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Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN)
Journal of Experimental Marine Biology and Ecology 305 (2004) 223 – 232 www.elsevier.com/locate/jembe
Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN) Mia Dahlstro¨m a,b,*, Henrik Jonsson b,1, Per R. Jonsson b, Hans Elwing a a
Department of Cell and Molecular Biology, Division of Interface Biophysics, Go¨teborg University, Box 462, SE-405 30 Go¨teborg, Sweden b Department of Marine Ecology, Tja¨rno¨ Marine Biological Laboratory, Go¨teborg University, SE-452 96 Stro¨mstad, Sweden Received 18 February 2003; received in revised form 24 October 2003; accepted 22 December 2003
Abstract Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase,
* Corresponding author. Department of Cell and Molecular Biology, Division of Interface Biophysics, Go¨teborg University, Box 462, SE-405 30 Go¨teborg, Sweden. Tel.: +46-526-68616; fax: +46-526-68607. E-mail address: [email protected] (M. Dahlstro¨m). 1 Present address: Department of Biological Oceanography, Baltic Sea Research Institute, Seestrasse 15, D18119 Warnemu¨nde, Germany. 0022-0981/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.jembe.2003.12.013
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i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules. D 2004 Published by Elsevier B.V. Keywords: Balanus improvisus; Settlement; Surface chemistry; Wettability
1. Introduction It has been demonstrated that the settlement in the field of the barnacle Balanus improvisus (Darwin) varies greatly both spatially and temporally and also, that settlement varies with respect to the physical properties of the surface, e.g. the surface topography (Berntsson, 2001; Berntsson and Jonsson, 2003; Berntsson et al., 2000). Thus, it has been established that B. improvisus has a clear preference for smooth substrata and also, that underlying behavioural responses cause the rejection of surfaces bearing a certain micro-geometry (Berntsson et al., 2000). However, the importance of inherent chemical properties of a potential settling site for B. improvisus has not been examined to the same extent. O’Connor and Richardson (1994) reported that cyprids of B. improvisus attach to a lesser extent to glass vials than to polystyrene dishes, however, no attempts of further characterising these surfaces were made. Previous reports on the settlement response in the field of marine invertebrate larvae to surfaces of varying wettability, show that the initial surface chemistry of a substratum for settlement is important for initial colonization but not in determining the overall structure of the macrofouling community (Roberts et al., 1991; Holm et al., 1997). Furthermore, laboratory studies of ascidians, bryozoans and the barnacle B. amphitrite have shown that the preferred wettability of a substrate for settlement displays some variation, with the barnacle B. amphitrite being the sole of these to have a clear preference for high wettability substrates (Mihm et al., 1981; Rittschof and Costlow, 1989; Gerhart et al., 1992; O’Connor and Richardson, 1994). Also, the tenacity of cyprid temporary adhesion of the barnacles B. amphitrite and Elminus modestus (Darwin) has been measured in respect to different chemical properties of the surface (Maki et al., 1994; Neal and Yule, 1994). Similar measurements have been conducted on cyprid larvae and juvenile barnacles of B. improvisus using low energy surfaces such as fluorinated silicone elastomeres and the more high energy surface poly (methyl methacrylate) (PMMA) where the tenacity of the temporary adhesion proved to be similar on both of these surfaces (Berglin et al., 2001). However, there are no reports on the overall settlement success of barnacle larvae of B. improvisus in response to surface chemistry, i.e. surfaces with high vs. low wettability. In previous studies we have used hydrophilic and hydrophobic dishes of polystyrene to monitor the accumulation of various settlement inhibiting compounds at the solid surface (Dahlstro¨m et al., 2000). However, during these assays we have observed that settlement of B. improvisus vary greatly in the control dishes between the two surfaces. Due to an extensive amount of data collected from settlement assays from late 1998 to early 2001 and also, due to the absence in the literature regarding reports of how the barnacle B. improvisus responds to surfaces of different inherent chemistry, we here present a brief report of the effect of initial surface chemistry on the settlement and
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metamorphosis of cyprids of B. improvisus, using highly polar to non-polar surfaces of both polystyrene and glass. These results are then combined with previous analyses of the chemical composition of these surfaces using X-ray photoelectron spectroscopy (XPS) (Dahlstro¨m et al., 2000). The results presented here are important both in view of preferences for settling sites in the field of B. improvisus where surfaces of various wettabilities are found and also, when using this species in laboratory screening assays.
