Article

Three Dimensional Tracking of Exploratory Behavior of Barnacle Cyprids Using Stereoscopy

Article

Three Dimensional Tracking of Exploratory Behavior of Barnacle Cyprids Using Stereoscopy

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Abstract

Surface exploration is a key step in the colonization of surfaces by sessile marine biofoulers. As many biofouling organisms can delay settlement until a suitable surface is encountered, colonization can comprise surface exploration and intermittent swimming. As such, the process is best followed in three dimensions. Here we present a low-cost transportable stereoscopic system consisting of two consumer camcorders. We apply this novel apparatus to behavioral analysis of barnacle larvae (≈800 μm length) during surface exploration and extract and analyze the three-dimensional patterns of movement. The resolution of the system and the accuracy of position determination are characterized. As a first practical result, three-dimensional swimming trajectories of the cypris larva of the barnacle Semibalanus balanoides are recorded in the vicinity of a glass surface and close to PEG2000-OH and C11NMe3⁺Cl− terminated self-assembled monolayers. Although less frequently used in biofouling experiments due to its short reproductive season, the selected model species [Marechal and Hellio (2011), Int Biodeterior Biodegrad, 65(1):92–101] has been used following a number of recent investigations on the settlement behavior on chemically different surfaces [Aldred et al. (2011), ACS Appl Mater Interfaces, 3(6):2085–2091]. Experiments were scheduled to match the availability of cyprids off the north east coast of England so that natural material could be used. In order to demonstrate the biological applicability of the system, analysis of parameters such as swimming direction, swimming velocity and swimming angle are performed. Electronic supplementary material The online version of this article (doi:10.1007/s13758-012-0050-x) contains supplementary material, which is available to authorized users.

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... Digital in-line holographic microscopy allowed 3-D trajectories to be determined for motile zoospores of the alga Ulva linza and provided insight into exploration and surface selection mechanisms (Leal-Taixé et al. 2009;Heydt et al. 2009Heydt et al. , 2012. While the technique is ideal to track small organisms down to the level of bacteria (Vater et al. 2014), stereoscopy is the technique of choice for larger organisms (Maleschlijski et al. 2012;. ...
... In this work a stereoscopic setup (Maleschlijski et al. 2012) was used to study the exploratory behaviour of cyprids in the vicinity of surfaces with different chemical terminations were compared. The advantage of this apparatus is that 3-D data can readily be extracted and the system provides a relatively broad field of view that allows the observation of a large part of the swimming arena. ...
... The cyprids were allowed to equilibrate for an additional 5 min before stereoscopic video recording began in order to minimize the convection from the inoculation procedure. The stereoscopy setup consisted of two synchronized consumer HD camcorders (Sony HDR-XR550, Sony Corp., Tokyo, Japan) (Maleschlijski et al. 2012). Both camcorders imaged the dish from above at a relative angle of ≈80°and 40 cm from the water surface. ...
Article
Testing of new coatings to control fouling frequently involves single-species laboratory bioassays. Barnacle cyprids are among the most widely used model organisms in marine biofouling research, and surfaces that inhibit their settlement are considered to be promising candidates for new coating concepts. An analysis of motility parameters (mean velocity and swimming area coefficient) and distribution of cyprids of Balanus amphitrite in different swimming regions in the vicinity of model surfaces (self-assembled monolayers) is presented. The data are correlated with the settlement preferences of cyprids on these surfaces. Cyprids were predominantly found in interfacial regions and the transition frequencies between swimming regions of different depths were determined.
... This study aimed to provide a framework for the classification of the pre-settlement behaviour of cyprids based on the acquisition of three-dimensional trajectories. For this purpose, the stereoscopic set-up described in Maleschlijski et al. [26] was applied, and the general pre-settlement behaviour was analysed. As it is known from previous studies that different chemical surface terminations can induce different motions [10,14], patterns above a range of surface chemistries were acquired to ensure that the most common ones were included. ...
... The stereoscopy set-up consisted of two synchronized consumer camcorders as described recently [26]. The cameras imaged the surface of interest from the top at a relative angle to each other of ca 808 and a distance to the water surface of ca 40 cm. ...
... Therefore, the two-dimensional coordinates were recorded in both camera perspectives and merged into three-dimensional coordinates for each object of interest. To apply the epipolar transformation and to allow the extraction of quantitative information from the data points, both cameras were calibrated using a calibration procedure as described in Maleschlijski et al. [26]. Here, the zvalues represented the distance to the surface, whereas the xand y-values denoted the position in the surface plane. ...
Article
Barnacle cyprids exhibit a complex swimming and exploratory behaviour on surfaces and settlement is a consequence of extensive surface probing and selection of suitable settlement sites. In this work, the behaviour of cyprids in their pre-settlement phase was studied by three-dimensional video stereoscopy. With this technique, three-dimensional trajectories were obtained that were quantitatively analysed. The velocity during vertical sinking of cyprids of Balanus amphitrite was used with a modified form of Stokes' law to calculate their mean body density. Furthermore, a classification of the swimming patterns allowed the extension of existing models describing cyprid locomotion and swimming behaviour. The patterns were characterized with respect to their occurrence, transition between patterns and their velocity distribution, and motions were identified that led to surface contacts. This analysis provides a classification framework, which can assist future attempts to identify behavioural responses of cyprids to specific settlement cues.
