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.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18-116 erg cm-2. To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm-2 for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm-2. The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Ys less than 50 erg cm-2), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (delta Fadh) show that delta Fadh decreases with increasing Ys, and equals zero around 45 erg cm-2 for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Variation in recruitment of the subtidal colonial ascidian Podoclavella cylindrica (Quoy & Gaimard): the rôle of substratum choice and early survival

The rôle of substratum choice and subsequent survival in determining the patterns of recruitment of the colonial ascidian Podoclavella cylindrica (Quoy & Gaimard) (Clavelinidae : Aplousobranchia) were examined. Larval settlement preferences on five substrata were determined by following individual larvae underwater and observing settlement behaviour on their first encounter with a substratum. The survival of settling larvae was monitored daily for 30 consecutive days in quadrats on the five substrata. Larvae settled preferentially on substrata that showed reduced mortality of settlers, hence recruitment was correlated with settlement. In almost all cases larvae rejected the unfouled surfaces of sponges, preferring those with epifauna, and settled in highest densities on bare space. Mortality of new recruits varied markedly between substrata but was very low on bare space. Juvenile mortality resulted from overgrowth by neighbouring invertebrates and from the removal of epifauna, associated with sponges, on which juveniles had settled.     

Self-decontaminating photocatalytic zinc oxide nanorod coatings for prevention of marine microfouling: a mesocosm study

The antifouling (AF) properties of zinc oxide (ZnO) nanorod coated glass substrata were investigated in an out-door mesocosm experiment under natural sunlight (14:10 light: dark photoperiod) over a period of five days. The total bacterial density (a six-fold reduction) and viability (a three-fold reduction) was significantly reduced by nanocoatings in the presence of sunlight. In the absence of sunlight, coated and control substrata were colonized equally by bacteria. MiSeq Illumina sequencing of 16S rRNA genes revealed distinct bacterial communities on the nanocoated and control substrata in the presence and absence of light. Diatom communities also varied on nanocoated substrata in the presence and the absence of light. The observed AF activity of the ZnO nanocoatings is attributed to the formation of reactive oxygen species (ROS) through photocatalysis in the presence of sunlight. These nanocoatings are a significant step towards the production of an environmentally friendly AF coating that utilizes a sustainable supply of sunlight.     

Seasonal variation in the effects of hard substratum biofilming on settlement of marine invertebrate larvae

The effect of season on "biofilming";, as a cue for the settlement of marine invertebrate larvae, was investigated in a long-term field study during the years 1992-1994. The series of settlement experiments was conducted in a tidal rapid on the west coast of Scotland, and involved manipulations of artificial panels. Biofilming of substrata, whilst excluding larval settlement, was achieved by the enclosure of panels within tight-fitting (but removable) mesh screens so that the number of settlers on filmed and unfilmed substrata were counted in the initial absence of other incumbent post-larvae. Depending on larval species, the effects of biofilming were found to be either facilitatory or inhibitory. Significant within- and between-species seasonal differences in the settlement responses were detected, and a reversal of the effect of biofilming on larval settlement response, from inhibitory to facilitatory and vice versa, was noted with season in the case of some taxonomic groups and species (e.g. Tubulipora sp., Plagioecia sp., Electra pilosa (L.)). The present study emphasizes the need for extended field studies of larval responses to environmental cues, when the focus of interest is in drawing general inferences about naturally occurring behavioural patterns at settlement.     

Post-settlement effects of habitat type and predator size on cannibalism of glaucothoe and juveniles of red king crab Paralithodes camtschaticus

Postlarval (glaucothoe) and juvenile (first crab stage, C1) red king crab Paralithodes camtschaticus actively select structurally complex substrata for settlement. Such habitats may provide them with shelter from predation during critical early stages. We tested this hypothesis by placing glaucothoe and juvenile crab in aquaria with or without natural or artificial habitats, and with or without predators (1-3-year-old red king crab) of two different sizes. Predators caused increased mortality of glaucothoe, but predator size, habitat presence and habitat type had no effect on survival. Predators caused significant mortality of C1 crabs in the absence of habitat, and mortality was inversely related to predator size. Density of glaucothoe on habitats was similar with or without predators, but density of C1 crab on habitats was higher than that of glaucothoe, and increased in the presence of large predators. Active selection for complex substrata by settling glaucothoe does not reduce cannibalism, but may pre-position them for improved survival after metamorphosis. In contrast, juvenile crabs modify their behavior to achieve higher densities in refuge habitats, which tends to dampen the effect of predation. These survival strategies may have evolved to compensate for the greater risk of predation in open habitats.     