2. Materials and methods 2.1. Larval rearing and settlement assays using polystyrene surfaces Cyprid larvae of B. improvisus were reared in a laboratory culture as described by Berntsson et al. (2000). Cyprids were used on their first or second day of moulting. The settlement assays were performed using Petri dishes of hydrophilised polystyrene (Nunc no 45-150326, F 48 mm) and pure polystyrene (Nunc no 240045, F 48 mm) containing 5– 8 ml filtered seawater (0.2 Am, salinity: 32 F 1x ) to which 15 – 30 cyprids were added. Hence, settlement assays were performed under static conditions as described by Rittschof et al. (1984, 1992). Dishes were maintained for 6 –8 days at room temperature temperature (20 F 2 jC) at a light/dark cycle of 9:15 h, after which the dishes were checked for (1) attached and metamorphosed individuals, (2) non-metamorphosed, living cyprids and (3) dead cyprids. In the work presented here, we use the term surface wettability to define which type of surface we refer to. The terms ‘‘surface wettability’’ and ‘‘surface free energy’’ both refer to polar dispersive forces at the surface, which, as implied by the definition, are affected by inherent surface chemistry as well as by physical forces. 2.2. Settlement assays using highly polar and non-polar glass surfaces In addition to the settlement experiments using Petri dishes of hydrophilic and hydrophobic polystyrene, we wished to further elucidate the importance of the underlying surface chemistry on the settlement of cyprid larvae of B. improvisus, by preparing highly hydrophilic and hydrophobic surfaces where the active groups at the surface were either hydroxyl groups or methyl groups. The controlled surface chemistry was achieved by using different washing procedures. Petri dishes of glass (F 60 mm, Go¨teborgs Termometer Fabrik) were subjected to a washing procedure to yield highly hydrophilic surfaces. The dishes were firstly washed in a beaker containing a mixture of mQ water, ammonium hydroxide (25%) and hydrogen peroxide (35%), in the proportions 5:1:1 at 80 jC. Then a second washing took place using a mixture of mQ water, hydrochloric acid (37%) and hydrogen peroxide (35%) in the proportions 6:1:1 at 80 jC as described by Elwing et al. (1987). A portion of these dishes was then subjected to a third washing procedure to yield methylised, highly hydrophobic surfaces. The dishes were immersed in a solution containing trichloro-ethylene (TCE) and dichloro-dimethylsilane (DDS) in the proportions 9:1. The dishes were used immediately in the cyprid settlement assay. However, prior to the use in the settlement assays, the polar dishes were thoroughly
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rinsed in mQ water ( 10) and the non-polar dishes were firstly rinsed in 96% ethanol ( 6) after which they were rinsed in mQ ( 4). The dishes were filled with 8.5 ml of FSW and 20– 30 cyprids were added to each dish. In each experiment 4 replicates of each treatment was used. The experiment was maintained for 6– 8 days. 2.3. Changes in wettability over time Also, dishes of hydrophilic and hydrophobic PS and glass surfaces subjected to the washing procedures described above were used in advancing contact angle measurements with mQ water to estimate the change in wettability over the time of the experiment. To these surfaces, FSW and cyprids were added and after 1, 2, 4 or 8 days the FSW and larvae, which had not attached or metamorphosed, were poured off. The dishes were then rinsed with mQ, shaken gently and then dried under nitrogen gas for 1 min each. Also, the initial wettability of clean dishes was measured (day 0). The contact angle measurements were performed immediately after the surfaces had been dried and the measurements were performed as described by Dahlgren and Sundqvist (1981). A constant volume (10 Al) of mQ water was applied to the surfaces and the drop diameter was measured in a stereo microscope. By regarding the drop as a truncated sphere, the measured diameter was used to calculate the contact angle, which is an appropriate wettability standard of any surface (Dahlgren and Sundqvist, 1981). In each treatment, four replicates were used and the advancing contact angle with water was measured four times at each surface. 2.4. Statistical methods Results are generally reported as means F standard error. Effects of the studied surface chemistry on cyprid settlement were tested using one-factor analysis of variance (ANOVA) with surface as a fixed factor. A two-factor ANOVA was used to test for the
Fig. 1. The effect of hydrophilic and hydrophobic polystyrene (PS) surfaces on the settlement of B. improvisus cyprid larvae. The response variables are percentage attached and metamorphosed, living cyprids and dead cyprids on each surface shown as means F SE (n = 51). *Indicates a significant effect of the treatment.
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Fig. 2. The interaction graph of the factors surface and batch. Regardless of which batch of larvae is used, there is a significant reduction of the settlement on hydrophilic PS surfaces as compared to hydrophobic PS surfaces. The response variable is percentage settled shown as means F SE (n = 5).
effect of larval batch on the results in the settlement assays with surface as fixed factor and batch as a random factor. In all tests an a of 0.05 was used.
3. Results 3.1. The settlement assay using hydrophobic and hydrophilic PS The effect of the different PS surfaces used in the experiments is presented in Fig. 1. The hydrophilic PS surfaces significantly reduced settlement as compared to the hydrophobic PS surfaces (one-factor ANOVA, F1,100 = 53.4, p < 0.05). When analyzing
Fig. 3. The effect of highly polar and non-polar glass surfaces on the settlement of cyprid larvae. Hydrophobic PS surfaces served as controls. The response variables are percentage attached and metamorphosed, living cyprids and dead cyprids on each type of surface shown as means F SE (n = 4).
