Barnacle settlement on rocky shores: Substratum preference and epibiosis on mussels

Journal of Experimental Marine Biology and Ecology 473 (2015) 195–201

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Barnacle settlement on rocky shores: Substratum preference and epibiosis on mussels Caroline Bell a,⁎, Christopher D. McQuaid a, Francesca Porri a,b a b

Coastal Research Group, Department of Zoology and Entomology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa South African Institute for Aquatic Biodiversity (SAIAB), Somerset Street, 6139 Grahamstown, South Africa

a r t i c l e

i n f o

Article history: Received 15 December 2014 Received in revised form 10 September 2015 Accepted 11 September 2015 Available online xxxx Keywords: Barnacle settlement Settlement Rocky shore Substratum choice

a b s t r a c t Understanding the dynamics of epibiosis is fundamental to understanding the role of biological interactions in the functioning of marine ecosystems. At many coastal sites, barnacles are abundant as epibionts on the shells of mussels. As they approach the shore, cypris larvae explore the environment, actively seeking suitable settlement sites and rejecting unsuitable substrata. Suitability involves micro-features of the surface, including its texture and contours, chemical signals and the presence of conspecifics. To investigate whether epibiosis on mussel shells indicates a preference for this substratum on the part of the barnacle Chthamalus dentatus, artificial plates were deployed at two rocky shore sites on the south coast of South Africa. These plates offered settling cyprids four habitat choices: a live mussel, the shell of a recently dead mussel, a fine resolution resin replica of a mussel shell to mimic architectural micro-surface, without the chemical characteristics of natural shells and a rock mimic, a surface covered in a film of hard plastic that resembled natural rock surface as closely as possible. Settling surfaces were photographed monthly, and new barnacles were counted and allocated to size classes, so that survival could be estimated over a period of several months. Barnacles showed a clear preference for the rock mimic surfaces, which on average supported double the number of settlers found on mussel replicas. These in turn were significantly higher than the numbers on live and dead mussel shell surfaces, implying that there are features of both dead and live mussel shells that deter barnacle settlement. In contrast, final abundances of adults showed no significant effect of settling surface. The results suggest that during settlement, cyprids avoid chemical cues from mussel shells that persist even after the death of the mussel. This suggests that the high numbers of barnacles often found on the surface of live mussels could be due to saturation of substratum. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Marine benthic organisms frequently show initial settlement patterns that are highly variable in space and time. Following settlement on a hard substratum, post-larval stages experience high rates of mortality through interactions with each other, as well as other species, while facing the effects of abiotic stressors (Pineda et al., 2009). As a result, post-settlement mortality (mortality immediately after settlement), is an important determinant of species success, defining the distinction between settlement and recruitment (Connell, 1985; Minchinton and Scheibling, 1993; Porri et al., 2006). Recruitment can be regarded as the rate at which juveniles join the adult population (i.e. become sexually mature), or as the number of juveniles that survive for a defined period of time after settlement (Bayne, 1964; Connell, 1985; Pineda, 2000). For benthic invertebrates, this period is usually a few days or weeks, after which mortality rates decline markedly

⁎ Corresponding author. E-mail address: [email protected] (C. Bell).

http://dx.doi.org/10.1016/j.jembe.2015.09.006 0022-0981/© 2015 Elsevier B.V. All rights reserved.

(Connell, 1985; Nakaoka, 1993; Pineda et al., 2009). In the case of sessile organisms, the selection of the settlement site is particularly critical as it has an enormous influence on post-settlement mortality and overall fitness. Barnacles are sessile and exhibit spatial patterns of settlement that reflect the effects of several, at times opposing, factors. These include a preference for cracks and pits (Crisp and Barnes, 1954), gregarious larval behaviour (Knight-Jones, 1953; Crisp and Meadows, 1962, 1963) and the need to maintain sufficient inter-individual distance to allow for growth within the maximum spacing that will allow internal fertilisation (Crisp, 1990). Barnacles are well known to show preferences for settlement sites (Crisp, 1961) and once cyprids reach the near shore environment, they actively seek out and evaluate the microenvironment, rejecting unsuitable positions (e.g. Rittschof et al., 1984). What constitutes a suitable position involves the micro features of the surface, its texture and contours and the presence of conspecifics. Surface topography can be classified according to scale, as texture (the irregularities of the surface that are smaller than the larvae) and contour (irregularities larger than the larvae) (Le Tourneux and Bourget, 1988). One measure of texture is roughness and most studies have shown that

