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Effect of ultrasound on cyprid footprint and juvenile barnacle adhesion on a fouling release material
Colloids and Surfaces B: Biointerfaces 115 (2014) 118–124
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Effect of ultrasound on cyprid footprint and juvenile barnacle adhesion on a fouling release material Shifeng Guo a,b,∗ , Boo Cheong Khoo a,d , Serena Lay Ming Teo c , Shaoping Zhong e , Chwee Teck Lim a,e,f , Heow Pueh Lee a,d a
Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore Institute of Materials Research and Engineering (IMRE), A*STAR, 3 Research Link, Singapore 117602, Singapore c Tropical Marine Science Institute, National University of Singapore, Singapore 119223, Singapore d National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiang Su 215123, People’s Republic of China e Department of Bioengineering, National University of Singapore, Singapore 117576, Singapore f Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore b
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 4 November 2013 Accepted 10 November 2013 Available online 23 November 2013 Keywords: Ultrasound AFM Barnacle cyprid footprint Settlement Adhesion strength Silicone substrates
a b s t r a c t In our earlier studies, we have demonstrated that low and high intensity ultrasound can prevent barnacle cyprid settlement. In this study, we found that ultrasound treatment reduced the adhesion of newly metamorphosed barnacles up to 2 days’ old. This was observed in the reduction of adhesion strength of the newly settled barnacles from ultrasound treated cyprids on silicone substrate compared to the adhesion strength of barnacles metamorphosed from cyprids not exposed to ultrasound. Atomic force microscopy (AFM) was used to analyze the effect of ultrasound on barnacle cyprid footprints (FPs), which are protein adhesives secreted when the larvae explore surfaces. The ultrasound treated cyprids were found to secrete less FPs, which appeared to spread a larger area than those generated by untreated cyprids. The evidence from this study suggests that ultrasound treatment results in a reduced cyprid settlement and footprint secretion, and may affect the subsequent recruitment of barnacles onto fouling release surfaces by reducing the ability of early settlement stage of barnacles (up to 2 days’ old) from firmly adhering to the substrates. Ultrasound therefore can be used in combination with fouling release coatings to offer a more efficient antifouling strategy. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Barnacles are a major problem in marine fouling due to their size, hard shell, and gregarious nature [1]. In earlier studies, it has been demonstrated that both low and high intensity ultrasound can prevent barnacle cyprid settlement [2–5]. The probable mechanism for the prevention of cyprid settlement using high intensity ultrasound was attributed to ultrasonic cavitation [3], and it was also shown that cavitation bubbles can be used to remove early stages of barnacle settlement [6]. In this paper, we further examine the feasibility of using ultrasound to control the barnacle fouling on fouling release surfaces. It has been reported that attachment strength plays a significant role in the recruitment of marine organisms onto a surfaces, and stronger forces are required to remove organisms on surfaces with higher settlement [7,8]. For example, the settlement of Mytilus
∗ Corresponding author at: Institute of Materials Research and Engineering (IMRE), A*STAR, 3 Research Link, Singapore 117602, Singapore. Tel.: +65 65148745. E-mail address: [email protected] (S. Guo). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.020
galloprovincialis was found to be positively correlated to adhesion strength [7]. Also, barnacle cyprids preferred to settle on the substrates where the possibility of subsequent removal is least likely to occur [8]. Fouling release coatings (FRCs) are now widely used as antifouling for ship hulls [9,10]. Hydrophobic FRCs was reported to be beneficial to vessels operating above 10 knots and resulted in less fuel consumption in comparison to biocide-containing selfpolishing coatings [11]. One of the main features of the FRCs is that the interfacial bond between the organism and coating is weak. As a result, the attached organisms are more easily removed by the hydrodynamic forces created from a vessel’s movement through the water, or by simple mechanical cleaning [9,12]. The cyprids and juvenile barnacles have been previously proposed as appropriate experimental models for the evaluation of antifouling performance [12]. The lifecycle of the barnacle, Amphibalanus amphitrite (=Balanus amphitrite), includes planktotrophic nauplius stages, a non-feeding cypris larval stage, and a sessile adult stage. The pre-settlement cypris stage actively explores surface and metamorphoses into the juvenile barnacle once a suitable site is found. Surface exploration is conducted using the antennules, in a form of bi-pedal ‘walking’, which appears to be
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affected by the surface texture, material properties, chemical clues, and presence of adult or cyprid nonspecific proteins [9,13]. During exploration, temporary proteins secretion is deposited as footprint (FP) through the antennular attachment discs and it has been implicated to act as settlement cues for other exploring cyprids [1,9]. The FP has been described as a temporary adhesive, as it enables reversible attachment to surfaces [14,15]. A settlement-inducing protein complex (SIPC), which also functions as a settlement cue, has been found in FPs [14,15]. It has also been reported that surfaces which are apt to the adsorption of FPs would lead to higher settlement [16]. Ultrasound treated cyprids have been shown to have an altered exploration behavior [2]. Since cyprids secrete temporary adhesive proteins during surface exploration, it is conceivable that the altered walking behavior may also be accompanied with change in the secreted FPs. Based on the preliminary experiments, juvenile barnacles metamorphosed from ultrasound treated cyprids were more easily detached. It is surmised that the adhesion strength may be reduced. Therefore, the effect of ultrasound on juvenile barnacle adhesion strength was evaluated using a nano-tensile tester. Also, since FPs play a significant role on cyprid settlement, the effect of ultrasound on the FPs was explored using AFM, with the objective of gaining a better understanding of the mechanism behind the ultrasound induced inhibitory effects. 2. Materials and methods 2.1. Barnacle culture Larvae of the barnacle Amphibalanus amphitrite were reared at 26 ◦ C, fed with an algal mixture of 1:1 (v/v) of Tetraselmis suecica and Chaetoceros muelleri at a density of approximately 5 × 105 cells/ml [17]. Barnacle larvae metamorphosed into cyprids within 5–7 days. The cyprids were stored at 4 ◦ C and used for experiments after 3 days. Juvenile barnacles metamorphosed from ultrasound treated and untreated cyprids were reared and fed daily with algal mixture of 1:1 (v/v) of Tetraselmis suecica and Chaetoceros muelleri at a density of approximately 5 × 105 cells/ml. 2.2. Ultrasonic experimental setup The ultrasonic experimental setup is same as that described in Guo et al. [3]. As low frequency of 23 kHz generated most significant effect on barnacle cyprid settlement and exploration behavior [2], in the present study, only this frequency was chosen, with the pressure set at 20 kPa. The cyprids were then subjected to ultrasound exposure for 5 min. 2.3. Surfaces choice for the barnacle adhesion and cyprid FPs study There are two surfaces used to study the effect of ultrasound on juvenile barnacle adhesion strength and cyprid footprints, respectively. The fouling release surfaces (silicone substrates) were used to evaluate the effect of ultrasound on barnacle adhesion strength. The surfaces were chosen because the interaction between juvenile barnacle cement and the surface is low. Therefore, the barnacles were easier to be fully detached, and the forces measured can be regarded as the adhesive forces. The fouling release surfaces used to evaluate barnacle adhesion strength were reported widely [9,12,18]. To study the effect of ultrasound on FPs, the NH2 terminated glass cover slips were used as the surfaces were easier for the FPs detection using AFM and shown more concentrated and distinct FP patterns [19,20].
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Fig. 1. The schematic of nano-tensile tester on juvenile barnacle detachment forces measurement.