... The stereoscopy setup consisted of two synchronized consumer camcorders as described recently [23]. The cameras imaged the surface of interest from the top at a relative angle to each other of ca. ...
... Therefore, the 2-D coordinates were recorded in both camera perspectives and merged into 3-D coordinates for each object of interest. To apply the epipolar transformation and to allow the extraction of quantitative information from the data points, both cameras were calibrated using a calibration object (see Fig. 1a), following the procedure described in Maleschlijski et al. [23]. The calibration object consisted of a precisely machined aluminum structure with rectangular posts of different height. ...
Article
Barnacle larvae (cyprids) explore surfaces to identify suitable settlement sites. This process is selective, and cyprids respond to numerous surface cues. To better understand the settlement process, it is desirable to simultaneously monitor both the surface exploration behavior and any close interactions with the surface. Stereoscopic 3D tracking of the cyprids provides quantitative access to surface exploration and pre-settlement rituals. Imaging surface plasmon resonance (SPR) reveals any interactions with the surfaces, such as surface inspection during bipedal walking and deposition of temporary adhesives. We report on a combination of both techniques to bring together information on swimming behavior in the vicinity of the interface and physical interactions of the cyprid with the surface. The technical requirements are described, and we applied the setup to cyprids of Balanus amphitrite. Initial data shows the applicability of the combined instrument to correlate exploration and touchdown events on surfaces with different chemical termination.
... Many surface topological and physicochemical properties, e.g., surface textures (Berntsson et al., 2000;Aldred et al., 2010;Chaw et al., 2011), hydrophobicity Phang et al., 2009;Guo et al., 2014), surface energy, and charge (Petrone et al., 2011;Di Fino et al., 2014) were also found to influence cyprid surface exploration. Using more complex video-/tracking-based approaches, the dynamic cyprid surface exploration process under different conditions was also monitored, offering a real-time and quantitative method of understanding the settling behaviors of a cyprid by measuring its swimming velocity, step length and duration, body movements, footprint deposition, and so on (Marechal et al., 2004;Andersson et al., 2009;Chaw and Birch, 2009;Maleschlijski et al., 2012;Aldred et al., 2013bAldred et al., , 2018Maleshlijski et al., 2016). ...
Article
Full-text available
Barnacles are notorious marine fouling organisms, whose life cycle initiates with the planktonic larva, followed by the free-swimming cyprid that voluntarily explores, and searches for an appropriate site to settle and metamorphoses into a sessile adult. Within this life cycle, both the cyprid and the adult barnacle deposit multi-protein adhesives for temporary or permanent underwater adhesion. Here, we present a comprehensive review of the biochemistries behind these different adhesion events in the life cycle of a barnacle. First, we introduce the multiple adhesion events and their corresponding adhesives from two complementary aspects: the in vivo synthesis, storage, and secretion as well as the in vitro morphology and biochemistry. The amino acid compositions, sequences, and structures of adult barnacle adhesive proteins are specifically highlighted. Second, we discuss the molecular mechanisms of adult barnacle underwater attachment in detail by analyzing the possible adhesive and cohesive roles of different adhesive proteins, and based on these analyses, we propose an update to the original barnacle underwater adhesion molecular model. We believe that this review can greatly promote the general understanding of the molecular mechanisms underlying the reversible and irreversible underwater adhesion of barnacles and their larvae. Such an understanding is the basis for the prevention of barnacle fouling on target surfaces as well as designing conceptually new barnacle-inspired artificial underwater adhesives.
... individuals and, thus, how useful a behavioural assay may be as an indicator of propensity to settle. Previous studies have been limited to interpretation of short snapshots of behaviour out of context [14,[16][17][18]20,21]. A significant advantage of our system is that it enables us to track multiple cyprids, simultaneously, throughout the entire pelagic-benthic transition to settlement, providing detail about the sequence of behaviours as well as the ability to compare between experimental conditions. ...
Article
For barnacle cypris larvae at the point of settlement, selection of an appropriate surface is critical. Since post-settlement relocation is usually impossible, barnacles have evolved finely tuned surface-sensing capabilities to identify suitable substrata, and a temporary adhesion system for extensive surface exploration. The pattern of exploratory behaviour appears complex and may last for several hours, imposing significant barriers to quantitative measurement. Here, we employ a novel tracking system that enables simultaneous analysis of the larval body movement of multiple individuals over their entire planktonic phase. For the first time, to our knowledge, we describe quantitatively the complete settlement process of cyprids as they explore and select surfaces for attachment. We confirm the 'classic' behaviours of wide searching, close searching and inspection that comprise a model originally proposed by Prof. Dennis Crisp FRS. Moreover, a short-term assay of cyprid body movement has identified inspection behaviour as the best indicator of propensity to settle, with more inspection-related movements occurring in conditions that also promote higher settlement. More than half a century after the model was first proposed by Crisp, there exists a precise method for quantifying cyprid settlement behaviour in wide-ranging investigations of barnacle ecology and applied studies of fouling management.
... The development of three-dimensional stereoscopic-tracking systems provided certainty regarding the vertical position of cyprids in the water column; however these systems are complex to implement and do not, per se, increase the ability of the experimenter to differentiate between exploratory and non-exploratory behaviour. The introduction of a novel method to track multiple cyprids simultaneously [27] was another notable advance, but the research has remained restricted by manual classification of cyprid behaviours [28]. Similarly, although Pradhan et al. [29] developed an automatic classification method to detect the wide and close search behaviour of a single cyprid, their imaging system was mounted horizontally and wide search behaviour was simply defined as when the cyprid moved approximately horizontally to the surface (a behaviour that often occurs during swimming). ...