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Table 1 The surfaces used in the settlement assays and the change in wettability over an 8-day period on of each surface, measured as advancing contact angle with water Surface
Advancing contact angle (Day 0)
Advancing contact angle (Day 8)
Active groups at the surface
Hydrophobic polystyrene (L) Hydrophilic polystyrene (I) Hydrophobic glass (L) Hydrophilic glass (H)
81.1 F 1.6 51.4 F 0.6 78.0 F 2.7 4.3 F 0.3
43.0 F 2.0 40.5 F 2.5 40.4 F 2.1 15.9 F 2.6
methyl (CH3) hydroxyl (OH), carboxyl (COOH) methyl (CH3) hydroxyl (OH)
The surfaces are designated Low-energy surfaces (L), Intermediate energy surfaces (I) and High-energy surfaces (H).
the effect of larval batch, using a two-factor ANOVA, the results show that there is no significant interaction between larval batch and type of surface and hence, larval batch did not significantly contribute to the differences obtained by using the two different surfaces in settlement assays (two-factor ANOVA, F4,20 = 0.98, p>0.05) (Fig. 2). 3.2. The settlement assay using highly polar to non-polar glass surfaces The results obtained from the settlement assay using highly polar to non-polar glass surfaces are presented in Fig. 3. The highly polar surfaces completely inhibited settlement for the whole length of the experiment (8 days) (one-factor ANOVA, F2,9 = 25.0, p < 0.05), whereas the settlement on highly non-polar surfaces did not significantly differ from the settlement on hydrophobic PS (Fig. 3). 3.3. Changes in wettability of the test surfaces over time The least initial wettability was displayed by the hydrophobic PS dishes, closely followed by the methylised glass dishes. The hydrophilic PS dishes displayed an
Fig. 4. The changes in wettability over an eight-day period (the length of the experiments), of the four test surfaces used in the settlement experiments. The response variable is the advancing contact angle with mQ on each type of surface shown as means F SE (n = 4).
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intermediary wettability while the highly polar glass dishes had the highest wettability (Table 1). It proved that the change in wettability over a period of 8 days (the length of the experiment) was greatest on the hydrophobic dishes, where the increase in wettability from day 0 to day 8 was 53% and 51.5%, respectively, for PS and glass surfaces (Fig. 4) (Table 1). There was a slight tendency also for hydrophilised PS dishes of becoming more wettable (Fig. 4) when subjected to FSW and cyprids while the hydrophilised glass surfaces did not become markedly dewetted. The contact angle in the hydrophilic glass surfaces increased from 4.3j to 15.9j, the latter which still represents a high wettability, i.e. high surface energy.
4. Discussion It has been shown that the wettability of solid surfaces affect initial adhesion and colonization of bacteria (Baier, 1984; Fletcher and Pringle, 1985; Meyer et al., 1988). A recent study also reports that spores of the green alga Enteromorpha are affected by wettability of a substrate for attachment (Callow et al., 2000). According to Rittschof et al. (1998) invertebrate larvae are also able to detect the wettability of a potential substrate for settlement. Several of those studies address the physicochemical aspects of adhesion. However, the present study indicate that two closely related species, B. improvisus and B. amphitrite, are oppositely affected by hydrophobic and hydrophilic surfaces and thus, elements of chemical recognition may be present besides simple adhesion effects. In the present study, we report results, which indicate that the degree of wettability is an important factor in the overall settlement success of B. improvisus cyprids. Interestingly, and in contrast to experiments performed with B. amphitrite (Rittschof and Costlow, 1989; Roberts et al., 1991; Gerhart et al., 1992) we found that in the experiments where cyprid settlement of B. improvisus was monitored on hydrophobic and hydrophilic PS surfaces, the settlement in the hydrophilic dishes was significantly reduced as compared to the hydrophobic PS dishes. When the experiments were terminated after 8 days, the average settlement success in dishes of hydrophobic PS dishes was 80% while the average settlement in hydrophilic PS was 50%, representing a 38% reduction of settlement. These results clearly indicate that an initial intermediary wettability has a strong impact on the overall settlement success of cyprids of B. improvisus. The negative effect of intermediately wettable substrates was found to be consistent across larval batches. The importance of surface wettability for cyprid settlement is further demonstrated by the results obtained from the experiments where larvae were subjected to glass dishes, which had been made highly polar. In this experiment, settlement was completely inhibited over an 8-day period. We conclude by these findings that the settlement of cyprids of the barnacle B. improvisus is strongly affected by wettability and that an increased wettability causes a total rejection of these surfaces. Interestingly, studies, which have attempted to measure the wettabilities, i.e the advancing contact angle, of natural substrates, e.g. filmed surfaces and algal surfaces, show that these range from highly wettable for filmed substrates (Maki et al., 1994) to quite hydrophobic for algal surfaces (Schmitt et al., 1995). The spatial and/or temporal scales on which wettability varies are therefore relevant to barnacles and also,