To prepare the NH2 terminated surfaces, the glass microscopy cover slips were firstly immersed in 5% Decon 90 solution and cleaned using ultrasonic clean-tank for 20 min. The use of Decon 90 solution to effectively clean surfaces for increasing surface density of silanol groups and/or prior to silanization has been well documented in the previous literature [21,22]. The slips were then rinsed thoroughly with ultrapure water and dried with nitrogen gas. The amino (NH2 ) terminated surfaces were obtained by immersing the cleaned slips in 5% 3-aminopropyl triethoxysilane (APTES) solution and were put in the shaker (GFP MBH, Germany) for 30 min. After that the surfaces were rinsed with ultrapure water thoroughly and dried using nitrogen gas. Static water contact angles (CA) of NH2 terminated surfaces and silicone substrates were measured at 25 ◦ C, using the sessile drop method with a 2 l water droplet, in a telescopic goniometer (model 100-00-(230), Rame-Hart, Inc., Mountain Lake, NJ, USA). To measure the contact angle of coated cover slips, eight samples were replicated and the averaged contact angle was 55◦ ± 2.4◦ and the value was approximately similar with reported results [19,20]. The contact angle of silicone substrates was 94◦ . 2.4. Cyprid settlement on silicone substrate Cyprid settlement was performed with a ‘no choice’ droplet assay, as described in Refs. [8,23]. The experiments were conducted on the medical grade silicone substrates (Bioplexus, USA). The material was cleaned following the provided instructions before usage. It was reported that the gregarious behavior of cyprids did not interfere with settlement rate at cyprid densities of 5–200 per 5 ml [24]. In our experiments, after ultrasound exposure, 500 l volume of filtered seawater (FSW), containing approximately 15–20 cyprids, was deposited on each silicone substrate (2 cm × 2 cm), forming a droplet on the substrate. The substrates were placed in Petri dishes and sealed with parafilm to prevent water evaporation. The assay was incubated at 26 ◦ C for 48 h, on a 15 h light and 9 h dark cycle. The cyprids were then examined under a stereo microscope (Nikon SMZ 1500, Japan), and the number of cyprids which had settled and metamorphosed into barnacles was enumerated. To evaluate the ultrasonic effect, eight replicate silicone substrates were prepared for both ultrasound treated and control cyprids. 2.5. Juvenile barnacle adhesion strength measurement Measurement of barnacle adhesive force was conducted using a calibrated Nano-tensile tester (Nano Bionix System, MTS, USA), and the schematic of the experimental setup is shown in Fig. 1. The silicone substrate (2 cm × 2 cm) with barnacles settled was clamped in the lower grip. A micro steel fiber with diameter of 80 m and length of 10 mm was clamped in the upper grip. The fiber was controlled and lowered until it was slightly above the top of barnacle. Then the tip of the fiber was wetted with a small drop of superglue (Selleys Pty Ltd, Australia) and lowered to touch the top of
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Fig. 2. The schematic of AFM on barnacle cyprid footprint scanning. (A) Cyprid explored the NH2 terminated cover slip and left footprint on it; (B) the morphology of footprint was scanned by AFM D3100.
barnacle shell [18]. The glue was allowed to dry for about 30 s and the force required to dislodge the barnacle was measured. To study the effect of pulling velocity on barnacle detachment force, two strain rates were chosen: one was 1.0 × 10−4 s−1 , representing low pulling velocity and the other was 1.0 × 10−2 s−1 , representing fast pulling velocity. The preliminary results revealed that no significant detachment forces were detected between the two pulling velocities, therefore, in our study, only strain rate of 1.0 × 10−2 s−1 was chosen. Similar results were found by Berglin et al. [18], who found that pulling velocities did not affect barnacle attachment force measurement. After detachment, the substrates were gently rinsed using ultra-pure distilled water and observed under a stereo microscope with image capture software (Nikon Instech Co., Ltd., Japan). Only the forces that completely detached the barnacles were recorded. If any visible amount of base plate of barnacle was still remained on the surface after pulling experiment, which indicates incomplete removal, the associate force was considered invalid and therefore discarded [25]. For each group barnacles, ten replicates were measured. Adhesion strength was calculated by dividing the measured force required to detach the barnacle by the basal area [26]. As such, the basal area of barnacle was estimated. The image of barnacle before experiment was photographed using a stereo microscope with an image capture software. The image was processed with ImageJ software (Version 1.43), which uses edge contrast to define the perimeter and thereby calculates the total basal area. Adhesion strength measurements were conducted on the newly metamorphosed barnacles (day 0) to day 8 barnacles. For the day 8 barnacles, partially barnacle base plates remained on the substrates after the experiments. Therefore, the measured values do not reflect the actual removal forces, but the data have been included for the discussion purposes.
as described in Ref. [27]. In this study, the AFM scanning was conducted using a Dimension D3100 atomic force microscope (Veeco/Digital Instruments (DI), Santa Barbara, CA). Since cyprid FP morphology showed no significant difference in air and in filtered sea water (FSW), but imaging in air provided higher resolution [19,20]. In the present study, the AFM scanning was conducted in air with tapping mode. The NH2 terminated cover slips were mounted on the glass sides prior to experiments. Cyprids were transferred to the modified cover slips using a micropipette. The walking cyprid was monitored by a stereo microscope equipped with video capture software NIS-Elements BR 3.2 (Nikon Instech Co., Ltd., Japan). The explored areas were marked on the back of glass slides and the videos of the ‘walking’ were captured for ease of FPs searching during AFM scanning. The cyprid was subsequently removed and the surface was rinsed with ultrapure distilled water and dried with nitrogen gas to minimize the contamination. Before exploration, cyprids were allowed 5 min’ acclimatization after ultrasound exposure and the observation time for both control and ultrasound treated cyprids were within 40 min. The FP images were taken in air using the intermittent contact mode with silicon cantilevers having a spring constant of 30 N/m approximately (Nanosensors, Germany). To evaluate the effect of ultrasound on cyprid FPs, six FPs of both control and ultrasound treated cyprids were analyzed and all the FPs were obtained from different individual cyprid. 2.7. Data analysis The statistical comparisons were performed using GraphPad Prism 5 (GraphPad Software Inc.). Barnacle adhesive forces/strength data were analyzed using a two-way analysis of variance (ANOVA) and the Bonferroni post tests were conducted to evaluate the influence of ultrasound and age effect. Data of settlement and cyprid FPs was analyzed using t-test. All data are reported as mean ± standard error (SE). For all comparisons, p-values ≤0.05 were considered as statistically significant. 3. Results 3.1. Cyprid settlement on silicone substrates The settlement of control and ultrasound treated cyprids is shown in Fig. 3. Significant differences were observed between ultrasound treated and control cyprids (t-test, p < 0.001). After ultrasound treatment with 23 kHz for 5 min at 20 kPa, the settlement was reduced significantly from 44% to 16% (Fig. 3).
2.6. Effect of ultrasound on cyprid footprints Enhanced FP absorption has been reported on the NH2 terminated glass microscopy cover slip [19,20]. Therefore, in this study, this particular surface was chosen to evaluate the effect of ultrasound on cyprid FPs. The schematic of AFM setup for cyprid FPs measurement is shown in Fig. 2 and the working principle and protocol are
Fig. 3. Cyprid settlement comparison. Error bars are standard errors. The asterisk represents statistically significant difference.
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3.3. Observation of barnacle images
Fig. 4. The adhesion strength comparison of juvenile barnacles metamorphosed from control and ultrasound treated cyprids. The asterisks represent statistically significant difference. The error bars are standard errors.
3.2. Effects of ultrasound on juvenile barnacle adhesion strength The detachment force of the settled juvenile barnacle was measured using a nano-tensile tester. The adhesion strength was calculated by dividing the detachment force over the basal area of juvenile barnacle. Both ultrasound treatment and barnacle age significantly affected the adhesion strength (two-way ANOVA, p < 0.05; Fig. 4). The results revealed that adhesion strength was significantly reduced after ultrasound exposure for the day 0 and day 2 barnacles (Fig. 4; Bonferroni post test, p < 0.01). For day 0 barnacles, the adhesion strength of the juvenile barnacles metamorphosed from control cyprids was 0.83 ± 0.05 × 105 Pa, while the adhesion strength of barnacles from ultrasound treated cyprids was 0.70 ± 0.03 × 105 Pa. The adhesion strength increased to 1.33 ± 0.11 × 105 Pa and 1.13 ± 0.06 × 105 Pa, respectively, for the day 2 barnacles. There was no difference detected after 4 days’ in culture (Bonferroni post test, p > 0.05; Fig. 4). The adhesion strength of barnacles was also found to increase significantly with age (Bonferroni post test, p < 0.01). The adhesion strength of day 6 barnacles was more than twice the value of day 2 barnacles (Fig. 4).