Article
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A focus on the development of nontoxic coatings to control marine biofouling has led to increasing interest in the settlement behaviour of fouling organisms. Barnacles pose a significant fouling challenge and accordingly the behaviour of their settlement-stage cypris larva (cyprid) has attracted much attention, yet remains poorly understood. Tracking technologies have been developed that quantify cyprid movement, but none have successfully automated data acquisition over the prolonged periods necessary to capture and identify the full repertoire of behaviours, from alighting on a surface to permanent attachment. Here we outline a new tracking system and a novel classification system for identifying and quantifying the exploratory behaviour of cyprids. The combined system enables, for the first time, tracking of multiple larvae, simultaneously, over long periods (hours), followed by automatic classification of typical cyprid behaviours into swimming, wide search, close search and inspection events. The system has been evaluated by comparing settlement behaviour in the light and dark (infrared illumination) and tracking one of a group of 25 cyprids from the water column to settlement over the course of 5 h. Having removed a significant technical barrier to progress in the field, it is anticipated that the system will accelerate our understanding of the process of surface selection and settlement by barnacles.
... Estimating and predicting trajectories in three-dimensional spaces based on two-dimensional projections available from one camera source is an open problem with wide-ranging applicability including entertainment [1], medicine [2], biology [3], physics [4], etc. Unfortunately, solving this problem is exceptionally difficult due to a variety of challenges, such as the variability of states of the trajectories, partial occlusions due to self articulation and layering of objects in the scene, and the loss of 3D information resulting from observing trajectories through 2D planar image projections. ...
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Estimation, recognition, and near-future prediction of 3D trajectories based on their two dimensional projections available from one camera source is an exceptionally difficult problem due to uncertainty in the trajectories and environment, high dimensionality of the specific trajectory states, lack of enough labeled data and so on. In this article, we propose a solution to solve this problem based on a novel deep learning model dubbed Disjunctive Factored Four-Way Conditional Restricted Boltzmann Machine (DFFW-CRBM). Our method improves state-of-the-art deep learning techniques for high dimensional time-series modeling by introducing a novel tensor factorization capable of driving forth order Boltzmann machines to considerably lower energy levels, at no computational costs. DFFW-CRBMs are capable of accurately estimating, recognizing, and performing near-future prediction of three-dimensional trajectories from their 2D projections while requiring limited amount of labeled data. We evaluate our method on both simulated and real-world data, showing its effectiveness in predicting and classifying complex ball trajectories and human activities.
... Automated tracking of the movement of small organisms in three dimensions allows the acquisition of a large number of trajectories, forming a reliable basis for the analysis of their dynamics. Several studies have developed methods to keep tracks of moving organisms, for instance fruit flies (Straw et al., 2011;Ardekani et al., 2013), sessile barnacles (Maleschlijski et al., 2012) or schooling fish (Butail and Paley, 2012). In ecotoxicology however, and despite the growing interest in using zooplankton behavior as an integrated measure of sub-lethal exposure to pollutants, quantitative characterization of three dimensional trajectories is uncommon, primarily because there is no standard technique to recover the position information of a large number of animals moving simultaneously. ...
... However, the missing third spatial component complicates an accurate quantitative data analysis since there are important characteristic values such as swimming velocities that cannot accurately be calculated. Recent work showed that 3D tracking of cyprids provides more detailed information, since it allows a clear distinction between sinking and swimming phases (Fig. 11) [102]. The data reveals that positively charged surfaces seem to cause longer periods of close surface inspection of cyprids of the barnacle Semibalanus balanoides than glass or PEG. ...
Article
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This review article summarizes some recent insights into the strategies used by marine organisms to select surfaces for colonization. While larger organisms rely on their sensory machinery to select surfaces, smaller microorganisms developed less complex but still effective ways to probe interfaces. Two examples, zoospores of algae and barnacle larvae, are discussed and both appear to have build-in test mechanisms to distinguish surfaces with different physicochemical properties. Some systematic studies on the influence of surface cues on exploration, settlement and adhesion are summarized. The intriguing notion that surface colonization resembles a parallelized surface sensing event is discussed towards its complementarity with conventional surface analytical tools. The strategy to populate only selected surfaces seems advantageous as waves, currents and storms constantly challenge adherent soft and hard fouling organism.
Chapter
This chapter summarises recent progress towards the characterisation of bioadhesives secreted by the larvae of marine invertebrates, with reference also to their subsequent developmental stages. These adhesives vary structurally and biochemically between species and between life stages to satisfy the requirements of the particular organism for permanent or temporary/reversible adhesion, often under hostile conditions. To date, a small number of bioadhesives have been described for the adult forms of marine invertebrates, while a functional understanding of larval adhesives remains elusive. Progress is essential, however, since the larval forms perform a key role in the fouling of marine structures, and their adhesives may have characteristics of interest for development of synthetic, bio-inspired glues. Despite recent advances in the fields of proteomics and genomics, major obstacles exist in the isolation and analysis of tiny quantities of larval adhesives, which have largely precluded these approaches. Further challenges relate to the in situ detection of larval adhesive materials, being usually secreted underwater and buried at the interface between a solid substrate and the organism’s body. Here, we discuss a range of novel experimental approaches that have surmounted these technical issues and provided useful insight into the morphology and composition of larval bioadhesives in situ. These involve imaging and spectroscopic approaches as well as nano-/micromechanical and surface-sensitive techniques that have enabled quantification of adhesion forces and surface adsorption of purified adhesive proteins.
