Surface Sensing and Settlement Strategies of Marine Biofouling Organisms
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.
1 Biological Systems Respond Sensitively to External Cues
Living organisms typically respond sensitively to numerous cues. Probably the most severe responses occur upon their exposure to harmful substances. Hence, toxicology is one of the oldest disciplines and since evolution of mankind, living organisms are used to determine toxicity of substances . Besides screening of drugs and active ingredients, response of small organisms is applied to monitor the existence of harmful substances in different water bodies. The conventional approach to monitor presence of toxins in rivers and estuaries applies physicochemical and analytical chemical techniques. However, the constantly increasing diversity of harmful substances and a range of potentially synergistic actions complicate a reliable assessment. Bioresponse-linked instrumental analytics bridges this gap to a certain extend as it links biomolecular recognition with chemical analysis . Other approaches make use of the fact that harmful substances such as certain metals frequently accumulate in biological organisms. Thus, chemical analysis of accumulation levels can drastically increase sensitivity of detection .
Besides approaches involving expensive and technically demanding chemical analysis, biological activity provides another natural and sensitive evidence for the presence of harmful substances. Changes in behavior occur fast and are thus especially suited for continuous biological monitoring of sudden increments in the concentration of harmful substances . Therefore, biological activity tests turned out to be complementary to trace analysis since water samples can not only be tested against known toxic contaminants, but also against other unknown substances. Within the framework for community action in the field of water policy of the European Commission such new tests are strongly encouraged .
In this sense, biological early warning systems are usually introduced to provide a “biological sum parameter” which points towards a possible pollution and thus triggers an extensive analytical program . Modern tests use bacteria, algae, waterfleas, fish or bivalves. A reliable operation is required at least for 1 week, the test setup needs to be easy to handle with less than 3 h of maintenance per week, and it should automatically detect alarm situations . Daphnia (water fleas) respond to toxins by changing their activity which is manifested in a change in motility [7, 8]. Such dynamical Daphnia tests were successfully applied at the Rhine river and seem to be especially useful to detect low concentrations of insecticides . The mussel activity test uses the opening and closing cycles of bivalves as sensitive indicator for the presence of harmful substances [4, 10]. These cycles can remotely be followed either by strain gauges or by electromagnetic induction . Since more than 10 years extensive data have been collected at different rivers (e.g. Rhine, Elbe, Danube) using the Dreissena-Monitor, an automated early warning system based on the opening and closing of the valves of 84 zebra mussels .
The fascinating selectivity and build-in sensory mechanisms of microorganisms do not only cause sensitive responses to toxins but also enables biofouling organisms to select surfaces and guide colonization. In this article some recent progress about the interaction of marine organisms with surfaces is reviewed. After a short outline describing the motivation to study cell-surface interactions in marine antifouling research, the behavior of zoospores of Ulva linza and barnacle cyprids are discussed in greater detail. For both species we summarize to which extend behavior and initial interactions correlate with surface properties. While biofouling organisms are frequently viewed as nuisance, this article highlights their intriguing skill to selectively colonize surfaces due to their ability to sense their properties.
2 Biofouling Research: The Quest for Environmentally Friendly Anti-fouling Coatings
Toxic anti-fouling is achieved by killing the organisms upon contact with the surface (a). Such action is usually achieved by embedding biocides into paints. Besides the restricted tin containing formulations based on TBT (tributyltin) and TPT (triphenyltin), especially copper is applied (usually as oxides) [15, 16]. Most of such metal-containing biocides are embedded into ablative coatings, which were first described by Holzapfel in 1904 . Even though ablative coatings  or self-polishing formulations  are able to limit the amount of deposited biocides into the environment, they cannot avoid the obvious environmental impact of the released toxins. Besides metal-based formulations, organic biocides are increasingly applied, either as active ingredient or as booster biocide. Some examples are zink pyrithiones (also used in anti-dandruff shampoos), isothiazolones, triazin-herbicides, dichloro-phenyl-dimethyl-urea (DCMU, Diuron), tetrachloro-isophthalonitrile, dichlorofuanid, zinc-ethylene-bis(dithio-carbamate), chlorothalonil, TCMS pyridine, and econea [13, 15, 16, 20].