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support the observed difference in response between B. improvisus and B. amphitrite. Indeed, differences in surface wettability could be one-component leading to a settlement decision. There are two major ways in which wettability might act on larval settlement. Firstly, larvae may perceive the chemistry of the surface by physically making contact with the surface. During this surface contact, which can take place with the antennules where aesthetasc structures, proposed to be situated on the fourth segment (Nott and Foster, 1969; Clare and Nott, 1994), can be employed in chemical recognition. Larvae may also, as often seen in the experimental dishes, turn dorsally to contact the surface, putatively employing the dorsally situated lattice organ which has been described by Hoeg et al. (1998) and which furthermore, are proposed to bear chemoreceptors. In essence, during the surface contact, molecular groups at the surface can be recognized either by their threedimensional pattern or by active groups at the surface, and thus, cyprids are able to estimate the quality of the surface-associated signal (Crisp, 1975; Crisp and Meadows, 1963; Nott and Foster, 1969; Gibson and Nott, 1971). Secondly, if the assumption is made that an unfavourable wettability instead impedes surface contact, the mechanism of action might be explained in terms of physical – chemical forces acting between the surface and the larva. Consequently, questions concerning the nature of these physical chemical-forces arise. A plausible account for the inhibition of surface contact is electrostatic repulsion between the surface of the cyprid and the surface of the dish. The surface chemistry of both the hydrophobic PS surfaces and the hydrophilic PS surfaces have previously been characterised using X-ray photoelectron spectroscopy (XPS) (Dahlstro¨m et al., 2000) and it was found that the hydrophilic surfaces bear mainly carboxylate groups at the surface. This chemical group has strong elements of negative charges. Furthermore, any surface, which is in contact with water has a tendency to become negatively charged, largely because cations present in the water have a greater tendency of being hydrated than anions. Hence, cations will remain in the aqueous phase while anions have a tendency to specifically adsorb to the surface (Shaw, 1991). The cyprid larva itself represents a surface which might become negatively charged by adsorbing anions present in the water and thus, electrostatic repulsive forces between the cyprid and the surface will prevent surface contact. Furthermore, it is possible that the highly wettable substrates inhibit surface contact and thereby settlement by charge repulsion between the active groups at the surface of the dish and protein side-chains in the temporary adhesive. An unfavourable wettability can pose problems in for example adhesion, where the effectiveness of the adhesives might be affected (Baier, 1984). However, this explanation does not account for the predisposition of B. amphitrite larvae of favouring highly wettable substrates, wherefore, we suggest that the differences in preference of substrate wettability between congeneric larvae of B. improvisus and B. amphitrite is most likely not a question of wettability acting on adhesion or adhesive forces, since studies have shown that the tenacity of cyprid temporary adhesion of B. amphitrite, is not affected by underlying surface wettability (Maki et al., 1994). Instead, we suggest that the difference in preference between B. improvisus and B. amphitrite is due to wettability being perceived as a cue in larval settlement and thus, evoke different responses in the two species on the basis of chemical recognition.
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Behavioural studies comparing the two species might reveal if components in larval behaviour are affected by different wettabilities, further demonstrating the importance of wettability in the settlement process. In conclusion, wettability might cause a biological inhibition by interacting with chemoreceptors when the larva is making surface contact, or the inhibition might be of a physical – chemical nature and thus, surface contact is impeded by repulsive chemical forces. We aim to further study the possible mechanism by which settlement of B. improvisus is inhibited by highly wettable substrates, by employing immunological staining of the foot-prints of cyprid temporary adhesive, which is left behind on surfaces explored by cyprid larvae.
Acknowledgements For financial support we thank the Carl Trygger Foundation, Birgit and Birger Wa˚hlstro¨ms Memorial Fund, the CF Lundstro¨m Foundation, SSF and MISTRA. We wish to thank Dr Karen Otto at the Dept of Cell and Molecular Biology, Go¨teborg University, Dr Kent Berntsson, Martin Sjo¨gren and Martin Ogemark at TMBL. [AU]
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Fletcher, M., Pringle, J.H., 1985. The effect of surface free energy and medium surface tension on bacterial attachment to solid surfaces. J. Colloid Interface Sci. 104, 5 – 14. Gerhart, D.J., Rittschof, D., Hooper, I.R., Eisenman, K., Meyer, A.E., Baier, R.E., Young, C., 1992. Rapid and inexpensive quantification of the combined polar components of surface wettability: application to biofouling. Biofouling 5, 251 – 259. Gibson, P.H., Nott, J.A., 1971. Concerning the fourth antennular segment of the cypris larvae of Balanus balanoides. In: Crisp, D.J. (Ed.), Proc. 4th Eur. Mar. Biol. Symp., Bangor 1969, Cambridge University Press, UK, pp. 227 – 236. Hoeg, J.T., Hosfeld, B., Gram-Jensen, P., 1998. TEM studies on the lattice organs of cirripede cypris larvae (Crustacea Thecostraca, Cirripedia). Zoomorphology 118, 195 – 205. Holm, E.R., Cannon, G., Roberts, D., Schmidt, A.R., Sutherland, J.P., Rittschof, D., 1997. The influence of initial surface chemistry on development of the fouling community at Beaufort, North Carolina. J. Exp. Mar. Biol. Ecol. 215, 189 – 203. Maki, J.S., Yule, A.B., Rittschof, D., Mitchell, R., 1994. The effect of bacterial films on the temporary adhesion and permanent fixation of cypris larvae, Balanus amphitrite Darwin. Biofouling 8, 121 – 131. Meyer, A.E., Baier, R.E., King, R.W., 1988. Initial fouling of nontoxic coatings in fresh, brackish and seawater. Can. J. Chem. Eng. 66, 55 – 62. Mihm, J.W., Banta, W.C., Loeb, G.I., 1981. Effects of adsorbed organic and primary fouling films on bryozoan settlement. J. Exp. Mar. Biol. Ecol. 54, 167 – 179. Neal, A.L., Yule, A.B., 1994. The interaction between Elminus modestus Darwin cyprids and biofilms och Deleya marina NCBM1877. J. Exp. Mar. Biol. Ecol. 176, 127 – 139. Nott, J.A., Foster, B.A., 1969. On the structure of the antennular attachment organ of the cypris larva of Balanus balanoides. Phil. Trans. R. Soc. 256, 115 – 133. O’Connor, N.J., Richardson, D.L., 1994. Comparative attachment of barnacle cyprids (Balanus amphitrite Darwin, 1854; B. improvisus Darwin, 1854; and B. eburneus Gould, 1841) to polystyrene and glass substrata. J. Exp. Mar. Biol. Ecol. 183, 213 – 225. Rittschof, D., Costlow, J.D., 1989. Bryozoan and barnacle settlement in relation