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Behavioral responses of aquatic organisms have received much less attention than developmental or reproductive ones due to the scarce presence of user-friendly tools for their acquisition. The technological development of data acquisition systems for quantifying behavior in the aquatic environment and the increase of studies on the understanding the relationship between the behavior of aquatic organisms and the physiological/ecological activities have generated renewed interest in using behavioral responses also in marine ecotoxicology. Recent reviews on freshwater environment show that behavioral end-points are comparatively fast and sensitive, and warrant further attention as tools for assessing the toxicological effects of environmental contaminants. In this mini-review, we perform a systematic analysis of the most recent works that have used marine invertebrate swimming alteration as behavioral end-point in ecotoxicological studies by assessing the differences between behavioral and acute responses in a wide range of species, in order to compare their sensitivity.
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Settlement of the planktonic dispersal stages of marine organisms is the crucial step for the development of marine biofouling. Four-dimensional holographic tracking reveals the mechanism by which algal spores select surfaces suitable for colonization. Quantitative analysis of the three dimensional swimming trajectories of motile spores of a macroalga (Ulva linza) in the vicinity of surfaces functionalized with different chemistries reveals that their search strategy and swimming behavior is correlated to the number of settled spores found in spore settlement bioassays conducted over 45 min. The spore motility and exploration behavior can be classified into different motion patterns, with their relative occurrence changing with the surface chemistry. Based on the detailed motility analysis we derived a model for the surface selection and settlement process of Ulva zoospores.
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Non-fouling surfaces that resist non-specific adsorption of proteins, bacteria, and higher organisms are of particular interest in diverse applications ranging from marine coatings to diagnostic devices and biomedical implants. Poly(ethylene glycol) (PEG) is the most frequently used polymer to impart surfaces with such non-fouling properties. Nevertheless, limitations in PEG stability have stimulated research on alternative polymers that are potentially more stable than PEG. Among them, we previously investigated poly(2-methyl-2-oxazoline) (PMOXA), a peptidomimetic polymer, and found that PMOXA shows excellent anti-fouling properties. Here, we compare the stability of films self-assembled from graft copolymers exposing a dense brush layer of PEG and PMOXA side chains, respectively, in physiological and oxidative media. Before media exposure both film types prevented the adsorption of full serum proteins to below the detection limit of optical waveguide in situ measurements. Before and after media exposure for up to 2 weeks, the total film thickness, chemical composition, and total adsorbed mass of the films were quantified using variable angle spectroscopic ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and optical waveguide lightmode spectroscopy (OWLS), respectively. We found (i) that PMOXA graft copolymer films were significantly more stable than PEG graft copolymer films and kept their protein-repellent properties under all investigated conditions and (ii) that film degradation was due to side chain degradation rather than due to copolymer desorption.
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Digital in-line holography is a microscopy technique which has gotten an increasing amount of attention over the last few years in the fields of microbiology, medicine and physics, as it provides an efficient way of measuring 3D microscopic data over time. In this paper we approach the challenges of a high throughput analysis of holographic microscopy data and present a system for detecting particles in 3D reconstructed holograms and their 3D trajectory estimation over time. Our main contribution is a robust method which evolves from the Hungarian bipartite weighted graph matching algorithm and allows us to deal with newly entering and leaving particles and compensate for missing data and outliers. In the experiments we compare our fully automatic system with manually labeled ground truth data and we can report an accuracy between 76% and 91%.
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Video microscopy of cyprids of Balanus amphitrite was used to monitor the action of antennular setae during the exploratory behaviour prior to attachment. In addition, SEM was used to provide a revised description of all antennular setae for that species. The videos describe if a particular seta touches the substratum and the area it can cover during surface exploration. On the fourth segment, the plumose terminal setae A and B are never in contact with the substratum, lack a terminal pore and it is argued that they sense hydrodynamic forces. The aesthetasc-like terminal seta D is likewise held free in the water at all times and it is speculated that it senses dissolved substances, but, since it contains a scolopale rod, it must also have a mechano-receptive function. All remaining antennular setae on the second, third and fourth segments have a terminal pore and it is argued that these are bimodal receptors with both chemo- and mechano-receptive modalities. These setae are also at one time or another in contact with the substratum, except perhaps for the small preaxial seta 2 and terminal seta C. The first seta to contact the surface during a tentative step is radial seta 5, which is longer than all other radial setae. All other setae on the second and third segment are only in contact after a step is completed. When the attachment disc touches the surface (=a step completed) the long and curved postaxial seta 2 (on the second segment) and postaxial seta 3 on the third segment are both flexed to either side of the antennule. This lateral displacement ensures that these two setae can touch large surface areas to either side of the appendage. The four subterminal setae on the fourth segment contact the surface both immediately before and after a step has been completed, and the constant flicking of the segment significantly increases the surface area tested by both these chemoreceptors and by terminal seta E, which can sweep up to 60 μm laterally from the attachment disc. The flicking of the fourth segment may also serve to dilute the boundary layer of chemoreceptors on the fourth segment such as the aesthetasc-like terminal seta D and thus facilitate the detection of new stimuli.