The images of barnacles before and after pulling experiments were captured using microscope equipped with image capturing software. The dislodgement areas were gently rinsed using deionised water (DI) water and imaged after drying. Since no difference in surface failure modes was detected between ultrasound treated and control barnacles, only the images of control barnacles are shown in Fig. 5. The newly metamorphosed barnacles to the day 6 barnacles can be completely removed, suggesting that the detachment occurred between barnacle adhesive cement and the substrates. Therefore, the values measured could be regarded as the adhesion strength. However, for the day 8 barnacles, partial barnacle base plates were still remained after detachment. This may suggest that partial cohesive failure occurred within barnacles (leaving the partial barnacle base plates on the substrate), which means that the adhesion strength between barnacle base plates and substrates exceeds the cohesive strength within the barnacles. Therefore, the strength measured for the day 8 barnacles could not be treated as the adhesion strength but lower than that value.
3.4. Effect of ultrasound on cyprid footprints The morphology of cyprid FPs was studied using AFM. The representative images of FPs obtained from control and ultrasound treated cyprids are shown in Fig. 6. Entire footprints and sections of footprints with higher magnification were imaged. For cyprids exposed to ultrasound, the FPs exhibited difference in morphology compared with that of control cyprids. The FPs from ultrasound treated cyprids had a larger spreading area than that secreted by control cyprids, however, FPs from control cyprids was found to be much thicker (t-test, p < 0.05; Fig. 6 and Table 1). The fibrillar and porous structure of the FP was observed for both ultrasound treated and control cyprids, however, less porous structures was found on control cyprids. The geometrical data extracted from AFM scanned FPs are presented in Table 1. The mean FP spreading area from ultrasound treated cyprids was 1142 ± 92 m2 , which was significantly larger
Fig. 5. Microscopy images of surfaces after removal of different age barnacles.
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Fig. 6. The morphological comparison of FP from control and ultrasound treated cyprids on NH2 terminated surfaces. (A) The FP of control cyprid; (B) the magnified FP of control cyprid; (C) the 3D structure of FP of control cyprid; (D) the FP of ultrasound treated cyprid; (E and F) the magnified and 3D structure of FP from ultrasound treated cyprids, respectively.
than the FP area of control cyprids of 778 ± 54 m2 (t-test, p < 0.05; Table 1). The root-mean-square (RMS) thickness of the FPs, calculated from AFM results, was found to be significant lower than that measured from the control cyprids (t-test, p < 0.05). The FP volume, which was used to quantify the amount of the temporary adhesive proteins secreted when cyprids explored the surfaces, was found to be significantly different between the ultrasound treated and control cyprids (t-test, p < 0.05; Table 1). The volume of FPs secreted by control cyprids was 12.6 ± 1.2 m3 , which was approximately one third more compared to the volume secreted by ultrasound treated cyprids (Table 1). 4. Discussion and conclusion Cyprid settlement on the silicone substrate was significantly inhibited with ultrasound treatment. The ability of ultrasound to prevent cyprid settlement has been previously reported [2,5], and it has been shown that ultrasound intensity exceeding the cavitation threshold could reduce cyprid settlement by cavitation generated forces [3,6]. One of the main features of fouling release coatings is to minimize the adhesion between the organism and the surface so that the hydrodynamic forces imposed by the ship moving through the water or a simple mechanical cleaning method will be able to remove the fouling [12]. However, these surfaces are highly susceptible to fouling when the vessels move slowly. We have previously reported that ultrasound can reduce cyprid settlement, and in this paper we demonstrate that ultrasound exposure also reduces the adhesion strength of the newly settled barnacles to fouling release silicone substrates up to 2 days’ old. Barnacle adhesion strength has attracted considerable scientific attention because of the strength and durability of the adhesive, which are practical concerns related to marine fouling [28]. The Table 1 The morphological information of FPs obtained by AFM. Six FPs of control and ultrasound treated cyprids were analyzed and the errors here are standard errors. Footprint type
Mean footprint area (m2 )
Mean footprint thickness (nm)
Mean footprint volume (m3 )
Ultrasound treated Control
1142 ± 92 778 ± 54
8.7 ± 0.6 16.3 ± 1.1
9.4 ± 0.62 12.6 ± 1.2
proteinaceous adhesive secreted by barnacles are able to cure underwater, providing a strong adhesion to the substrate [29]. The barnacle adhesion strength measurement in this study was conducted on the fouling release coatings using a nano-tensile tester on different age barnacles and the surface failure modes were observed using microscope. The results showed that the newly metamorphosed juvenile barnacles (up to 6 days’ old) from control and ultrasound treated cyprids can be completely detached. The measured forces can be regarded as the adhesive forces as the detachment was completely. However, for the day 8 barnacles (Fig. 6), the base plates were still observed on the substrates after the pulling experiments. Therefore, in this case, the forces measured could not be regarded as adhesive forces associated with attachment to the substrate [30]. No force difference was detected between control and ultrasound treated barnacles at older ages. The transition of surface failure modes may be explained that as the barnacle grows, greater calcification has occurred with more cement laid down and the properties of the adult barnacle cement may also have changed with time. The adhesion strength of the newly metamorphosed barnacles (day 0) was affected by ultrasound and it was lower (0.70 × 105 Pa) than that of untreated control barnacles (0.83 × 105 Pa). However, this difference in adhesion strength diminished after 4 days’ in culture, suggesting that the animals are able to recover from the treatment to secrete normal glues. Reduced adhesion strength may alter the settlement and recruitment of marine organisms on to surfaces, and it has been reported that marine organisms prefer to settle on sites with minimal possibility of removal [7,8]. In our study, we also observed a reduced settlement of ultrasound treated cyprids and lower adhesion strength of ultrasound treated barnacles. Also, it was found that barnacle was more susceptible to removal at their younger stages. The longer the attachment duration of barnacles, the greater the drag force required to detach them [31,32]. The possible explanations of the age-related changes are: older stages have larger and thus perhaps stronger adherent areas which may be increasingly calcified, and provide a firmer attachment. Additionally, increased adhesion strength may simply reflect an increase in the amount of adhesive as attachment duration increases; and further “curing” of the adhesive, which leads to increased strength [32].