A perfect alternative to ablative biocidal coatings would be inert foul-inhibiting surfaces (Fig. 1b). These anti-fouling surfaces are the most elegant, environmentally benign and desirable solution. Such inert coatings have been identified for a number of well-defined surfaces in short term, single species assays. Especially ethylene glycol (EG)x-containing coatings have been used in the biomedical area [21, 22, 23, 24] and have recently been investigated with respect to their marine anti-fouling potential [25, 26, 27, 28, 29, 30, 31, 32, 33]. However, the degradation of the ethylene-glycol-containing chemistries makes them unsuitable for long-term anti-fouling applications [34, 35]. Other promising approaches involve the use of amphiphilic [26, 27, 29, 36] or zwitterionic chemistries [37, 38, 39, 40]. Even though fouling inhibition is the most desirable way of avoiding biofouling, the development of such inert, non-toxic, and long-term stable coatings remains to be the most challenging of the three approaches.
The third possibility consists not in preventing, but in removing all unwanted fouling by creating a “self-cleaning” surface. The removal of foulers from so-called fouling-release coatings is achieved by the hydrodynamic drag caused by the movement of the vessel (Fig. 1c). Such coatings are usually based on silicone elastomers or fluoropolymers which do not inhibit settlement of biofouling organisms and thus biomass accumulates. However, the weak attachment strength to these polymeric materials allows fast-moving ships to self clean simply by the shear force present during their movement through the ocean [13, 41, 42, 43, 44]. Modern fouling-release coatings have self-cleaning ability even below 15 knots (e.g. Intersleek 900 requires >10 knots, Hempasil X3 87500 >8 knots) . Especially the combination of fouling-release with mechanical cleaning techniques such as hull grooming seem to be promising hybrid approaches for the future [46, 47].
The development of any of these three types of antifouling coatings frequently involves laboratory assays to quantify the effectiveness of the coatings towards reduction of surface colonization and/or adhesion strength of single species under well-defined conditions. Many of such biofouling laboratory assays focus on the sessile spore or larvae stage rather than the adult, macroscopically visible biofouling organism [14, 48, 49, 50]. In general, the applied assays can be subdivided into biomass accumulation measurements (settlement assays), adhesion strength measurements, and tracking experiments to quantify exploration behavior. All three measurements reflect the responses or interactions of microorganisms and larvae with surfaces. Prior to permanent adhesion, motile and highly selective species are able to explore the surface and commitment to permanent adhesion only occurs if positive cues are sensed. Two remarkably selective species that have extensively been investigated with respect to their selection of surfaces are zoospores of the green algae Ulva linza and barnacle larvae.
3 Zoospores of the Green Alga Ulva Linza—Swimming and Selective Plants
3.1 Holographic 3D Tracking of Zoospores Reveals How Spores Select a Suitable Location for Settlement
3.2 Swimming Zoospores Explore Surfaces and Respond to Surface Cues
However, the surface does not only influence the swimming speed of the spores, but also their behavior. Iken et al.  observed that the presence of a surface induces a motion behavior termed gyration of the brown algae Hincksia irregularis that differs from those ones observed in solution and is characterized by intense exploration and occasional surface contacts. Such patterns are also observed by holographic microscopy for the green algae Ulva linza and schematically depicted in Fig. 5c . Within the gyration motion pattern (pattern 1), two extreme cases of motion can be subdivided: hit and run (pattern 2), which describes a single surface contact after which the spores immediately left the surface; and hit and stick (pattern 3), that describes the situation whereby, as soon as spores contacted the surface, they immediately stop swimming and stick to the surface. The term “sticking” means that the spores remain motionless at a distinct point on the surface, but they have not yet undergone the process of initiating irreversible settlement, i.e. shedding their flagella and discharge of adhesive. As shown in Fig. 5d, the occurrence of the different motion patterns depends on the surface chemistry. Gyration is detected as the dominant pattern on PEG and on AWG. However, on PEG the probability of observing a hit and run event is nearly twice as high (44 %) compared to AWG, indicating that the PEG surfaces are less attractive to the spores. The situation is different on FOTS and spores exploring the surface show predominantly the hit and stick behavior. A hit and stick pattern never occurred on PEG and AWG. The high probability of observing a hit and stick pattern indicates that the pristine and hydrophobic fluorinated surface attracted spores. The occurrence of the different motion patterns associated with attractive or repulsive properties of the surface correlates well with the integral assay and the deceleration analysis.