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In this paper an approach for motion capture of dressed people is presented. A cloth draping method is incorporated in a silhouette based motion capture system. This leads to a simultaneous estimation of pose, joint angles, cloth draping parameters and wind forces. An error functional is formalized to minimize the involved parameters simultaneously. This allows for reconstruction of the underlying kinematic structure, even though it is covered with fabrics. Finally, a quantitative error analysis is performed. Pose results are compared with results obtained from a commercially available marker based tracking system. The deviations have a magnitude of three degrees which indicates a reasonably stable approach.
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The surface interactions of exploring cyprids of the barnacle Semibalanus balanoides were studied in situ using imaging surface plasmon resonance. It was demonstrated how the deposition of a proteinaceous adhesive could be followed in real time as the cyprids explored and temporarily attached to a surface. Furthermore, the amount of protein left on the surface when the cyprids moved on could be quantified. Clear differences were demonstrated between an oligo(ethyleneglycol) coated surface and a bare gold substrate. It is anticipated that this technique will be a valuable tool in the development of novel surface chemistries that can prevent biofouling.
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The shallow depth of field of conventional microscopy hampers analyses of 3D swimming behavior of fast dinoflagellates, whose motility influences macroassemblages of these cells into often-observed dense "blooms." The present analysis of cinematic digital holographic microscopy data enables simultaneous tracking and characterization of swimming of thousands of cells within dense suspensions. We focus on Karlodinium veneficum and Pfiesteria piscicida, mixotrophic and heterotrophic dinoflagellates, respectively, and their preys. Nearest-neighbor distance analysis shows that predator and prey cells are randomly distributed relative to themselves, but, in mixed culture, each predator clusters around its respective prey. Both dinoflagellate species exhibit complex highly variable swimming behavior as characterized by radius and pitch of helical swimming trajectories and by translational and angular velocity. K. veneficum moves in both left- and right-hand helices, whereas P. piscicida swims only in right-hand helices. When presented with its prey (Storeatula major), the slower K. veneficum reduces its velocity, radius, and pitch but increases its angular velocity, changes that reduce its hydrodynamic signature while still scanning its environment as "a spinning antenna." Conversely, the faster P. piscicida increases its speed, radius, and angular velocity but slightly reduces its pitch when exposed to prey (Rhodomonas sp.), suggesting the preferred predation tactics of an "active hunter."
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Larvae of many benthic invertebrates settle on surfaces where they metamorphose into juveniles if suitable substrata are available, and are responsible for the major costs of biofouling. When assessing new formulations or compounds for potential antifouling (AF) application, constraints such as seasonal availability may restrict most bioassays to relatively few taxa and species. For example, amongst barnacles, Amphibalanus amphitrite is popular as a test organism but is it really representative of other barnacle species? In order to test this hypothesis, we have chosen to work with marine natural extracts as a probe. Indeed, one substitution technology to toxic metal-based coatings to control fouling is the development of AF coatings with active compounds derived from marine organisms or analogues of the lead compounds. In this study, the AF activity and toxicity of extracts from 30 algae from the North East Atlantic coast were investigated for their potential anti-settlement activities against larvae of two species of barnacle, A. amphitrite and Semibalanus balanoides. As a trend, most of the active extracts displayed activity towards S. balanoides, only few displayed targeted activity against A. amphitrite, or against both species. In order to better understand if this tendency could be linked to chemical ecology, surface extracts were prepared on a selection of species. The results highlight that surface extracts of algae all displayed highest levels of activity than total extracts when tested on S. balanoides. This difference illustrates that specific compounds in their ecological context can have potentially a better efficacy on target species.
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The aim of this work was to develop techniques for the real time filming of Semibalanus balanoides cyprid larvae in the field in order to describe the exploratory behaviour of S. balanoides cyprids under natural field conditions. The underwater camera system consisted of a high resolution, remotely controlled colour camera attached to a cradle which could be deployed from a pier. A light utilising a battery of far‐red light‐emitting diodes (λmin = 615 nm) was used for filming at night. Transparent acrylic treated with barnacle settlement factor and smooth green polyester tiles were used as targets. From the films the tracks of the exploring cyprids were digitised from the 30 min trials. During five, 30 min trials a total of 1014 cyprids explored the surface. The numbers of cyprids exploring the surface varied from 49 to 522 cyprids per trial, and a Monte‐Carlo randomisation model showed that larval supply was random or aggregated. The mean exploration time spent on the target was 163 s with a mean distance travelled on the target of 8.5 cm. Exploratory track length was related to water current velocity by a negative curvilinear regression (R = 0.92, F = 45.65, p < 0.05). There were significant differences in exploratory behaviour between day and night for total distance travelled (p < 0.001), straight line distance (p < 0.001) and velocity (p < 0.001) and the mean heading angle of the exploratory track (p < 0.01). The results are discussed in relation to settlement patterns and the screening of antifouling surfaces.