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We also investigated how ultrasound induces biological changes during cyprid settlement. Different FP morphologies have previously been observed when FP was deposited on surfaces with different properties using AFM [19], and it is hypothesized that a highly charged bioadhesive can displace water and spread more easily on a hydrophobic surfaces. In this study, it was observed that the FP morphology differed significantly between ultrasound treated and control cyprids (Fig. 6). The FP area from ultrasound treated cyprids was larger than that of control cyprids. The thickness of FPs, however, was half of that of control cyprids, and the estimated volume of FPs was one third less than the FPs from control cyprids. It is possible that the larger area of FPs from ultrasound treated cyprids could be the result of sliding of the antennules while in contact with the substrates during ultrasound treatment. In our earlier study of ultrasound on cyprid exploration behavior [2], cyprids were observed to exhibit an unstable ‘walking’ behavior after ultrasound treatment. A change in the temporary adhesive secretion could also have resulted from ultrasonic induced impacts on the larvae’s physiology. The effect of ultrasound on the protein secretion has been reported. In a study of ultrasound on photodynamic antimicrobial therapy, it was found that ultrasound can reduce cell surface protein secretion [33]; whereas the exposure of ultrasound on the enhancement of adhesion protein secretion in mesenchymal stem was also reported [34]. Since FPs contains settlement pheromones [14,15,20], this reduction of FP density and volume may also reduce settlement as hypothesized by [15], which suggested that lower FP concentration on hydrophobic surfaces results in less settlement. Another possibility is that the exposure of ultrasound induces physical damage to the cyprid’s internal organs and subsequently affects cyprid FPs secretion and settlement. The ability of ultrasound to reduce cyprid settlement as well as to reduce the adhesion of juvenile barnacles (up to 2 days’ old) can be useful for enhancing the performance of fouling release coatings to prevent fouling. The hydrodynamic forces imposed by a vessel moving through the water is usually sufficient to remove any hard fouling adhering to fouling release coatings. However, these surfaces are less effective to fouling release when the vessels move slowly. We have demonstrated that ultrasound can reduce cyprid settlement, as well as reduce the adhesion strength of newly settled barnacles. It seems plausible that a combination of antifouling strategy consisting of ultrasound method and fouling release coatings may provide a more effective and economical method to reduce barnacle based marine biofouling. Based on our study, it is suggested that ultrasound may be a promising strategy for the barnacle induced marine fouling prevention. The ability of acoustic/vibration on the control of barnacle induced marine fouling has also been reported [35], which found that vibration at a specific frequency band significantly reduced barnacle attachment in the field tests, but did not affect other marine organisms’ attachment. Similar results were also reported by Guo et al. [36], which found low intensity ultrasound in the frequency range of 20–25 kHz significantly reduced cyprid settlement than higher frequencies, based on the laboratory tests. Ultrasonic Antifouling (UK) and Shipsonic (Netherlands) have commercialized ultrasound-based products, which are targeted for marine fouling prevention on berthed pleasure craft. Although ultrasound shows great effect on the barnacle based marine fouling prevention, a thorough assessment of the effect of ultrasound on the marine environment would be prudent. For example, the biological impact of ultrasound on other marine organisms needs to be properly addressed, and the energy consumption also needs careful consideration. Although above mentioned issues needs to be addressed, the ultrasound based strategies process several benefits over biocide strategies. Ultrasound application does not generate a cumulative effect, whereas biocide-based antifouling coatings systematically release of toxic molecules from the coating’s surface
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into the ocean which may endanger other marine organisms. In addition, ultrasound can be applied in a highly controlled manner. For example, ultrasound could be implemented in a regimented pulsed fashion, or could be turned on while in port and turned off once a ship has reached cruising speed, thus potentially saving power and other resources [36]. Ultrasound may also be applied to surfaces where there is low liquid shear forces, and fouling release coatings have limited effectiveness. Last but not least, ultrasound not only prevents cyprid settlement, but also reduces the adhesion strength of newly metamorphosed barnacles. The combination of FRCs and ultrasound may provide a more efficient antifouling strategy. Acknowledgments This work was supported by the NUS graduate research scholarship for the first author. The authors would like to thank Ms. Serina Siew Chen Lee from the Tropical Marine Science Institute (TMSI) for assistance with the culture of cyprids and Dr. In Yee Phang from the Institute of Materials Research and Engineering (IMRE) for the guidance of sample preparation. The authors also like to thank Dr. Zhengfeng Wang from the Singapore Institute of Manufacturing Research (SIMTECH) and Dr. Deny Hartono from the Department of Chemical and Biomolecular Engineering of NUS for the surface modification. References [1] D.J. Crip, G. Walker, G.A. Young, A.B. Yule, J. Colloid Interface. Sci. 104 (1985) 40–50. [2] S.F. Guo, H.P. Lee, K.C. Chaw, J. Miklas, S.L.M. Teo, G.H. Dickinson, W.R. Birch, B.C. Khoo, Biofouling 27 (2011) 185–192. [3] S. Guo, H.P. Lee, B.C. Khoo, J. Exp. Mar. Biol. Ecol. 409 (2011) 253–258. [4] N. Seth, P. Chakravarty, L. Khandeparker, A.C. Anil, A.B. Pandit, Mar. Biol. Assoc. J. U.K. 90 (2010) 1475–1482. [5] H. Kitamura, K. Takahashi, D. Kanamaru, Mar. Fouling 12 (1995) 9–13. [6] S. Guo, B.C. Khoo, S.L.M. Teo, H.P. Lee, Colloids Surf. B 109 (2013) 219–227. [7] C. Carl, A.J. Ppple, B.A. Sexton, F.L. Glenn, M.J. Vucko, M.R. Williams, S. Whalan, R.D. Nys, Biofouling 28 (2012) 175–186. [8] N. Aldred, A. Scardino, A. Cavaco, R. de Nys, A.S. Clare, Biofouling 26 (2010) 287–299. [9] N. Aldred, A.S. Clare, Biofouling 24 (2008) 351–363. [10] D. Rittschof, B. Orihuela, T. Harder, S. Stafslien, B. Chisholm, G.H. Dickinson, PLoS One 6 (2011) e16487. [11] P. Buskens, M. Wouters, C. Rentrop, Z. Veroon, J. Coat. Technol. Res. 1 (2012) 1–8. [12] A.I. Larsson, L.M. Thorngren, L.M. Granhag, M. Berglin, J. Exp. Mar. Biol. Ecol. 392 (2010) 107–114. [13] J.F. Schumacher, N. Aldred, M.E. Callow, J.A. Finlay, J.A. Callow, A.S. Clare, A.B. Brennan, Biofouling 23 (2007) 307–317. [14] C. Dreanno, R.R. Kirby, A.S. Clare, Biol. Lett. 2 (2006) 423–425. [15] K. Matsumura, M. Nagano, Y. Kato-Yoshinaga, M. Yamazaki, A.S. Clare, N. Fusetani, Proc. R. Soc. Lond. Ser. B 265 (1998) 1825–1830. [16] I.Y. Phang, K.C. Chaw, S.S. Choo, R.K. Kang, S.S. Lee, W.R. Birch, S.L.M. Teo, G.J. Vancso, Biofouling 25 (2009) 139–147. [17] D. Rittschof, C.H. Lai, L.M. Kok, S.L.M. Teo, Biofouling 19 (2003) 207–212. [18] M. Berglin, A. Larsson, P.R. Jonsson, P. Gatenholm, J. Adhes. Sci. Technol. 15 (2001) 1485–1502. [19] I.Y. Phang, N. Aldred, A.S. Clare, G.J. Vancso, J. R. Soc. Interface 5 (2008) 397–401. [20] I.Y. Phang, N. Aldred, X.Y. Ling, J. Huskens, G.J. Vancso, J. R. Soc. Interface 43 (2009) 285–296. [21] X.Y. Bi, D. Hartono, K.L/ Yang, Sens. Actuator B. Chem. 127 (2007) 406–413. [22] D. Hartono, X.Y. Bi, K.L. Yang, L.Y.L. Yung, Adv. Funct. Mater. 18 (2008) 2938–2945. [23] R.E. Pérez-Roa, M.A. Anderson, D. Rittschof, B. Orihuela, D. Wendt, G.L. Kowalke, D.R. Noguera, Biofouling 24 (2008) 177–184. [24] V. Thiyagarajan, T. Harder, P.Y. Qian, J. Exp. Mar. Biol. Ecol. 274 (2002) 65–74. [25] D. Rittschof, B. Orihuela, S. Stafslien, J. Daniels, D. Christianson, B. Chisholm, E. Holm, Biofouling 24 (2008) 1–9. [26] C.J. Kavanagh, M.P. Schultz, G.W. Swain, J. Stein, K. Truby, C.D. Wood, Biofouling 17 (2001) 155–167. [27] I.Y. Phang, N. Aldred, A.S. Clare, G.J. Vancso, NanoS 1 (2007) 36–41. [28] G.H. Dickinson, I.E. Vega, K.J. Wahl, B. Orihuela, V. Beyley, E.N.K. Rodriguez, K. Richard, K.J. Bonaventura, D. Rittschof, J. Exp. Biol. 21 (2009) 3499–3510. [29] K. Kamino, K. Inoue, T. Maruyama, N. Takamatsu, S. Harayama, Y. Shizuri, J. Biol. Chem. 35 (2010) 27360–27365. [30] D.E. Wendt, G.L. Kowalke, J. Kim, I.L. Singer, Biofouling 22 (2006) 1–9.