to initial surface wettability: a comparison of laboratory and field studies. In: Ros, J.D. (Ed.), Topics in Marine Biology. Proc. 22nd Eur. Mar. Biol. Symp., vol. 53. Instituto de Ciencias del Mar, Barcelona, Spain, pp. 411 – 416. Rittschof, D., Branscomb, E.S., Costlow, J.D., 1984. Settlement and behavior in relation to flow and surface in larval barnacle, Balanus amphitrite Darwin. J. Exp. Mar. Biol. Ecol. 82, 131 – 146. Rittschof, D., Clare, A.S., Gerhart, D.J., Avelin, S.M., Bonaventura, J., 1992. Barnacle in vitro assays for biologically active substances: toxicity and settlement inhibition assays using mass cultured Balanus amphitrite amphitrite Darwin. Biofouling 6, 115 – 122. Rittschof, D., Forward Jr., R.B., Cannon, G., Welch, J.M., McClary Jr., M., Holm, E.R., Clare, A.S., Conova, S., McKelvey, L.M., Bryan, P., Van Dover, C.L., 1998. Cues and context: larval responses to physical and chemical cues. Biofouling 12, 31 – 44. Roberts, D., Rittschof, D., Holm, E., Schmidt, A.R., 1991. Factors influencing initial larval settlement: temporal, spatial and surface molecular components. J. Exp. Mar. Biol. Ecol. 150, 203 – 211. Schmitt, T.M., Hay, M.E., Lindquist, N., 1995. Constraints on chemically mediated coevolution: multiple functions for seaweed secondary metabolites. Ecology 76, 107 – 123. Shaw, D., 1991. Introduction to Colloid and Surface Chemistry, 4th ed. Butterworth-Heinemann. Reed Elsevier Plc Group, Oxford.
Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN) Mia Dahlstro¨m a,b,*, Henrik Jonsson b,1, Per R. Jonsson b, Hans Elwing a a
Department of Cell and Molecular Biology, Division of Interface Biophysics, Go¨teborg University, Box 462, SE-405 30 Go¨teborg, Sweden b Department of Marine Ecology, Tja¨rno¨ Marine Biological Laboratory, Go¨teborg University, SE-452 96 Stro¨mstad, Sweden Received 18 February 2003; received in revised form 24 October 2003; accepted 22 December 2003
Abstract Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase,
* Corresponding author. Department of Cell and Molecular Biology, Division of Interface Biophysics, Go¨teborg University, Box 462, SE-405 30 Go¨teborg, Sweden. Tel.: +46-526-68616; fax: +46-526-68607. E-mail address: [email protected] (M. Dahlstro¨m). 1 Present address: Department of Biological Oceanography, Baltic Sea Research Institute, Seestrasse 15, D18119 Warnemu¨nde, Germany. 0022-0981/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.jembe.2003.12.013
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i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules. D 2004 Published by Elsevier B.V. Keywords: Balanus improvisus; Settlement; Surface chemistry; Wettability
1. Introduction It has been demonstrated that the settlement in the field of the barnacle Balanus improvisus (Darwin) varies greatly both spatially and temporally and also, that settlement varies with respect to the physical properties of the surface, e.g. the surface topography (Berntsson, 2001; Berntsson and Jonsson, 2003; Berntsson et al., 2000). Thus, it has been established that B. improvisus has a clear preference for smooth substrata and also, that underlying behavioural responses cause the rejection of surfaces bearing a certain micro-geometry (Berntsson et al., 2000). However, the importance of inherent chemical properties of a potential settling site for B. improvisus has not been examined to the same extent. O’Connor and Richardson (1994) reported that cyprids of B. improvisus attach to a lesser extent to glass vials than to polystyrene dishes, however, no attempts of further characterising these surfaces were made. Previous reports on the settlement response in the field of marine invertebrate larvae to surfaces of varying wettability, show that the initial surface chemistry of a substratum for settlement is important for initial colonization but not in determining the overall structure of the macrofouling community (Roberts et al., 1991; Holm et al., 1997). Furthermore, laboratory studies of ascidians, bryozoans and the barnacle B. amphitrite have shown that the preferred wettability of a substrate for settlement displays some variation, with the barnacle B. amphitrite being the sole of these to have a clear preference for high wettability substrates (Mihm et al., 1981; Rittschof and Costlow, 1989; Gerhart et al., 1992; O’Connor and Richardson, 1994). Also, the tenacity of cyprid temporary adhesion of the barnacles B. amphitrite and Elminus modestus (Darwin) has been measured in respect to different chemical properties of the surface (Maki et al., 1994; Neal and Yule, 1994). Similar measurements have been conducted on cyprid larvae and juvenile barnacles of B. improvisus using low energy surfaces such as fluorinated silicone elastomeres and the more high energy surface poly (methyl methacrylate) (PMMA) where the tenacity of the temporary adhesion proved to be similar on both of these surfaces (Berglin et al., 2001). However, there are no reports on the overall settlement success of barnacle larvae of B. improvisus in response to surface chemistry, i.e. surfaces with high vs. low wettability. In previous studies we have used hydrophilic and hydrophobic dishes of polystyrene to monitor the accumulation of various settlement inhibiting compounds at the solid surface (Dahlstro¨m et al., 2000). However, during these assays we have observed that settlement of B. improvisus vary greatly in the control dishes between the two surfaces. Due to an extensive amount of data collected from settlement assays from late 1998 to early 2001 and also, due to the absence in the literature regarding reports of how the barnacle B. improvisus responds to surfaces of different inherent chemistry, we here present a brief report of the effect of initial surface chemistry on the settlement and
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metamorphosis of cyprids of B. improvisus, using highly polar to non-polar surfaces of both polystyrene and glass. These results are then combined with previous analyses of the chemical composition of these surfaces using X-ray photoelectron spectroscopy (XPS) (Dahlstro¨m et al., 2000). The results presented here are important both in view of preferences for settling sites in the field of B. improvisus where surfaces of various wettabilities are found and also, when using this species in laboratory screening assays.