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Heterosaccus lunatus parasitizes the portunid crab, Charybdis callianassa, in Moreton Bay, Queensland, Australia. Larval broods, released during July and August (males only), were allowed to develop through to cyprids (lecithotrophic development). Speeds of all the developmental stages—nauplius I to nauplius IV and the cyprids were measured by timing continuously swimming larvae over 5 mm distances under a microscope. The absolute speeds of the naupliar stages were similar (3–4 mm s−1), but the cyprids were significantly faster at [approximate]10 mm s−1. These speeds were compared with published speeds of cirripede and copepod nauplii and other cirripede cyprids. Relative speeds (body lengths s−1) showed surprising parity, which for the cyprids probably reflects the similar efficiency of the fusiform shape and thoracopod propulsion for all sizes of this highly specialized larval form. The lecithotrophic H. lunatus nauplii have rather minimalistic appendages (compared with planktotrophic nauplii) which were examined at the light microscope and scanning electron microscope levels. The natatory setae were found to be plumo-denticulate.
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This paper describes the use of surface plasmon resonance spectroscopy and self-assembled monolayers (SAMs) of alkanethiols on gold to evaluate the ability of surfaces terminating in different combinations of charged groups to resist the nonspecific adsorption of proteins from aqueous buffer. Mixed SAMs formed from a 1:1 combination of a thiol terminated in a trimethylammonium group and a thiol terminated in a sulfonate group adsorbed less than 1% of a monolayer of two proteins with different characteristics: fibrinogen and lysozyme. Single-component SAMs formed from thiols terminating in groups combining a positively charged moiety and a negatively charged moiety were also capable of resisting the adsorption of proteins. Single-component SAMs presenting single charges adsorbed nearly a full monolayer of protein. The amount of protein that adsorbed to mixed zwitterionic SAMs did not depend on the ionic strength or the pH of the buffer in which the protein was dissolved. The amount of protein that adsorbed to single-component zwitterionic SAMs increased as the ionic strength of the buffer decreased; it also decreased as the pH of the buffer increased (at constant ionic strength). Single-component zwitterionic SAMs composed of thiols terminating in N,N-dimethyl-amino-propane-1-sulfonic acid (-N + (CH3)2CH2CH2CH2SO3 -) groups were substantially more effective at resisting adsorption of fibrinogen and lysozyme from buffer at physiological ionic strength and pH than single-component zwitterionic SAMs composed of thiols terminating in phosphoric acid 2-trimethylamino-ethyl ester (-OP(O)2 -OCH2CH2N + (CH3)3). Several of these zwitterionic SAMs were comparable to the best known systems for resisting nonspecific adsorption of protein.
Article
The swimming patterns of zoospores of the green alga Ulva linza in the vicinity of a surface were investigated by digital in-line holography. Full 3D motion patterns were retrieved from measurements and the traces obtained were compared with known swimming patterns of spores of the brown alga Hincksia irregularis and the green alga Ulva linza as seen in a conventional optical microscope. Quantitative information such as swimming velocity was calculated from the 3D traces. The results demonstrate the potential of digital in-line holography to image and quantify exploratory patterns of behavior of motile spores close to surfaces. This technique can give detailed insight into mechanisms of surface colonization by spores and larvae of fouling organisms in response to changes in surface properties.
Article
1. Extensive research has been carried out on the biological, physical and chemical characteristics of surfaces that promote, or prevent, marine fouling. The texture of the substratum is probably the most important factor affecting settlement of the Acorn Barnacle Semibalanus balanoides in the field. The aim of this study was to examine the relationship between fractal and Euclidean descriptions of substratum surface complexity and settlement of the Acorn Barnacle. 2. Replicate settlement panels with smooth, fine, medium and coarse surfaces were manufactured using a precise and accurate technique. Two-dimensional profiles were measured using a laser profilometer, and two surface complexity indices, potential settling sites (PSS) and the Minkowski fractal dimension (MFD) were calculated. PSS is a Euclidean measure of the surface complexity taking into account the body size and settlement behaviour of the barnacle larvae, whereas MFD is a fractal dimension related to the complexity of the surface at a variety of scales. 3. In a field experiment, settlement density of S. balanoides was positively related to both PSS and MFD. To break the correlation between PSS and MFD, surfaces were modified by sandblasting with either 64–125-μm or 250–500-μm grains. This decreased MFD but did not affect PSS. 4. A further field experiment found that this erosion using small-scale sand particles had no effect on settlement of the barnacle. It was concluded that S. balanoides larvae were responding to characteristics of the surface related to PSS and not MFD. 5. Although a correlation was found between species abundance and the fractal complexity of its habitat, further experimentation showed that there was no causality in this relationship. Many surveys, with no experimentation, of aquatic and terrestrial communities have shown relationships between organisms and their habitat and assumed causality; however, care must be taken in interpretation of such studies.
Article
The imminent ban of environmentally harmful tributyltin (TBT)-based paint products has been the cause of a major change in the antifouling paint industry. In the past decade, several tin-free products have reached the commercial market, and claimed their effectiveness as regards the prevention of marine biofouling on ships in an environmentally friendly manner. The main objective of this review is to describe these products in as much detail as possible based on the knowledge available in the open literature. This knowledge has been supplemented by means of performance data provided, upon request, by some of the paint-producing companies. An exhaustive review of the historical development of antifouling systems and a detailed characterisation of sea water are also included. The need for studies on the behaviour of chemically active paints under different sea water conditions is emphasised. In addition, the most common booster biocides used to replace TBT-containing compounds are listed and described. It must be stressed that there is still a lack of knowledge of their potential environmental side effects.