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[31] J.D. Zardus, B.T. Nedved, Y. Huang, C. Tran, M.G. Hadfield, Biol. Bull. 214 (2008) 91–98. [32] J.E. Eckman, W.B. Savidge, T.F. Gross, J. Mar. Biol. 107 (1990) 111–118. [33] K. Ishibashi, K. Shimada, T. Kawato, S. Kaji, M. Maeno, Appl. Environ. Microbiol. 3 (2010) 751–756.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effect of ultrasound on cyprid footprint and juvenile barnacle adhesion on a fouling release material Shifeng Guo a,b,∗ , Boo Cheong Khoo a,d , Serena Lay Ming Teo c , Shaoping Zhong e , Chwee Teck Lim a,e,f , Heow Pueh Lee a,d a
Department of Mechanical Engineering, National University of Singapore, Singapore 117576, Singapore Institute of Materials Research and Engineering (IMRE), A*STAR, 3 Research Link, Singapore 117602, Singapore c Tropical Marine Science Institute, National University of Singapore, Singapore 119223, Singapore d National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiang Su 215123, People’s Republic of China e Department of Bioengineering, National University of Singapore, Singapore 117576, Singapore f Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore b
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 4 November 2013 Accepted 10 November 2013 Available online 23 November 2013 Keywords: Ultrasound AFM Barnacle cyprid footprint Settlement Adhesion strength Silicone substrates
a b s t r a c t In our earlier studies, we have demonstrated that low and high intensity ultrasound can prevent barnacle cyprid settlement. In this study, we found that ultrasound treatment reduced the adhesion of newly metamorphosed barnacles up to 2 days’ old. This was observed in the reduction of adhesion strength of the newly settled barnacles from ultrasound treated cyprids on silicone substrate compared to the adhesion strength of barnacles metamorphosed from cyprids not exposed to ultrasound. Atomic force microscopy (AFM) was used to analyze the effect of ultrasound on barnacle cyprid footprints (FPs), which are protein adhesives secreted when the larvae explore surfaces. The ultrasound treated cyprids were found to secrete less FPs, which appeared to spread a larger area than those generated by untreated cyprids. The evidence from this study suggests that ultrasound treatment results in a reduced cyprid settlement and footprint secretion, and may affect the subsequent recruitment of barnacles onto fouling release surfaces by reducing the ability of early settlement stage of barnacles (up to 2 days’ old) from firmly adhering to the substrates. Ultrasound therefore can be used in combination with fouling release coatings to offer a more efficient antifouling strategy. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Barnacles are a major problem in marine fouling due to their size, hard shell, and gregarious nature [1]. In earlier studies, it has been demonstrated that both low and high intensity ultrasound can prevent barnacle cyprid settlement [2–5]. The probable mechanism for the prevention of cyprid settlement using high intensity ultrasound was attributed to ultrasonic cavitation [3], and it was also shown that cavitation bubbles can be used to remove early stages of barnacle settlement [6]. In this paper, we further examine the feasibility of using ultrasound to control the barnacle fouling on fouling release surfaces. It has been reported that attachment strength plays a significant role in the recruitment of marine organisms onto a surfaces, and stronger forces are required to remove organisms on surfaces with higher settlement [7,8]. For example, the settlement of Mytilus
∗ Corresponding author at: Institute of Materials Research and Engineering (IMRE), A*STAR, 3 Research Link, Singapore 117602, Singapore. Tel.: +65 65148745. E-mail address: [email protected] (S. Guo). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.020
galloprovincialis was found to be positively correlated to adhesion strength [7]. Also, barnacle cyprids preferred to settle on the substrates where the possibility of subsequent removal is least likely to occur [8]. Fouling release coatings (FRCs) are now widely used as antifouling for ship hulls [9,10]. Hydrophobic FRCs was reported to be beneficial to vessels operating above 10 knots and resulted in less fuel consumption in comparison to biocide-containing selfpolishing coatings [11]. One of the main features of the FRCs is that the interfacial bond between the organism and coating is weak. As a result, the attached organisms are more easily removed by the hydrodynamic forces created from a vessel’s movement through the water, or by simple mechanical cleaning [9,12]. The cyprids and juvenile barnacles have been previously proposed as appropriate experimental models for the evaluation of antifouling performance [12]. The lifecycle of the barnacle, Amphibalanus amphitrite (=Balanus amphitrite), includes planktotrophic nauplius stages, a non-feeding cypris larval stage, and a sessile adult stage. The pre-settlement cypris stage actively explores surface and metamorphoses into the juvenile barnacle once a suitable site is found. Surface exploration is conducted using the antennules, in a form of bi-pedal ‘walking’, which appears to be
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affected by the surface texture, material properties, chemical clues, and presence of adult or cyprid nonspecific proteins [9,13]. During exploration, temporary proteins secretion is deposited as footprint (FP) through the antennular attachment discs and it has been implicated to act as settlement cues for other exploring cyprids [1,9]. The FP has been described as a temporary adhesive, as it enables reversible attachment to surfaces [14,15]. A settlement-inducing protein complex (SIPC), which also functions as a settlement cue, has been found in FPs [14,15]. It has also been reported that surfaces which are apt to the adsorption of FPs would lead to higher settlement [16]. Ultrasound treated cyprids have been shown to have an altered exploration behavior [2]. Since cyprids secrete temporary adhesive proteins during surface exploration, it is conceivable that the altered walking behavior may also be accompanied with change in the secreted FPs. Based on the preliminary experiments, juvenile barnacles metamorphosed from ultrasound treated cyprids were more easily detached. It is surmised that the adhesion strength may be reduced. Therefore, the effect of ultrasound on juvenile barnacle adhesion strength was evaluated using a nano-tensile tester. Also, since FPs play a significant role on cyprid settlement, the effect of ultrasound on the FPs was explored using AFM, with the objective of gaining a better understanding of the mechanism behind the ultrasound induced inhibitory effects. 2. Materials and methods 2.1. Barnacle culture Larvae of the barnacle Amphibalanus amphitrite were reared at 26 ◦ C, fed with an algal mixture of 1:1 (v/v) of Tetraselmis suecica and Chaetoceros muelleri at a density of approximately 5 × 105 cells/ml [17]. Barnacle larvae metamorphosed into cyprids within 5–7 days. The cyprids were stored at 4 ◦ C and used for experiments after 3 days. Juvenile barnacles metamorphosed from ultrasound treated and untreated cyprids were reared and fed daily with algal mixture of 1:1 (v/v) of Tetraselmis suecica and Chaetoceros muelleri at a density of approximately 5 × 105 cells/ml. 2.2. Ultrasonic experimental setup The ultrasonic experimental setup is same as that described in Guo et al. [3]. As low frequency of 23 kHz generated most significant effect on barnacle cyprid settlement and exploration behavior [2], in the present study, only this frequency was chosen, with the pressure set at 20 kPa. The cyprids were then subjected to ultrasound exposure for 5 min. 2.3. Surfaces choice for the barnacle adhesion and cyprid FPs study There are two surfaces used to study the effect of ultrasound on juvenile barnacle adhesion strength and cyprid footprints, respectively. The fouling release surfaces (silicone substrates) were used to evaluate the effect of ultrasound on barnacle adhesion strength. The surfaces were chosen because the interaction between juvenile barnacle cement and the surface is low. Therefore, the barnacles were easier to be fully detached, and the forces measured can be regarded as the adhesive forces. The fouling release surfaces used to evaluate barnacle adhesion strength were reported widely [9,12,18]. To study the effect of ultrasound on FPs, the NH2 terminated glass cover slips were used as the surfaces were easier for the FPs detection using AFM and shown more concentrated and distinct FP patterns [19,20].
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Fig. 1. The schematic of nano-tensile tester on juvenile barnacle detachment forces measurement.