2. Materials and methods 2.1. Larval rearing and settlement assays using polystyrene surfaces Cyprid larvae of B. improvisus were reared in a laboratory culture as described by Berntsson et al. (2000). Cyprids were used on their first or second day of moulting. The settlement assays were performed using Petri dishes of hydrophilised polystyrene (Nunc no 45-150326, F 48 mm) and pure polystyrene (Nunc no 240045, F 48 mm) containing 5– 8 ml filtered seawater (0.2 Am, salinity: 32 F 1x ) to which 15 – 30 cyprids were added. Hence, settlement assays were performed under static conditions as described by Rittschof et al. (1984, 1992). Dishes were maintained for 6 –8 days at room temperature temperature (20 F 2 jC) at a light/dark cycle of 9:15 h, after which the dishes were checked for (1) attached and metamorphosed individuals, (2) non-metamorphosed, living cyprids and (3) dead cyprids. In the work presented here, we use the term surface wettability to define which type of surface we refer to. The terms ‘‘surface wettability’’ and ‘‘surface free energy’’ both refer to polar dispersive forces at the surface, which, as implied by the definition, are affected by inherent surface chemistry as well as by physical forces. 2.2. Settlement assays using highly polar and non-polar glass surfaces In addition to the settlement experiments using Petri dishes of hydrophilic and hydrophobic polystyrene, we wished to further elucidate the importance of the underlying surface chemistry on the settlement of cyprid larvae of B. improvisus, by preparing highly hydrophilic and hydrophobic surfaces where the active groups at the surface were either hydroxyl groups or methyl groups. The controlled surface chemistry was achieved by using different washing procedures. Petri dishes of glass (F 60 mm, Go¨teborgs Termometer Fabrik) were subjected to a washing procedure to yield highly hydrophilic surfaces. The dishes were firstly washed in a beaker containing a mixture of mQ water, ammonium hydroxide (25%) and hydrogen peroxide (35%), in the proportions 5:1:1 at 80 jC. Then a second washing took place using a mixture of mQ water, hydrochloric acid (37%) and hydrogen peroxide (35%) in the proportions 6:1:1 at 80 jC as described by Elwing et al. (1987). A portion of these dishes was then subjected to a third washing procedure to yield methylised, highly hydrophobic surfaces. The dishes were immersed in a solution containing trichloro-ethylene (TCE) and dichloro-dimethylsilane (DDS) in the proportions 9:1. The dishes were used immediately in the cyprid settlement assay. However, prior to the use in the settlement assays, the polar dishes were thoroughly
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rinsed in mQ water ( 10) and the non-polar dishes were firstly rinsed in 96% ethanol ( 6) after which they were rinsed in mQ ( 4). The dishes were filled with 8.5 ml of FSW and 20– 30 cyprids were added to each dish. In each experiment 4 replicates of each treatment was used. The experiment was maintained for 6– 8 days. 2.3. Changes in wettability over time Also, dishes of hydrophilic and hydrophobic PS and glass surfaces subjected to the washing procedures described above were used in advancing contact angle measurements with mQ water to estimate the change in wettability over the time of the experiment. To these surfaces, FSW and cyprids were added and after 1, 2, 4 or 8 days the FSW and larvae, which had not attached or metamorphosed, were poured off. The dishes were then rinsed with mQ, shaken gently and then dried under nitrogen gas for 1 min each. Also, the initial wettability of clean dishes was measured (day 0). The contact angle measurements were performed immediately after the surfaces had been dried and the measurements were performed as described by Dahlgren and Sundqvist (1981). A constant volume (10 Al) of mQ water was applied to the surfaces and the drop diameter was measured in a stereo microscope. By regarding the drop as a truncated sphere, the measured diameter was used to calculate the contact angle, which is an appropriate wettability standard of any surface (Dahlgren and Sundqvist, 1981). In each treatment, four replicates were used and the advancing contact angle with water was measured four times at each surface. 2.4. Statistical methods Results are generally reported as means F standard error. Effects of the studied surface chemistry on cyprid settlement were tested using one-factor analysis of variance (ANOVA) with surface as a fixed factor. A two-factor ANOVA was used to test for the
Fig. 1. The effect of hydrophilic and hydrophobic polystyrene (PS) surfaces on the settlement of B. improvisus cyprid larvae. The response variables are percentage attached and metamorphosed, living cyprids and dead cyprids on each surface shown as means F SE (n = 51). *Indicates a significant effect of the treatment.
