in marine sediments is also presented, suggesting that these chemicals may serve a natural role as chemical cues for settlement and metamorphosis for Capitella larvae in the marine environment.     

Marine Biofilms as Mediators of Colonization by Marine Macroorganisms: Implications for Antifouling and Aquaculture

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.     

Settlement inhibition of fouling invertebrate larvae by metabolites of the marine bacterium halomonas marina within a polyurethane coating

The marine bacterium, Halomonas marina (ATCC 27129), was shown to inhibit settlement and development of the sessile invertebrates Balanus amphitrite and Bugula neritina. Different bacterial treatments were employed to investigate this interaction. Filmed bacteria and liquid suspensions of whole cells, lysed cells and culture filtrate all reduced settlement of B. amphitrite. Polyurethane coatings containing whole cells were partially inhibitory while lysed cells caused complete inhibition of B. amphitrite larval settlement. In contrast, culture filtrate in a polyurethane matrix stimulated settlement of B. amphitrite larvae. Whole cells, culture filtrate, and lysed cells embedded in a polyurethane coating also controlled B. neritina settlement and maturation.     

CHEMICAL MEDIATION OF COLONIZATION OF SEAWEED SURFACES1

The surfaces of macroalgal thalli are colonized by planktonic propagules (larvae, spores, cells, etc.) from a wide diversity of eukaryotes and prokaryotes. Colonization (here defined broadly to include processes such as settlement, attachment, metamorphosis, biofilm formation, and infection) of seaweed surfaces can be both induced and inhibited by metabolites produced at those surfaces. However, detailed examples of chemically mediated interactions at seaweed surfaces for which chemical cues have been characterized, quantified in situ, a biological effect determined, and the consequences to the demography of the seaweeds or colonizers demonstrated are very rare. Here we briefly review the literature on both deterrents (“natural antifoulants”) and inducers of colonization and on interactions at seaweed surfaces between the hosts and associated bacterial biofilms. One theme that emerges is the strong need to integrate ecology, cell biology, and chemistry to understand the distribution of surface‐active molecules in situ and their ecological consequences. This multidisciplinary approach is further emphasized for research on biofilms on seaweeds, where recently developed molecular tools for characterizing bacterial communities are opening up an entire new area of marine chemical ecology. Finally, we emphasize an integrated approach to the topic, as we believe that many aspects of somewhat disparate fields including, for example, induction of larval settlement, algal pathogenesis, and the molecular biology of bacterial signaling can be usefully viewed within the overall framework of chemical mediation of surface colonization.