To prepare the NH2 terminated surfaces, the glass microscopy cover slips were firstly immersed in 5% Decon 90 solution and cleaned using ultrasonic clean-tank for 20 min. The use of Decon 90 solution to effectively clean surfaces for increasing surface density of silanol groups and/or prior to silanization has been well documented in the previous literature [21,22]. The slips were then rinsed thoroughly with ultrapure water and dried with nitrogen gas. The amino (NH2 ) terminated surfaces were obtained by immersing the cleaned slips in 5% 3-aminopropyl triethoxysilane (APTES) solution and were put in the shaker (GFP MBH, Germany) for 30 min. After that the surfaces were rinsed with ultrapure water thoroughly and dried using nitrogen gas. Static water contact angles (CA) of NH2 terminated surfaces and silicone substrates were measured at 25 ◦ C, using the sessile drop method with a 2 l water droplet, in a telescopic goniometer (model 100-00-(230), Rame-Hart, Inc., Mountain Lake, NJ, USA). To measure the contact angle of coated cover slips, eight samples were replicated and the averaged contact angle was 55◦ ± 2.4◦ and the value was approximately similar with reported results [19,20]. The contact angle of silicone substrates was 94◦ . 2.4. Cyprid settlement on silicone substrate Cyprid settlement was performed with a ‘no choice’ droplet assay, as described in Refs. [8,23]. The experiments were conducted on the medical grade silicone substrates (Bioplexus, USA). The material was cleaned following the provided instructions before usage. It was reported that the gregarious behavior of cyprids did not interfere with settlement rate at cyprid densities of 5–200 per 5 ml [24]. In our experiments, after ultrasound exposure, 500 l volume of filtered seawater (FSW), containing approximately 15–20 cyprids, was deposited on each silicone substrate (2 cm × 2 cm), forming a droplet on the substrate. The substrates were placed in Petri dishes and sealed with parafilm to prevent water evaporation. The assay was incubated at 26 ◦ C for 48 h, on a 15 h light and 9 h dark cycle. The cyprids were then examined under a stereo microscope (Nikon SMZ 1500, Japan), and the number of cyprids which had settled and metamorphosed into barnacles was enumerated. To evaluate the ultrasonic effect, eight replicate silicone substrates were prepared for both ultrasound treated and control cyprids. 2.5. Juvenile barnacle adhesion strength measurement Measurement of barnacle adhesive force was conducted using a calibrated Nano-tensile tester (Nano Bionix System, MTS, USA), and the schematic of the experimental setup is shown in Fig. 1. The silicone substrate (2 cm × 2 cm) with barnacles settled was clamped in the lower grip. A micro steel fiber with diameter of 80 m and length of 10 mm was clamped in the upper grip. The fiber was controlled and lowered until it was slightly above the top of barnacle. Then the tip of the fiber was wetted with a small drop of superglue (Selleys Pty Ltd, Australia) and lowered to touch the top of
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Fig. 2. The schematic of AFM on barnacle cyprid footprint scanning. (A) Cyprid explored the NH2 terminated cover slip and left footprint on it; (B) the morphology of footprint was scanned by AFM D3100.
barnacle shell [18]. The glue was allowed to dry for about 30 s and the force required to dislodge the barnacle was measured. To study the effect of pulling velocity on barnacle detachment force, two strain rates were chosen: one was 1.0 × 10−4 s−1 , representing low pulling velocity and the other was 1.0 × 10−2 s−1 , representing fast pulling velocity. The preliminary results revealed that no significant detachment forces were detected between the two pulling velocities, therefore, in our study, only strain rate of 1.0 × 10−2 s−1 was chosen. Similar results were found by Berglin et al. [18], who found that pulling velocities did not affect barnacle attachment force measurement. After detachment, the substrates were gently rinsed using ultra-pure distilled water and observed under a stereo microscope with image capture software (Nikon Instech Co., Ltd., Japan). Only the forces that completely detached the barnacles were recorded. If any visible amount of base plate of barnacle was still remained on the surface after pulling experiment, which indicates incomplete removal, the associate force was considered invalid and therefore discarded [25]. For each group barnacles, ten replicates were measured. Adhesion strength was calculated by dividing the measured force required to detach the barnacle by the basal area [26]. As such, the basal area of barnacle was estimated. The image of barnacle before experiment was photographed using a stereo microscope with an image capture software. The image was processed with ImageJ software (Version 1.43), which uses edge contrast to define the perimeter and thereby calculates the total basal area. Adhesion strength measurements were conducted on the newly metamorphosed barnacles (day 0) to day 8 barnacles. For the day 8 barnacles, partially barnacle base plates remained on the substrates after the experiments. Therefore, the measured values do not reflect the actual removal forces, but the data have been included for the discussion purposes.
as described in Ref. [27]. In this study, the AFM scanning was conducted using a Dimension D3100 atomic force microscope (Veeco/Digital Instruments (DI), Santa Barbara, CA). Since cyprid FP morphology showed no significant difference in air and in filtered sea water (FSW), but imaging in air provided higher resolution [19,20]. In the present study, the AFM scanning was conducted in air with tapping mode. The NH2 terminated cover slips were mounted on the glass sides prior to experiments. Cyprids were transferred to the modified cover slips using a micropipette. The walking cyprid was monitored by a stereo microscope equipped with video capture software NIS-Elements BR 3.2 (Nikon Instech Co., Ltd., Japan). The explored areas were marked on the back of glass slides and the videos of the ‘walking’ were captured for ease of FPs searching during AFM scanning. The cyprid was subsequently removed and the surface was rinsed with ultrapure distilled water and dried with nitrogen gas to minimize the contamination. Before exploration, cyprids were allowed 5 min’ acclimatization after ultrasound exposure and the observation time for both control and ultrasound treated cyprids were within 40 min. The FP images were taken in air using the intermittent contact mode with silicon cantilevers having a spring constant of 30 N/m approximately (Nanosensors, Germany). To evaluate the effect of ultrasound on cyprid FPs, six FPs of both control and ultrasound treated cyprids were analyzed and all the FPs were obtained from different individual cyprid. 2.7. Data analysis The statistical comparisons were performed using GraphPad Prism 5 (GraphPad Software Inc.). Barnacle adhesive forces/strength data were analyzed using a two-way analysis of variance (ANOVA) and the Bonferroni post tests were conducted to evaluate the influence of ultrasound and age effect. Data of settlement and cyprid FPs was analyzed using t-test. All data are reported as mean ± standard error (SE). For all comparisons, p-values ≤0.05 were considered as statistically significant. 3. Results 3.1. Cyprid settlement on silicone substrates The settlement of control and ultrasound treated cyprids is shown in Fig. 3. Significant differences were observed between ultrasound treated and control cyprids (t-test, p < 0.001). After ultrasound treatment with 23 kHz for 5 min at 20 kPa, the settlement was reduced significantly from 44% to 16% (Fig. 3).
2.6. Effect of ultrasound on cyprid footprints Enhanced FP absorption has been reported on the NH2 terminated glass microscopy cover slip [19,20]. Therefore, in this study, this particular surface was chosen to evaluate the effect of ultrasound on cyprid FPs. The schematic of AFM setup for cyprid FPs measurement is shown in Fig. 2 and the working principle and protocol are
Fig. 3. Cyprid settlement comparison. Error bars are standard errors. The asterisk represents statistically significant difference.
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3.3. Observation of barnacle images
Fig. 4. The adhesion strength comparison of juvenile barnacles metamorphosed from control and ultrasound treated cyprids. The asterisks represent statistically significant difference. The error bars are standard errors.
3.2. Effects of ultrasound on juvenile barnacle adhesion strength The detachment force of the settled juvenile barnacle was measured using a nano-tensile tester. The adhesion strength was calculated by dividing the detachment force over the basal area of juvenile barnacle. Both ultrasound treatment and barnacle age significantly affected the adhesion strength (two-way ANOVA, p < 0.05; Fig. 4). The results revealed that adhesion strength was significantly reduced after ultrasound exposure for the day 0 and day 2 barnacles (Fig. 4; Bonferroni post test, p < 0.01). For day 0 barnacles, the adhesion strength of the juvenile barnacles metamorphosed from control cyprids was 0.83 ± 0.05 × 105 Pa, while the adhesion strength of barnacles from ultrasound treated cyprids was 0.70 ± 0.03 × 105 Pa. The adhesion strength increased to 1.33 ± 0.11 × 105 Pa and 1.13 ± 0.06 × 105 Pa, respectively, for the day 2 barnacles. There was no difference detected after 4 days’ in culture (Bonferroni post test, p > 0.05; Fig. 4). The adhesion strength of barnacles was also found to increase significantly with age (Bonferroni post test, p < 0.01). The adhesion strength of day 6 barnacles was more than twice the value of day 2 barnacles (Fig. 4).