3.3 The Spinning Motion Tests the Strength of Temporary Adhesion
The accumulation kinetics of spores is not only a consequence of deceleration and exploration patterns as both can change with time. Such changes are most obvious on very hydrophobic surfaces (i.e. FOTS) as the probability to observe patterns indicative of an attractive surface vanish over time . On such hydrophobic surfaces, conditioning films are formed within hours and Thomé et al.  revealed that the presence of such overlayers decreases the settlement rates of Ulva zoospores by ≈50 %. Thus it is likely that surface conditioning affects the deceleration and the probability to observe certain motion patterns. However, Ulva zoospores have a second build-in sensory mechanism that involves the spinning motion (Fig. 4d).
Irrespectively of whether spores got stuck on the surface as a result of hit and stick motion or gyration, soon after the surface contact a spinning motion is started. This motion involves a rapid spinning of the spores around a temporary anchoring point on the surface (Fig. 4d). This spinning motion can take up to several minutes, but most spores (>95 %) leave the surface soon after spinning is initiated and continue exploration. Only a minority of spores (<5 %) spins for a longer time and finally settles permanently, which involves secretion of adhesive and shedding of the flagella [52, 53]. The duration of the spinning phase depends on the surface chemistry and spinning is longer on FOTS than on the less attractive AWG surface . Only those spores that spin long enough initiate permanent settlement. It appears as if the spinning motion exerts a force on the temporary surface contact and only if the spore-surface contact is strong enough, the spinning process reaches the required critical duration to trigger the permanent secretion of adhesive. The duration of the spinning phase may thus reflect the strength of the initial temporary bond to the surface. This strategy seems advantageous since it may reduce the likelihood of spores committing to permanent settlement on surfaces to which they adhere weakly, as they immediately leave such surfaces after initiation of spinning. Therefore, spores use a sophisticated spinning mechanism to probe the stability of the cell-surface contact in order to restrict permanent settlement to those surfaces providing a stable anchoring point. This mechanism complements surface selection by exploration behavior.
3.4 Surface Cues Can Trigger Permanent Adhesion of Zoospores of Ulva linza
The deceleration, the behavioral response, and the spinning phase finally determine the kinetics by which spores colonize a surface. This, in turn, is affected by the chemical termination of the surface [35, 64]. The Callow group established an assay that allows spores to settle within 45 min to surfaces in order to compare the spore accumulation rate on different surfaces and thus to discriminate their non-fouling potential . A vast number of experiments demonstrated that the settlement kinetics of zoospores of Ulva is affected by a number of physical and chemical surface cues, such as wettability [31, 64, 67, 68], topography [69, 70, 71, 72], and charge [73, 74].
A comparison of receding contact angle against spore settlement has been done for a number of amphiphilic and non-amphiphilic polymer surfaces by Grozea and Walker . The study clearly shows that receding contact angle is not the only surface property that mediates spore settlement and there exist classes of surfaces where such a correlation is not valid. A similar observation has been made by the Grunze group, who found that surfaces with similar wettability can show different settlement of zoospores [33, 82]. In this study, ethylene glycols with different chain length and thus decreasing packing density were used [82, 83]. Monte Carlo simulations revealed that such a decreased packing density facilitates penetration of water into the thin films, providing the necessary steric freedom for a stable binding of water [84, 85]. Despite the different hydration, all of the tested surfaces have a similar water contact angle [82, 83]. Ulva zoospore experiments show that spores adhere much weaker on well hydrated surfaces , an observation in line with the protein resistance of the surfaces . The fact that changing hydration continuously alters the inertness of a surface was finally proven by Christophis et al.  who used a microfluidic experiment to show that the adhesion strength of cells gradually decreases with increasing ethylene glycol chain length.
The selected overview on settlement data shows that the accumulation rate of spores on surfaces is not determined by one surface property alone but results as a combination of different properties. The sensory mechanism of spores thus responds to each surface property in a different way. When viewing colonization of surfaces by spores as highly parallelized surface sensing event, the relative contribution of the different physicochemical properties on the sensing process seems to be of major relevance, but yet needs to be fully understood. In a way, the situation is similar to protein affinity assays that also not always correlate with single surface properties. It seems to be rather the interaction strength that results from the combined physicochemical properties of the surface that finally determines adhesion and potential degeneration of proteins or, in the discussed example, settlement of algal spores.