Article
Gibbs surface energy has long been considered to be an important parameter in the design of fouling-resistant surfaces for marine applications. Rigorous testing of the hypothesis that settlement is related to Gibbs surface energy however has never been accomplished, due mainly to practical limitations imposed by the necessary combination of surface engineering and biological evaluation methods. In this article, the effects of surface charge and Gibbs surface energy on the settlement of cyprids of an important fouling barnacle, Balanus amphitrite, were evaluated. Settlement assays were conducted on a range of self-assembled monolayers (SAMs) (CH(3)-, OH-, COOH-, N(CH(3))(3) (+)-, NH(2)-terminated), presented in gold-coated polystyrene well plates, varying in terms of their surface charge and Gibbs surface energy. Contrary to contemporary theory, settlement was not increased by high-energy surfaces, rather the opposite was found to be the case with cyprids settling in greater numbers on a low-energy CH(3)- SAM compared to a high-energy OH- SAM. Settlement was also greater on negatively-charged SAMs, compared to neutral and positively-charged SAMs. These findings are discussed in the context of data drawn from surfaces that varied in multiple characteristics simultaneously, as have been used previously for such experiments. The finding that surface charge, rather than total surface energy, may be responsible for surface selection by cyprids, will have significant implications for the design of future fouling-resistant materials.
Article
A three-camera stereoscopy setup is presented that allows to reconstruct the trajectories of particles in dusty plasmas under microgravity. The calibration procedure for the three-camera setup takes the special circumstances into account that occur in close-range imaging of small particles. Additionally, a reconstruction algorithm is presented that is based on the epipolar geometry and delivers the essential particle correspondences. Further improvements are achieved by analyzing the dynamic particle behavior. Two applications of our calibration and reconstruction procedure are presented: A two-dimensional dust structure in the laboratory with a large percentage of hidden particles, and particles inside the void of a dust cloud under microgravity.
Article
From macro- to nanoscales, adhesion phenomena are all-pervasive in nature yet remain poorly understood. In recent years, studies of biological adhesion mechanisms, terrestrial and marine, have provided inspiration for "biomimetic" adhesion strategies and important insights for the development of fouling-resistant materials. Although the focus of most contemporary bioadhesion research is on large organisms such as marine mussels, insects and geckos, adhesion events on the micro/nanoscale are critical to our understanding of important underlying mechanisms. Observing and quantifying adhesion at this scale is particularly relevant for the development of biomedical implants and in the prevention of marine biofouling. However, such characterization has so far been restricted by insufficient quantities of material for biochemical analysis and the limitations of contemporary imaging techniques. Here, we introduce a recently developed optical method that allows precise determination of adhesive deposition by microscale organisms in situ and in real time; a capability not before demonstrated. In this extended study we used the cypris larvae of barnacles and a combination of conventional and imaging surface plasmon resonance techniques to observe and quantify adhesive deposition onto a range of model surfaces (CH(3)-, COOH-, NH(3)-, and mPEG-terminated SAMs and a PEGMA/HEMA hydrogel). We then correlated this deposition to passive adsorption of a putatively adhesive protein from barnacles. In this way, we were able to rank surfaces in order of effectiveness for preventing barnacle cyprid exploration and demonstrate the importance of observing the natural process of adhesion, rather than predicting surface effects from a model system. As well as contributing fundamentally to the knowledge on the adhesion and adhesives of barnacle larvae, a potential target for future biomimetic glues, this method also provides a versatile technique for laboratory testing of fouling-resistant chemistries.
Article
'Marine biofouling', the undesired growth of marine organisms such as microorganisms, barnacles and seaweeds on submerged surfaces, is a global problem for maritime industries, with both economic and environmental penalties. The primary strategy for combating marine fouling is to use biocide-containing paints, but environmental concerns and legislation are driving science and technology towards non-biocidal solutions based solely on physico-chemical and materials properties of coatings. Advances in nanotechnology and polymer science, and the development of novel surface designs 'bioinspired' by nature, are expected to have a significant impact on the development of a new generation of environmentally friendly marine coatings.
Article
Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.
Article
The settlement and colonization of marine organisms on submerged man-made surfaces is a major economic problem for many marine industries. The most apparent detrimental effects of biofouling are increased fuel consumption of ships, clogging of membranes and heat exchangers, disabled underwater sensors, and growth of biofoulers in aquaculture systems. The presently common-but environmentally very problematic-way to deal with marine biofouling is to incorporate biocides, which use biocidal products in the surface coatings to kill the colonizing organisms, into the surface coatings. Since the implementation of the International Maritime Organization Treaty on biocides in 2008, the use of tributyltin (TBT) is restricted and thus environmentally benign but effective surface coatings are required. In this short review, we summarize the different strategies which are pursued in academia and industry to better understand the mechanisms of biofouling and to develop strategies which can be used for industrial products. Our focus will be on chemically "inert" model surface coatings, in particular oligo- and poly(ethylene glycol) (OEG and PEG) functionalized surface films. The reasons for choosing this class of chemistry as an example are three-fold: Firstly, experiments on spore settlement on OEG and PEG coatings help to understand the mechanism of non-fouling of highly hydrated interfaces; secondly, these studies defy the common assumption that surface hydrophilicity-as measured by water contact angles-is an unambiguous and predictive tool to determine the fouling behavior on the surface; and thirdly, choosing this system is a good example for "interfacial systems chemistry": it connects the behavior of unicellular marine organisms with the antifouling properties of a hydrated surface coating with structural and electronic properties as derived from ab initio quantum mechanical calculations using the electronic wave functions of oxygen, hydrogen, and carbon. This short review is written to outline for non-experts the hierarchical structure in length- and timescale of marine biofouling and the role of surface chemistry in fouling prevention. Experts in the field are referred to more specialized recent reviews.