Effects of substratum and conspecific adults on the metamorphosis of Chasmagnathus granulata (Dana) (Decapoda: Grapsidae) megalopae

The ability of marine invertebrate larvae to delay their metamorphosis in the absence of adequate environmental cues has been reported for numerous sedentary and sessile species. In the present study, the effect of various substrata and the presence of conspecific adults on the metamorphosis of a mobile species, the crab Chasmagnathus granulata, was evaluated. The duration of the megalopa stage in experiments with six different substrata and in the presence or absence of conspecific adults was compared in a laboratory study. In addition, the influence of natural substrata was compared with that of artificial substrata of similar grain size or texture. In a further experiment, the two most effective cues (natural mud and conspecific adults) were tested as single vs. combined factors. Natural mud and unidentified chemical cues from conspecific adults had the strongest accelerating effects on development duration to metamorphosis. With the exception of nylon threads (artificial filamentous substratum), none of the artificial substrata had a significant effect on the duration of the megalopa stage. Simultaneous exposure to natural mud and water containing chemical cues from conspecific adults accelerated metamorphosis more than each of these factors separately. Megalopae that were reared without a substratum (control) delayed their metamorphosis by 29% (about 3 days) compared with those in simultaneous contact with natural mud and rearing water of adult conspecifics. The results indicate that the metamorphosis of the megalopa of C. granulata is influenced by the presence or absence of environmental stimuli that are associated with the preferred adult habitat.     

Effects of adsorbed organic and primary fouling films on bryozoan settlement

Marine primary fouling films, which consist of molecular organic and microbial components, have been reported to facilitate colonization of immersed surfaces by marine fouling organisms. Larvae of the cosmopolitan fouling bryozoan Bugula neritina (Linnaeus) were offered various substrata for attachment and metamorphosis. The materials were offered (a) after detergent washing, (b) after sorption of dissolved organic molecular films, and (c) after formation of primary films consisting of both microbial and adsorbed organic material. Wettability of the substrata by sea water was determined by contact angle measurements for each substratum. On washed substrata, attachment was favored with contact angles greater than ≈45° (cos contact angle <0.7). Adsorbed surface films had no effect on the low settlement of larvae on glass and high settlement on plastics. Microbial primary films, however, made glass attractive and plastics unattractive. These settlement preference changes did not correlate with the changes in wettability observed on these substrata. Dispersion of larvae over the settlement surface was random except on wettable surfaces coated with bacterial films, where settlement was strongly clustered (contagious).     

Larval settlement preferences and post-settlement survival of the threatened Caribbean corals Acropora palmata and A. cervicornis

The settlement specificity of two threatened Caribbean corals, Acropora palmata and A. cervicornis, was tested by measuring their rates of larval metamorphosis in response to crustose coralline algae (CCA) and other substrata. In the no-choice experiments, the coral larvae were placed in six treatments: filtered seawater (FSW), a fragment of biofilmed dead skeleton of A. palmata, or a fragment of one of four species of CCA (Hydrolithon boergesenii, Porolithon pachydermum, Paragoniolithon solubile, and Titanoderma prototypum). Within each CCA treatment, there were three different substrata on which to settle and metamorphose: (1) the CCA surface, (2) the rock under the CCA, or (3) the plastic dish. The 5-day-old larvae of both A. palmata and A. cervicornis had similar rates of total metamorphosis (all substrata combined) in every treatment (excluding FSW) even in the absence of CCA. However, there were differences in larval behavior among the CCA species since the larvae settled and metamorphosed on different substrata in the presence of different CCA species. In the no-choice experiments the larvae of both corals had higher rates of metamorphosis on the top surfaces of H. boergesenii and/or T. prototypum than on P. pachydermum. In the choice experiments, the coral larvae were offered two species of CCA in the same dish. When given a choice, both species of coral larvae had more settlement and metamorphosis on the surface of H. boergesenii or T. prototypum or clean rock than onto the surface of P. solubile. After 6 weeks in the field, transplanted A. palmata recruits had approximately 15% survival on both T. prototypum and H. boergesenii, but A. cervicornis recruits only survived on T. prototypum (13%). Some, but not all, CCA species facilitated the larval settlement and post-settlement survival of these two threatened corals, highlighting the importance of benthic community composition for successful coral recruitment.     