The images of barnacles before and after pulling experiments were captured using microscope equipped with image capturing software. The dislodgement areas were gently rinsed using deionised water (DI) water and imaged after drying. Since no difference in surface failure modes was detected between ultrasound treated and control barnacles, only the images of control barnacles are shown in Fig. 5. The newly metamorphosed barnacles to the day 6 barnacles can be completely removed, suggesting that the detachment occurred between barnacle adhesive cement and the substrates. Therefore, the values measured could be regarded as the adhesion strength. However, for the day 8 barnacles, partial barnacle base plates were still remained after detachment. This may suggest that partial cohesive failure occurred within barnacles (leaving the partial barnacle base plates on the substrate), which means that the adhesion strength between barnacle base plates and substrates exceeds the cohesive strength within the barnacles. Therefore, the strength measured for the day 8 barnacles could not be treated as the adhesion strength but lower than that value.
3.4. Effect of ultrasound on cyprid footprints The morphology of cyprid FPs was studied using AFM. The representative images of FPs obtained from control and ultrasound treated cyprids are shown in Fig. 6. Entire footprints and sections of footprints with higher magnification were imaged. For cyprids exposed to ultrasound, the FPs exhibited difference in morphology compared with that of control cyprids. The FPs from ultrasound treated cyprids had a larger spreading area than that secreted by control cyprids, however, FPs from control cyprids was found to be much thicker (t-test, p < 0.05; Fig. 6 and Table 1). The fibrillar and porous structure of the FP was observed for both ultrasound treated and control cyprids, however, less porous structures was found on control cyprids. The geometrical data extracted from AFM scanned FPs are presented in Table 1. The mean FP spreading area from ultrasound treated cyprids was 1142 ± 92 m2 , which was significantly larger
Fig. 5. Microscopy images of surfaces after removal of different age barnacles.
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Fig. 6. The morphological comparison of FP from control and ultrasound treated cyprids on NH2 terminated surfaces. (A) The FP of control cyprid; (B) the magnified FP of control cyprid; (C) the 3D structure of FP of control cyprid; (D) the FP of ultrasound treated cyprid; (E and F) the magnified and 3D structure of FP from ultrasound treated cyprids, respectively.
than the FP area of control cyprids of 778 ± 54 m2 (t-test, p < 0.05; Table 1). The root-mean-square (RMS) thickness of the FPs, calculated from AFM results, was found to be significant lower than that measured from the control cyprids (t-test, p < 0.05). The FP volume, which was used to quantify the amount of the temporary adhesive proteins secreted when cyprids explored the surfaces, was found to be significantly different between the ultrasound treated and control cyprids (t-test, p < 0.05; Table 1). The volume of FPs secreted by control cyprids was 12.6 ± 1.2 m3 , which was approximately one third more compared to the volume secreted by ultrasound treated cyprids (Table 1). 4. Discussion and conclusion Cyprid settlement on the silicone substrate was significantly inhibited with ultrasound treatment. The ability of ultrasound to prevent cyprid settlement has been previously reported [2,5], and it has been shown that ultrasound intensity exceeding the cavitation threshold could reduce cyprid settlement by cavitation generated forces [3,6]. One of the main features of fouling release coatings is to minimize the adhesion between the organism and the surface so that the hydrodynamic forces imposed by the ship moving through the water or a simple mechanical cleaning method will be able to remove the fouling [12]. However, these surfaces are highly susceptible to fouling when the vessels move slowly. We have previously reported that ultrasound can reduce cyprid settlement, and in this paper we demonstrate that ultrasound exposure also reduces the adhesion strength of the newly settled barnacles to fouling release silicone substrates up to 2 days’ old. Barnacle adhesion strength has attracted considerable scientific attention because of the strength and durability of the adhesive, which are practical concerns related to marine fouling [28]. The Table 1 The morphological information of FPs obtained by AFM. Six FPs of control and ultrasound treated cyprids were analyzed and the errors here are standard errors. Footprint type
Mean footprint area (m2 )
Mean footprint thickness (nm)
Mean footprint volume (m3 )
Ultrasound treated Control
1142 ± 92 778 ± 54
8.7 ± 0.6 16.3 ± 1.1
9.4 ± 0.62 12.6 ± 1.2
proteinaceous adhesive secreted by barnacles are able to cure underwater, providing a strong adhesion to the substrate [29]. The barnacle adhesion strength measurement in this study was conducted on the fouling release coatings using a nano-tensile tester on different age barnacles and the surface failure modes were observed using microscope. The results showed that the newly metamorphosed juvenile barnacles (up to 6 days’ old) from control and ultrasound treated cyprids can be completely detached. The measured forces can be regarded as the adhesive forces as the detachment was completely. However, for the day 8 barnacles (Fig. 6), the base plates were still observed on the substrates after the pulling experiments. Therefore, in this case, the forces measured could not be regarded as adhesive forces associated with attachment to the substrate [30]. No force difference was detected between control and ultrasound treated barnacles at older ages. The transition of surface failure modes may be explained that as the barnacle grows, greater calcification has occurred with more cement laid down and the properties of the adult barnacle cement may also have changed with time. The adhesion strength of the newly metamorphosed barnacles (day 0) was affected by ultrasound and it was lower (0.70 × 105 Pa) than that of untreated control barnacles (0.83 × 105 Pa). However, this difference in adhesion strength diminished after 4 days’ in culture, suggesting that the animals are able to recover from the treatment to secrete normal glues. Reduced adhesion strength may alter the settlement and recruitment of marine organisms on to surfaces, and it has been reported that marine organisms prefer to settle on sites with minimal possibility of removal [7,8]. In our study, we also observed a reduced settlement of ultrasound treated cyprids and lower adhesion strength of ultrasound treated barnacles. Also, it was found that barnacle was more susceptible to removal at their younger stages. The longer the attachment duration of barnacles, the greater the drag force required to detach them [31,32]. The possible explanations of the age-related changes are: older stages have larger and thus perhaps stronger adherent areas which may be increasingly calcified, and provide a firmer attachment. Additionally, increased adhesion strength may simply reflect an increase in the amount of adhesive as attachment duration increases; and further “curing” of the adhesive, which leads to increased strength [32].