Article
This work describes the fabrication, characterization, and protein adsorption behavior of charged polymer gradients. The thin gradient films were fabricated by a two-step technique using UV-initiated free-radical polymerization in a reactor with a moving shutter. A homogeneous layer of cationic poly(2-aminoethyl methacrylate hydrochloride) was first formed, followed by a layer of oppositely charged poly(2-carboxyethyl acrylate) with a continuously increasing thickness. Adsorption from protein solutions as well as human blood plasma was investigated by imaging surface plasmon resonance and infrared microscopy. The results showed excessive protein adsorption in the areas where one of the polymers dominated the composition, while there was a clear minimum at an intermediate position of the gradient. The charge of the surface was estimated by direct force measurements and found to correlate well with the protein adsorption, showing the lowest net charge in the same area as the protein adsorption minimum. We therefore hypothesize that a combination of the charged polymers, in the right proportions, can result in a protein-resistant surface due to balanced charges.
Article
We describe a simple holographic method that has enabled us to capture as a single data set the trajectories of micrometer-sized objects suspended in water. By subtracting consecutive holograms of a particle suspension and then adding these difference holograms, we constructed a final data set that contains the time evolution of the particle trajectories free from spurious background interference effects. The method is illustrated by a recording of the motion of 5-10-microm diameter algae in water.
Article
The ship resistance penalties of slime, shell and weed are discussed in turn. Methods to measure the hard paint roughness of antifouling coatings are recapitulated. The determination of a satisfactory roughness parameter from correlations with measured roughness functions is described. This in turn, allows a relationship between ship added friction and roughness height to be found. This recapitulation allows consideration of using the same route for a surface with filamentous fouling. Consideration is given to low surface energy coatings and their roughness idiosyncrasies. The determination of economic penalties is discussed, both for a particular ship and globally.
Article
The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.
Article
Over the last decade, approaches to the development of surfaces that perturb settlement and/or adhesion by barnacles have diversified substantially. Although, previously, coatings research focussed almost exclusively on biocidal technologies and low modulus, low surface-free-energy 'fouling-release' materials, novel strategies to control surface colonisation are now receiving significant attention. It is timely, therefore, to review the current 'state of knowledge' regarding fouling-resistant surface characteristics and their mechanisms of action against settling larvae of barnacles. The role of the barnacle in marine fouling is discussed here in the context of its life cycle and the behavioural ecology of its cypris larva. The temporary and permanent adhesion mechanisms of cyprids are covered in detail and an overview of adult barnacle adhesion is presented. Recent legislation has directed academic research firmly towards environmentally inert marine coatings, so the actions of traditional biocides on barnacles are not described here. Instead, the discussion is restricted to those surface modifications that interfere with settlement-site selection and adhesion of barnacle cypris larvae; specifically, textural engineering of surfaces, development of inert 'non-fouling' surfaces and the use of enzymes in antifouling.
Marine aquarium blog. http://www.jonolavsakvarium.com/blog/200805/barnacle01.jpg
  • Bjørndal
doi:10.1016/j.porgcoat
  • Dm Yebra
  • S Kiil
  • Dam
  • Johansen
Deutscher Verlag der Wissenschaften
  • H W Brauner
  • Blaschke
  • Kinematik Und Quaternionen
Marine aquarium blog
  • Jo Bjørndal
  • B Rosenhahn
  • U Kersting
  • K Powell
  • R Klette
  • G Klette
  • H-P Seidel
Rosenhahn B, Kersting U, Powell K, Klette R, Klette G, Seidel H-P (2007) Mach Vis Appl 18(1):25–40. doi:10.1007/s00138006-0046-y
  • M Himpel
  • B Buttenschon
  • A Melzer
Himpel M, Buttenschon B, Melzer A (2011) Rev Sci Instrum 82(5):053706
  • N Otsu
Otsu N (1979) IEEE Trans Syst Man Cybern 9(1):62-66
Preis geb. DM 20,40. ZAMM
  • Deutscher Verlag Der Wissenschaften
Deutscher Verlag der Wissenschaften. Preis geb. DM 20,40. ZAMM. J Appl Math Mech/Zeitschrift für Angewandte Mathematik und Mechanik 42 (7-8):366-367. doi:10.1002/zamm. 19620420724
Rosenhahn B Classification of Swimming Microorganisms Motion Patterns in 4D Digital In-Line Holography Data
  • L Leal-Taixé
  • M Heydt
  • S Weisse
  • A Rosenhahn
Leal-Taixé L, Heydt M, Weisse S, Rosenhahn A, Rosenhahn B Classification of Swimming Microorganisms Motion Patterns in 4D Digital In-Line Holography Data. In: 32nd Annual Symposium of the German Association for Pattern Recognition (DAGM), 2010. Lecture Notes in Computer Science. Springer, pp 283-292