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Fig. 2. The interaction graph of the factors surface and batch. Regardless of which batch of larvae is used, there is a significant reduction of the settlement on hydrophilic PS surfaces as compared to hydrophobic PS surfaces. The response variable is percentage settled shown as means F SE (n = 5).
effect of larval batch on the results in the settlement assays with surface as fixed factor and batch as a random factor. In all tests an a of 0.05 was used.
3. Results 3.1. The settlement assay using hydrophobic and hydrophilic PS The effect of the different PS surfaces used in the experiments is presented in Fig. 1. The hydrophilic PS surfaces significantly reduced settlement as compared to the hydrophobic PS surfaces (one-factor ANOVA, F1,100 = 53.4, p < 0.05). When analyzing
Fig. 3. The effect of highly polar and non-polar glass surfaces on the settlement of cyprid larvae. Hydrophobic PS surfaces served as controls. The response variables are percentage attached and metamorphosed, living cyprids and dead cyprids on each type of surface shown as means F SE (n = 4).
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Table 1 The surfaces used in the settlement assays and the change in wettability over an 8-day period on of each surface, measured as advancing contact angle with water Surface
Advancing contact angle (Day 0)
Advancing contact angle (Day 8)
Active groups at the surface
Hydrophobic polystyrene (L) Hydrophilic polystyrene (I) Hydrophobic glass (L) Hydrophilic glass (H)
81.1 F 1.6 51.4 F 0.6 78.0 F 2.7 4.3 F 0.3
43.0 F 2.0 40.5 F 2.5 40.4 F 2.1 15.9 F 2.6
methyl (CH3) hydroxyl (OH), carboxyl (COOH) methyl (CH3) hydroxyl (OH)
The surfaces are designated Low-energy surfaces (L), Intermediate energy surfaces (I) and High-energy surfaces (H).
the effect of larval batch, using a two-factor ANOVA, the results show that there is no significant interaction between larval batch and type of surface and hence, larval batch did not significantly contribute to the differences obtained by using the two different surfaces in settlement assays (two-factor ANOVA, F4,20 = 0.98, p>0.05) (Fig. 2). 3.2. The settlement assay using highly polar to non-polar glass surfaces The results obtained from the settlement assay using highly polar to non-polar glass surfaces are presented in Fig. 3. The highly polar surfaces completely inhibited settlement for the whole length of the experiment (8 days) (one-factor ANOVA, F2,9 = 25.0, p < 0.05), whereas the settlement on highly non-polar surfaces did not significantly differ from the settlement on hydrophobic PS (Fig. 3). 3.3. Changes in wettability of the test surfaces over time The least initial wettability was displayed by the hydrophobic PS dishes, closely followed by the methylised glass dishes. The hydrophilic PS dishes displayed an
Fig. 4. The changes in wettability over an eight-day period (the length of the experiments), of the four test surfaces used in the settlement experiments. The response variable is the advancing contact angle with mQ on each type of surface shown as means F SE (n = 4).
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intermediary wettability while the highly polar glass dishes had the highest wettability (Table 1). It proved that the change in wettability over a period of 8 days (the length of the experiment) was greatest on the hydrophobic dishes, where the increase in wettability from day 0 to day 8 was 53% and 51.5%, respectively, for PS and glass surfaces (Fig. 4) (Table 1). There was a slight tendency also for hydrophilised PS dishes of becoming more wettable (Fig. 4) when subjected to FSW and cyprids while the hydrophilised glass surfaces did not become markedly dewetted. The contact angle in the hydrophilic glass surfaces increased from 4.3j to 15.9j, the latter which still represents a high wettability, i.e. high surface energy.