Adhesion of oral streptococci from a flowing suspension to uncoated and albumin-coated surfaces

A flow cell system was developed which allowed the study of bacterial adhesion to solid substrata at well-defined shear rates. In addition, the system enabled the solid surfaces to be coated with a proteinaceous film under exactly the same shear conditions. In this flow cell system, adhesion of three strains of oral streptococci from a phosphate-buffered solution onto three different substrata was studied as a function of time in the absence and presence of a bovine serum albumin (BSA) coating at a shear rate of 21 s-1. To obtain a wide range in surface free energies (gamma) representative strains (gamma b 38-117 mJ m-2) and solid substrata (gamma s 20-109 mJ m-2) were selected. The number of bacteria adhering was counted microscopically. In the absence of a BSA coating a linear relation was found between the number of bacteria adhering at saturation (nb,s) and the calculated interfacial free energy of adhesion (delta Fadh) for each of the three strains. In the presence of a BSA coating the number of bacteria adhering was greatly decreased in all cases. However, despite the presence of the BSA coating there was still a linear relation between the number of bacteria adhering at saturation and the interfacial free energy of adhesion, calculated on the basis of the surface free energy of the uncoated substrata. It can be concluded that the bare, uncoated substratum still influenced bacterial adhesion in spite of the marked influence of a BSA coating.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.     

Do Predators Condition the Distribution of Prey within Micro Habitats? An Experiment with Stoneflies (Plecoptera)

The selection of habitat by macroinvertebrates living in running waters may be influenced by the physical characteristics of the substratum, as well as by the presence of other species. In this study, an artificial river with three different substrata (pebbles, detritus, and leaves) was utilized to analyze the microhabitat preference of two Plecoptera prey species (Amphinemura sulcicollis and Brachyptera risi), both in absence and in presence of a Plecoptera predator species (Perla marginata). In the absence of predators, both prey species showed a clear preference for the leaf microhabitat. When the predators were present, only Brachyptera risi showed a change of microhabitat selection, with a decrease of leaves and an increase of pebbles and detritus utilization. Amphinemura sulcicollis did not change their substratum utilization. This study demonstrates that the presence of a predator may affect microhabitat selection through a switch from the preferred to the less preferred substrata, although not all species change their habitat utilization in response to predator presence. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Reproductive seasonality,settlement, and post-settlement mortality of Pocillopora damicornis (Linnaeus), at Lizard Island,Great Barrier Reef

Pocillopora damicornis (Linnaeus) has seasonal gametogenesis and planula release at Lizard Island, Great Barrier Reef, in contrast with several previous reports on the species at other locations. The number of planulae released and gonad development varied considerably among colonies sampled at the same time, but reproductive activity occurred predominantly in winter. P. damicornis planulae settled preferentially on algal-covered substrata, rather than bare coral substrata, but showed subsequent mortality inversely related to this settlement preference. Competition with algae and biological disturbance contribute to spat mortality at different stages of settlement and growth.     

Substratum preferences and planulae settling of two red sea alcyonaceans: Xenia macrospiculata Gohar and Parerythropodium fulvum fulvum (Forskl)

The settling behaviour and substratum preferences of the planulae of the Red Sea soft corals Xenia macrospiculata Gohar and Parerythropodium fulvum fulvum (Forskl) were examined in the laboratory. The planulae of the two species have a short pelagic phase and they tend to settle immediately upon leaving the parent colonies. Mucous secretion is used by the larvae for crawling and adhering to the substratum. They exhibit an aggregated pattern of settlement. The developing polyps are found in depressions or pits of the substratum. The planulae preferentially settle on rough substrata and avoid smooth surfaces. They search for substrata covered with an organic coating, composed of turf or crustose coralline algae. Such substrata create better conditions for larval settlement and metamorphosis. The planulae of P. f. fulvum exhibit a striking preference for upside-down attachment on undersides of the substrata, while Xenia macrospiculata utilizes both substratum faces for settlement. Light intensity seems insignificant in determining attachment sites. The findings of the experiments correspond well with the distributional patterns of juveniles of the two species as found in the natural environment. The specific requirements for settling of both species increase their chances of successful development and thus enhance their survival.     

No effect of naturally-occurring rock types on settlement or survival in the intertidal barnacle, Tesseropora rosea (Krauss)

The settlement and early survival of young barnacles, Tesseropora rosea (Krauss), on four different, naturally-occurring rock substrata were investigated in a four-factor experiment. Three factors assessed variation on three spatial scales: among shores on the New South Wales coast in eastern Australia i.e., 10 to 600 km apart; among sites within these shores i.e., 20 to 50 m apart; within these sites, i.e., < 3 m². Variation on these scales was usually significant for settlement density and proportional survival 30–60 days post-settlement. The fourth factor, different rock types, showed no consistent trends for settlement density or proportional survival. Previously observed differences in these phenomena among shores cannot, therefore, be ascribed to differences in rock substrata.     