3.5 Settlement and Adhesion Strength
As revealed by the holography study summarized above, spores select surfaces by different ways of active probing. This involves a spinning phase which is used to test adhesion strength to a surface. The accumulation kinetics that leads to the spore biomass data in Fig. 6 should be a direct consequence from this selection process. One could now ask, how well is the spinning phase capable to predict adhesion strength of settled spores, i.e. how reliable is this mechanism. Experiments in calibrated flow channels allow to measure the removal of spores from surfaces and thus to discriminate between weakly and strongly sticking spores [87, 88]. For the series of alkyl terminated OEG SAMs with different wettability (used in Fig. 6a), the removal is easier from surfaces with low settlement . This means that spores accumulate only on those surfaces where stable anchoring is possible. A similar correlation has been found comparing amphiphilic and other polymeric materials by the Ober group . However, in other examples such as on mixed aliphatic SAMs, opposite trends are observed . In some cases such as hexaethylene glycols, even highly gregarious behavior is observed, although attachment strength is extremely weak . The contrary examples show that the rate of spore accumulation does not always correlate with adhesion strength. One of the many possible reasons for the observed discrepancies could be a different composition of the temporary and the permanent adhesive.
Summarizing, despite their small size and their limited sensory abilities, spores show a surprisingly sophisticated mechanism for selecting surfaces. Although they might be viewed as living surface analytical tool, further research is required for a more detailed interpretation of the obtained data and a better understanding of the correlation between interface properties, spore behavior, settlement and adhesion strength.
4 Barnacle Cyprids: Motile and Selective Larvae as Early Stage of Hard Fouling
4.1 Settlement of Barnacle Cyprids
4.2 Behavior of Barnacle Cyprids on Surfaces and Response to Surface Cues
4.3 Field Studies of Surface Exploration and Settlement Behavior
The behavior of wild cyprid larvae of Semibalanus balanoides in situ in the ocean close to different surface textures treated and untreated with crude conspecific adult extract (AE) has been studied by Prendergast et al. . The treatment with AE produces an increase in the number of cyprids arriving on the surface both, within the first minute and after a longer time. Results furthermore suggest that cyprids tend to explore smooth surfaces longer and leave rough surfaces earlier. This means that during the exploration phase cyprids were not only sensitive to the surface chemistry but also to the surface topography as they directly respond by changing their behavior. As pointed out by Aldred et al. , those topographies reducing adhesion are less likely to be selected for settlement and metamorphosis. Probably, the sensing during the exploration phase and the observed responses are directly connected with the strong influence of surface morphology on the probability of cyprids to settle and metamorphose [99, 100, 101].
4.4 3D Tracking of Barnacle Cyprids
4.5 A Closer View on Surface Exploration: “Walking” Cyprids
Tracking of cyprids under flow allowed to understand the influence of the surface properties on exploration under dynamic conditions . As cyprids use antennules to attach, detach, and reattach to surfaces, Chaw and Birch evaluated the “walking” behavior of Amphibalanus amphitrite by measuring the step length and the duration required to carry the steps out. In the absence of water flow, both parameters are significantly influenced by the surface properties. The mean step length on hydrophilic surfaces (bare glass and –NH2 functionalized glass) is larger than on hydrophobic surfaces (–CH3 functionalized glass). In turn, the step duration is longer on the hydrophilic surfaces than on the hydrophilic ones. Consequently, the longer step duration and shorter steps leads to a slower motion on the hydrophobic surfaces, while the opposite is observed for the hydrophilic coatings .
If a water flow is applied and shear forces are present, cyprids actively respond by altering their exploration behavior . On hydrophilic surfaces, the step length remains unchanged, but the step duration increases. In contrast, larger step lengths and shorter step durations are observed on hydrophobic surfaces. Especially the shorter steps can be connected with the requirement to re-generate surface contacts more frequently as the temporary anchoring point is challenged by the presence of shear. It was also found that behavior of cyprids depended on the age and discrimination power between hydrophilic and hydrophobic surfaces is lost when older cyprids are used .