4. Discussion It has been shown that the wettability of solid surfaces affect initial adhesion and colonization of bacteria (Baier, 1984; Fletcher and Pringle, 1985; Meyer et al., 1988). A recent study also reports that spores of the green alga Enteromorpha are affected by wettability of a substrate for attachment (Callow et al., 2000). According to Rittschof et al. (1998) invertebrate larvae are also able to detect the wettability of a potential substrate for settlement. Several of those studies address the physicochemical aspects of adhesion. However, the present study indicate that two closely related species, B. improvisus and B. amphitrite, are oppositely affected by hydrophobic and hydrophilic surfaces and thus, elements of chemical recognition may be present besides simple adhesion effects. In the present study, we report results, which indicate that the degree of wettability is an important factor in the overall settlement success of B. improvisus cyprids. Interestingly, and in contrast to experiments performed with B. amphitrite (Rittschof and Costlow, 1989; Roberts et al., 1991; Gerhart et al., 1992) we found that in the experiments where cyprid settlement of B. improvisus was monitored on hydrophobic and hydrophilic PS surfaces, the settlement in the hydrophilic dishes was significantly reduced as compared to the hydrophobic PS dishes. When the experiments were terminated after 8 days, the average settlement success in dishes of hydrophobic PS dishes was 80% while the average settlement in hydrophilic PS was 50%, representing a 38% reduction of settlement. These results clearly indicate that an initial intermediary wettability has a strong impact on the overall settlement success of cyprids of B. improvisus. The negative effect of intermediately wettable substrates was found to be consistent across larval batches. The importance of surface wettability for cyprid settlement is further demonstrated by the results obtained from the experiments where larvae were subjected to glass dishes, which had been made highly polar. In this experiment, settlement was completely inhibited over an 8-day period. We conclude by these findings that the settlement of cyprids of the barnacle B. improvisus is strongly affected by wettability and that an increased wettability causes a total rejection of these surfaces. Interestingly, studies, which have attempted to measure the wettabilities, i.e the advancing contact angle, of natural substrates, e.g. filmed surfaces and algal surfaces, show that these range from highly wettable for filmed substrates (Maki et al., 1994) to quite hydrophobic for algal surfaces (Schmitt et al., 1995). The spatial and/or temporal scales on which wettability varies are therefore relevant to barnacles and also,
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support the observed difference in response between B. improvisus and B. amphitrite. Indeed, differences in surface wettability could be one-component leading to a settlement decision. There are two major ways in which wettability might act on larval settlement. Firstly, larvae may perceive the chemistry of the surface by physically making contact with the surface. During this surface contact, which can take place with the antennules where aesthetasc structures, proposed to be situated on the fourth segment (Nott and Foster, 1969; Clare and Nott, 1994), can be employed in chemical recognition. Larvae may also, as often seen in the experimental dishes, turn dorsally to contact the surface, putatively employing the dorsally situated lattice organ which has been described by Hoeg et al. (1998) and which furthermore, are proposed to bear chemoreceptors. In essence, during the surface contact, molecular groups at the surface can be recognized either by their threedimensional pattern or by active groups at the surface, and thus, cyprids are able to estimate the quality of the surface-associated signal (Crisp, 1975; Crisp and Meadows, 1963; Nott and Foster, 1969; Gibson and Nott, 1971). Secondly, if the assumption is made that an unfavourable wettability instead impedes surface contact, the mechanism of action might be explained in terms of physical – chemical forces acting between the surface and the larva. Consequently, questions concerning the nature of these physical chemical-forces arise. A plausible account for the inhibition of surface contact is electrostatic repulsion between the surface of the cyprid and the surface of the dish. The surface chemistry of both the hydrophobic PS surfaces and the hydrophilic PS surfaces have previously been characterised using X-ray photoelectron spectroscopy (XPS) (Dahlstro¨m et al., 2000) and it was found that the hydrophilic surfaces bear mainly carboxylate groups at the surface. This chemical group has strong elements of negative charges. Furthermore, any surface, which is in contact with water has a tendency to become negatively charged, largely because cations present in the water have a greater tendency of being hydrated than anions. Hence, cations will remain in the aqueous phase while anions have a tendency to specifically adsorb to the surface (Shaw, 1991). The cyprid larva itself represents a surface which might become negatively charged by adsorbing anions present in the water and thus, electrostatic repulsive forces between the cyprid and the surface will prevent surface contact. Furthermore, it is possible that the highly wettable substrates inhibit surface contact and thereby settlement by charge repulsion between the active groups at the surface of the dish and protein side-chains in the temporary adhesive. An unfavourable wettability can pose problems in for example adhesion, where the effectiveness of the adhesives might be affected (Baier, 1984). However, this explanation does not account for the predisposition of B. amphitrite larvae of favouring highly wettable substrates, wherefore, we suggest that the differences in preference of substrate wettability between congeneric larvae of B. improvisus and B. amphitrite is most likely not a question of wettability acting on adhesion or adhesive forces, since studies have shown that the tenacity of cyprid temporary adhesion of B. amphitrite, is not affected by underlying surface wettability (Maki et al., 1994). Instead, we suggest that the difference in preference between B. improvisus and B. amphitrite is due to wettability being perceived as a cue in larval settlement and thus, evoke different responses in the two species on the basis of chemical recognition.
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Behavioural studies comparing the two species might reveal if components in larval behaviour are affected by different wettabilities, further demonstrating the importance of wettability in the settlement process. In conclusion, wettability might cause a biological inhibition by interacting with chemoreceptors when the larva is making surface contact, or the inhibition might be of a physical – chemical nature and thus, surface contact is impeded by repulsive chemical forces. We aim to further study the possible mechanism by which settlement of B. improvisus is inhibited by highly wettable substrates, by employing immunological staining of the foot-prints of cyprid temporary adhesive, which is left behind on surfaces explored by cyprid larvae.
Acknowledgements For financial support we thank the Carl Trygger Foundation, Birgit and Birger Wa˚hlstro¨ms Memorial Fund, the CF Lundstro¨m Foundation, SSF and MISTRA. We wish to thank Dr Karen Otto at the Dept of Cell and Molecular Biology, Go¨teborg University, Dr Kent Berntsson, Martin Sjo¨gren and Martin Ogemark at TMBL. [AU]
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