Linear scan voltammetric indirect determination of Al(III) by the catalytic cathodic response of norepinephrine at the hanging mercury drop electrode

The biological effects of aluminum (Al) have received much attention in recent years. Al is of basic relevance as concern with its reactivity and bioavailability. In this paper, the electrochemical behaviors of norepinephrine (NE) in the absence and presence of Al(III) at the hanging mercury drop electrode have been studied and applied to the practical analysis. Highly selective catalytic cathodic peak of NE is yielded by linear scan voltammetry (LSV) at -1.32 V (vs. SCE). A linear relationship holds between the cathodic peak current and the Al(III) concentration. It has been successfully applied to the determination of Al(III) in real waters and synthetic biological samples with satisfying results, which are in accordance with those obtained by ICP-AES method. The electrochemical properties and the mechanisms of the peaks in the presence and absence of Al(III) have been explored. The results show that they are irreversible adsorptive hydrogen catalytic waves. These studies not only enrich the methods of determining Al, but also lay foundations of further understanding of the mechanisms of neurodementia.     

Coral larvae settle at a higher frequency on red surfaces

Although chemical cues serve as the primary determinants of larval settlement and metamorphosis, light is also known to influence the behavior and the settlement of coral planulae. For example, Porites astreoides planulae settle preferentially on unconditioned red substrata. In order to test whether this behavior was a response to color and whether other species also demonstrate color preference, settlement choice experiments were conducted with P. astreoides and Acropora palmata. In these experiments, larvae were offered various types of plastic substrata representing three to seven different color choices. Both species consistently settled on red (or red and orange) substrata at a higher frequency than other colors. In one experiment, P. astreoides settled on 100% of red, plastic cable ties but failed to settle on green or white substrata. In a second experiment, 24% of larvae settled on red buttons, more than settled on six other colors combined. A. palmata settled on 80% of red and of orange cables ties but failed to settle on blue in one experiment and settled on a greater proportion of red acrylic squares than on four other colors or limestone controls in a second experiment. The consistency of the response across a variety of plastic materials suggests the response is related to long-wavelength photosensitivity. Fluorescence and reflectance spectra of experimental substrata demonstrated that the preferred substrata had spectra dominated by wavelengths greater than 550 nm with little or no reflection or emission of shorter wavelengths. These results suggest that some species of coral larvae may use spectral cues for fine-scale habitat selection during settlement. This behavior may be an adaptation to promote settlement in crustose coralline algae (CCA)-dominated habitats facilitating juvenile survival.     

Adding coral rubble to substrata enhances settlement of Pocillopora?damicornis larvae

Settlement preferences of Pocillopora damicornis larvae were examined on artificial substrata. Planulation of P. damicornis followed a lunar cycle and the release of larvae occurred after new moon. P. damicornis larvae had the highest rates of settlement within 3 days of being presented settlement substrata. Cumulative settlement gradually increased from 3 to 8 days, and post-settlement mortality was most frequent after 8 days. Settlement experiments showed greatest settlement preference to cement tiles containing 10% coral rubble. This study suggests that physical cues are important in the settlement process, which may be useful for coral reef rehabilitation projects.     

Settlement and post-settlement processes limit the abundance of the geniculate coralline alga Calliarthron on subtidal walls

Cover of geniculate coralline algae is significantly greater on subtidal platforms than on vertical walls in a central California kelp forest. To determine the cause(s) of this pattern, settlement and growth of Calliarthron were compared on horizontal and vertical substrata attached to platforms or walls. Recruitment on horizontal substrata attached to reef walls indicated that spores were abundant along the walls. Densities of recruits were significantly smaller on vertical substrata attached to walls than on horizontal substrata attached to platforms, indicating that Calliarthron settlement is reduced on vertical substrata. Growth of basal crusts and the densities of fronds were also significantly greater on horizontal substrata attached to platforms than on vertical substrata attached to walls. Irradiance reaching wall surfaces was less than irradiance reaching platform surfaces. We suggest that light reaching wall surface is low enough to affect Calliarthron growth. These results indicate that the low abundance of Calliarthron on subtidal walls is a result of poor recruitment and slow growth.     

Influence of rocky substrata on three-dimensional sponge cells model development

Many marine and freshwater organisms are rocky bottom dwellers, and the mineralogical composition of the substratum is known to potentially condition their biology and ecology. In this work, we propose the use of 3D sponge cellular aggregates, called primmorphs, as suitable models for a multidisciplinary study of the mechanisms which regulate the biological responses triggered by the contact with different inorganic substrata. In our experiments, primmorphs obtained from the marine sponge Petrosia ficiformis (Poiret, 1789) were reared on calcium carbonate or on quartzitic substrata, respectively, and their morphological development was described. In parallel, the quantitative expression levels of two genes, silicatein and heat shock protein 70 (HSP70), were evaluated. The first gene is strictly correlated to spiculogenesis and sponge growth, while the second is an important indicator of stress. The results achieved with this in vitro model clearly demonstrate that quartzitic substrata determine the increase of silicatein gene expression, a lower expression of HSP70 gene, and a remarkable difference in primmorphs morphology compared to the analogous samples grown on marble.