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We also investigated how ultrasound induces biological changes during cyprid settlement. Different FP morphologies have previously been observed when FP was deposited on surfaces with different properties using AFM [19], and it is hypothesized that a highly charged bioadhesive can displace water and spread more easily on a hydrophobic surfaces. In this study, it was observed that the FP morphology differed significantly between ultrasound treated and control cyprids (Fig. 6). The FP area from ultrasound treated cyprids was larger than that of control cyprids. The thickness of FPs, however, was half of that of control cyprids, and the estimated volume of FPs was one third less than the FPs from control cyprids. It is possible that the larger area of FPs from ultrasound treated cyprids could be the result of sliding of the antennules while in contact with the substrates during ultrasound treatment. In our earlier study of ultrasound on cyprid exploration behavior [2], cyprids were observed to exhibit an unstable ‘walking’ behavior after ultrasound treatment. A change in the temporary adhesive secretion could also have resulted from ultrasonic induced impacts on the larvae’s physiology. The effect of ultrasound on the protein secretion has been reported. In a study of ultrasound on photodynamic antimicrobial therapy, it was found that ultrasound can reduce cell surface protein secretion [33]; whereas the exposure of ultrasound on the enhancement of adhesion protein secretion in mesenchymal stem was also reported [34]. Since FPs contains settlement pheromones [14,15,20], this reduction of FP density and volume may also reduce settlement as hypothesized by [15], which suggested that lower FP concentration on hydrophobic surfaces results in less settlement. Another possibility is that the exposure of ultrasound induces physical damage to the cyprid’s internal organs and subsequently affects cyprid FPs secretion and settlement. The ability of ultrasound to reduce cyprid settlement as well as to reduce the adhesion of juvenile barnacles (up to 2 days’ old) can be useful for enhancing the performance of fouling release coatings to prevent fouling. The hydrodynamic forces imposed by a vessel moving through the water is usually sufficient to remove any hard fouling adhering to fouling release coatings. However, these surfaces are less effective to fouling release when the vessels move slowly. We have demonstrated that ultrasound can reduce cyprid settlement, as well as reduce the adhesion strength of newly settled barnacles. It seems plausible that a combination of antifouling strategy consisting of ultrasound method and fouling release coatings may provide a more effective and economical method to reduce barnacle based marine biofouling. Based on our study, it is suggested that ultrasound may be a promising strategy for the barnacle induced marine fouling prevention. The ability of acoustic/vibration on the control of barnacle induced marine fouling has also been reported [35], which found that vibration at a specific frequency band significantly reduced barnacle attachment in the field tests, but did not affect other marine organisms’ attachment. Similar results were also reported by Guo et al. [36], which found low intensity ultrasound in the frequency range of 20–25 kHz significantly reduced cyprid settlement than higher frequencies, based on the laboratory tests. Ultrasonic Antifouling (UK) and Shipsonic (Netherlands) have commercialized ultrasound-based products, which are targeted for marine fouling prevention on berthed pleasure craft. Although ultrasound shows great effect on the barnacle based marine fouling prevention, a thorough assessment of the effect of ultrasound on the marine environment would be prudent. For example, the biological impact of ultrasound on other marine organisms needs to be properly addressed, and the energy consumption also needs careful consideration. Although above mentioned issues needs to be addressed, the ultrasound based strategies process several benefits over biocide strategies. Ultrasound application does not generate a cumulative effect, whereas biocide-based antifouling coatings systematically release of toxic molecules from the coating’s surface
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into the ocean which may endanger other marine organisms. In addition, ultrasound can be applied in a highly controlled manner. For example, ultrasound could be implemented in a regimented pulsed fashion, or could be turned on while in port and turned off once a ship has reached cruising speed, thus potentially saving power and other resources [36]. Ultrasound may also be applied to surfaces where there is low liquid shear forces, and fouling release coatings have limited effectiveness. Last but not least, ultrasound not only prevents cyprid settlement, but also reduces the adhesion strength of newly metamorphosed barnacles. The combination of FRCs and ultrasound may provide a more efficient antifouling strategy. Acknowledgments This work was supported by the NUS graduate research scholarship for the first author. The authors would like to thank Ms. Serina Siew Chen Lee from the Tropical Marine Science Institute (TMSI) for assistance with the culture of cyprids and Dr. In Yee Phang from the Institute of Materials Research and Engineering (IMRE) for the guidance of sample preparation. The authors also like to thank Dr. Zhengfeng Wang from the Singapore Institute of Manufacturing Research (SIMTECH) and Dr. Deny Hartono from the Department of Chemical and Biomolecular Engineering of NUS for the surface modification. References [1] D.J. Crip, G. Walker, G.A. Young, A.B. Yule, J. Colloid Interface. Sci. 104 (1985) 40–50. [2] S.F. Guo, H.P. Lee, K.C. Chaw, J. Miklas, S.L.M. Teo, G.H. Dickinson, W.R. Birch, B.C. Khoo, Biofouling 27 (2011) 185–192. [3] S. Guo, H.P. Lee, B.C. Khoo, J. Exp. Mar. Biol. Ecol. 409 (2011) 253–258. [4] N. Seth, P. Chakravarty, L. Khandeparker, A.C. Anil, A.B. Pandit, Mar. Biol. Assoc. J. U.K. 90 (2010) 1475–1482. [5] H. Kitamura, K. Takahashi, D. Kanamaru, Mar. Fouling 12 (1995) 9–13. [6] S. Guo, B.C. Khoo, S.L.M. Teo, H.P. Lee, Colloids Surf. B 109 (2013) 219–227. [7] C. Carl, A.J. Ppple, B.A. Sexton, F.L. Glenn, M.J. Vucko, M.R. Williams, S. Whalan, R.D. Nys, Biofouling 28 (2012) 175–186. [8] N. Aldred, A. Scardino, A. Cavaco, R. de Nys, A.S. Clare, Biofouling 26 (2010) 287–299. [9] N. Aldred, A.S. Clare, Biofouling 24 (2008) 351–363. [10] D. Rittschof, B. Orihuela, T. Harder, S. Stafslien, B. Chisholm, G.H. Dickinson, PLoS One 6 (2011) e16487. [11] P. Buskens, M. Wouters, C. Rentrop, Z. Veroon, J. Coat. Technol. Res. 1 (2012) 1–8. [12] A.I. Larsson, L.M. Thorngren, L.M. Granhag, M. Berglin, J. Exp. Mar. Biol. Ecol. 392 (2010) 107–114. [13] J.F. Schumacher, N. Aldred, M.E. Callow, J.A. Finlay, J.A. Callow, A.S. Clare, A.B. Brennan, Biofouling 23 (2007) 307–317. [14] C. Dreanno, R.R. Kirby, A.S. Clare, Biol. Lett. 2 (2006) 423–425. [15] K. Matsumura, M. Nagano, Y. Kato-Yoshinaga, M. Yamazaki, A.S. Clare, N. Fusetani, Proc. R. Soc. Lond. Ser. B 265 (1998) 1825–1830. [16] I.Y. Phang, K.C. Chaw, S.S. Choo, R.K. Kang, S.S. Lee, W.R. Birch, S.L.M. Teo, G.J. Vancso, Biofouling 25 (2009) 139–147. [17] D. Rittschof, C.H. Lai, L.M. Kok, S.L.M. Teo, Biofouling 19 (2003) 207–212. [18] M. Berglin, A. Larsson, P.R. Jonsson, P. Gatenholm, J. Adhes. Sci. Technol. 15 (2001) 1485–1502. [19] I.Y. Phang, N. Aldred, A.S. Clare, G.J. Vancso, J. R. Soc. Interface 5 (2008) 397–401. [20] I.Y. Phang, N. Aldred, X.Y. Ling, J. Huskens, G.J. Vancso, J. R. Soc. Interface 43 (2009) 285–296. [21] X.Y. Bi, D. Hartono, K.L/ Yang, Sens. Actuator B. Chem. 127 (2007) 406–413. [22] D. Hartono, X.Y. Bi, K.L. Yang, L.Y.L. Yung, Adv. Funct. Mater. 18 (2008) 2938–2945. [23] R.E. Pérez-Roa, M.A. Anderson, D. Rittschof, B. Orihuela, D. Wendt, G.L. Kowalke, D.R. Noguera, Biofouling 24 (2008) 177–184. [24] V. Thiyagarajan, T. Harder, P.Y. Qian, J. Exp. Mar. Biol. Ecol. 274 (2002) 65–74. [25] D. Rittschof, B. Orihuela, S. Stafslien, J. Daniels, D. Christianson, B. Chisholm, E. Holm, Biofouling 24 (2008) 1–9. [26] C.J. Kavanagh, M.P. Schultz, G.W. Swain, J. Stein, K. Truby, C.D. Wood, Biofouling 17 (2001) 155–167. [27] I.Y. Phang, N. Aldred, A.S. Clare, G.J. Vancso, NanoS 1 (2007) 36–41. [28] G.H. Dickinson, I.E. Vega, K.J. Wahl, B. Orihuela, V. Beyley, E.N.K. Rodriguez, K. Richard, K.J. Bonaventura, D. Rittschof, J. Exp. Biol. 21 (2009) 3499–3510. [29] K. Kamino, K. Inoue, T. Maruyama, N. Takamatsu, S. Harayama, Y. Shizuri, J. Biol. Chem. 35 (2010) 27360–27365. [30] D.E. Wendt, G.L. Kowalke, J. Kim, I.L. Singer, Biofouling 22 (2006) 1–9.
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