As already observed by Schumacher et al. , Aldred et al.  and in field experiments by Prendergast et al. , surface morphology affects exploration and settlement. Chaw et al.  described the behavior of Amphibalanus amphitrite over a pattern of cylindrical micropillars with heights of 5 and 30 μm, a separation of 10 μm and diameters ranging from 5 to 100 μm. Only the higher pillars significantly influence cyprid exploration. Temporary attachment mainly occurs in the voids or at the sides of the pillars rather than on their top. The 30 μm high and 5 μm thin pillars offer a very small contact area for the attachement discs of the cyprids, resulting in a strong reduction of the step length and a large increase of the step duration (at least 50 % compared to other diameters and smooth surfaces).
4.6 Footprints of Walking Cyprids Visualized by Imaging Surface Plasmon Resonance
During the “walking phase”, temporary contacts with the surface are established by the two antennules . The antennules touch the surface via attachment disks that facilitate bipedal walking over the surface. The attachment disk itself is covered with small cuticular villi and pores allow the secretion of a ‘temporary adhesive’ composed majorly of proteins [91, 105]. The antennules do not only moderate adhesion but also serve as sensory organ. Small setae are present as mechanosensors to perceive surface properties. During exploration and intense inspection, cyprids can use a ‘temporary adhesive’ to interact with the surface. As consequence, exploring barnacle cyprids may leave footprints that contain pheromones .
Summarizing, barnacle cyprids exhibit a selective mechanism to determine where to settle. This selection process involves initial contacts by walking and exerting of local forces on the temporary adhesive. As consequence, one observes behavioral responses and eventually commitment to settlement. As in the case of Ulva spores, more studies are needed for a better understanding of the surface properties involved, but also surface colonization by cyprids of barnacles can be viewed as collective surface sensing event.
5 Summary and Outlook
Some recent results on the interaction of algal zoospores and barnacle cyprids with well-characterized surfaces were summarized with examples of how the sessile stages of marine organisms respond to the properties of surfaces by changing their exploration behavior. Ulva zoospores established a remarkable strategy to test surfaces, involving deceleration close to the surface and a subsequent spinning behavior to probe cell-surface contact. Both, the deceleration and the duration of the spinning phase depend on the surface properties. This surprisingly sophisticated mechanism leads to different accumulation kinetics on chemically different surfaces. Interestingly, hydrophilic, well-hydrated surfaces seem to reduce settlement and adhesion strength, while hydrophobic, weakly hydrated surfaces encourage settlement with strong spore adhesion. However, this picture is merely a black and white picture as e.g. trends in amphiphilic coatings are more challenging to understand. Especially the combination of behavioral studies, spinning phase analysis, and potential future combination with SPR could serve to understand this open question. Surface colonization can be considered as parallelized surface sensing event and the general perspective to have many little and independent surface sensors is intriguing as each of them is capable of attaching and exerting a force on a temporary adhesive. However, for a future application, e.g. for multiplexed testing of interaction forces, more knowledge about the complementarity of spore settlement, protein affinity and physicochemical surface properties need to be derived.
A similar conclusion can be drawn for exploration behavior of barnacle cyprids. Settlement preferences are different for different species and are guided by the physicochemical properties of the surfaces. Cyprids distinguish surface topographies and select those morphologies for settlement and metamorphosis that allow thorough adhesion. Tracking reveals behavioral responses and velocities close to chemically and morphologically different surfaces. Especially 3D tracking has great potential as it allows direct imaging of larvae responses not only in the laboratory but also in the natural habitat, the real ocean environment. In particular, the correlative analysis of the “walking” behavior and footprint deposition as accessible with iSPR seems to be very promising and can be expected to contribute to understand surface selection, settlement and adhesion of cyprids.
We expect three research and application fields to be relevant in the future: First of all, 3D tracking and time resolved, interface sensitive surface analysis techniques will allow to understand surface selection strategies of marine biofouling organisms. Secondly, the highly parallelized surface selection that involves active surface sensing could be applied to test surfaces with respect to their inert properties. However, this application requires more knowledge about which physicochemical surface properties are probed in such an experiment. The third point is rather a consequence as these new techniques will help to identify novel surface coatings that aim on reduced adhesion and thus enhanced foul released properties.
We appreciate helpful comments on the manuscript from Michael Grunze. We acknowledge funding by the ONR project N000141210498, the Grant Agreement number 237997 (SEACOAT) of the European Community’s Seventh Framework Programme FP7/2007-2013 and the DFG grant RO 2524/2-2.
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