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surface roughness generally promotes barnacle settlement (see review in Berntsson et al., 2000a). There are also chemical cues to settlement, for example from conspecifics (Knight-Jones, 1953; Crisp and Meadows, 1962, 1963; Chabot and Bourget, 1988; Raimondi, 1988), and final larval choice at settlement will involve a combination of chemical and physical cues (Wethey, 1984). Selection of microhabitat, at scales of heterogeneity larger than 10 cm, in combination with the presence of conspecifics, at scales smaller than 1.5 cm, seems to drive settlement of Semibalanus balanoides (Chabot and Bourget, 1988). A more cryptic factor influencing barnacle settlement may be the bacterial film that develops on surfaces that have been immersed in the sea and many marine larvae settle readily on such surfaces (Zobell and Allen, 1935; Tighe-Ford et al., 1970), though Maki et al. (1988) and Nasrolahi et al. (2012) found that bacterial films generally inhibited the attachment of barnacle larvae. Larval age is important, with older larvae attaching more readily to clean surfaces that lack a biofilm than younger individuals, perhaps an example of the desperate larva hypothesis (Knight-Jones, 1951, 1953). By increasing surface complexity, the shells of bivalves in the intertidal increase the available surface for larval settlement. Despite the fact that shells offer substantial space, mytilids often appear to support fewer fouling organisms than adjacent non-biological substrata (Wahl et al., 1998; Bers and Wahl, 2004). One possible explanation is that many marine species have evolved behavioural, chemical, physical or mechanical defence mechanisms to prevent or minimise epibiotic settlement (Wahl, 1989). Given the possibility of harmful effects of epibiosis, one might expect surfaces of endemic species to evolve preventative measures to inhibit the settlement of local epibionts, while cosmopolitan species should show a generalised antifouling strategy (Bers et al., 2006). The texture of the periostracum influences the antifouling capacity of the shells of the mussel Mytilus edulis (Wahl et al., 1998; Bers and Wahl, 2004) and Mytilus galloprovincialis (Scardino et al., 2003) and there are indications of adaptation to local and abundant epibionts (Bers et al., 2006). The periostracum can inhibit damage to mussel shells by boring endolithic organisms (Kaehler, 1999) and general fitness is improved by an intact periostracum texture, which reduces the incidence of epibiosis on M. galloprovincialis (Scardino and de Nys, 2004). It is clear that settlement is affected by factors that operate across a wide range of scales, from kilometres to sub-millimetres (Pineda, 2000), and settlement of barnacles may be limited by multiple elements, from planktonic larval supply (Buschbaum, 2000), linked to oceanic and tidal features (Raimondi, 1990; Pineda, 1994; Hills and Thomason, 1996), to micro-heterogeneity of the available surface (Le Tourneux and Bourget, 1988). The choice of settlement sites by sessile organisms on rocky shores is especially important, as it determines their final position, directly influencing the structure of the intertidal assemblage (reviewed by Chan and Høeg, 2015). In the case of epibiosis, this also affects biological interactions within the community, including those between epibiont and host (Laihonen and Furman, 1986; Garner and Litvaitis, 2013). In order to clarify whether the selection of mussels as a settlement substratum by barnacles is due to active choice, and how such choices affect survival, we addressed the question of whether the physical and chemical properties of mussel shells influence barnacle settlement and their survival to adulthood. We hypothesised that the surface texture of mussel shells and chemical properties of their periostracum would deter barnacle settlement and reduce barnacle survival. 2. Methods Artificial plates were deployed at two rocky shores separated by approximately 10 km on the south coast of South Africa: Beacon Isle (34°3̍ 35̎ S; 23°22̍ 49̎ E) and Keurboomstrand (34°0′ 18″ S; 23° 27′ 30″ E). Both sites are situated within Plettenberg Bay and mussels at both sites experience high levels of epibiosis by barnacles (per. obs.).

2.1. Experimental set up The following four treatments (termed settling surfaces, hereafter) were fixed to perspex plates for attachment to the shore: live mussels, dead mussels (shell only), resin shells (fine-resolution replicas of shells to mimic the architectural micro-surface but not the chemical characteristics of natural shells) and a rock mimic. Settling surfaces were deployed haphazardly within the mid mussel zone. Live mussels were collected two days prior to field deployment and kept alive in aerated sea water, at ambient temperature (21–25 °C), with the water being changed daily. The dead shell surface was prepared by scraping out the soft tissue, drying the shells and filling the two halves with twocomponent epoxy adhesive (Abe epidermix 372). The valves were glued back together using clear, two-component epoxy (Alcolin rapidepoxy) and left for 24 h to set. This was done approximately 24 h before deployment to ensure the periostracum was as intact as possible and its chemical characteristics still present. Resin replicas were made from natural mussel shells from which the soft tissues had been removed. This methodology has been successfully applied to separate the effect of microstructure and shell chemistry (Marrs et al., 1995; Bers et al., 2006; Bers et al., 2010). The shells were then gently washed and dried. Moulds were made of the outer surfaces of the two valves separately, using silicone (Loctite, RTV Silicone 587, Blue 80 ml Tube). These moulds were left to set for 20 to 30 min at room temperature. Resin was mixed at the ratio of 5 ml of catalyst to 300 ml of resin, kept bubble-free by slowly stirring for five minutes. The resin was then carefully poured into the silicone moulds and left overnight to cure at room temperature. Once solid, the resin casts were removed from the moulds and the two halves of the replica mussel were glued together as above. Live, dead and replica mussels were glued onto clear, perspex plates that had been roughened for better attachment. The rock mimic surfaces were settlement plates that had a film of hard plastic (3M™ Safety-Walk™, Medium duty, Grey) that resembled natural rock surface as closely as possible, attached to them. All settlement plates were 8.0 × 5.5 cm in size and the mussels used for all settling surfaces were Perna perna of 3.0–5.0 cm shell length. Settlement plates were attached to the rocks using battery powered drills and self-tapping screws. Prior to the attachment of the settlement plates, the immediate area around each plate was scraped clean of any barnacles or mussels to avoid attraction by conspecifics. On 8 March 2012, at Beacon Isle, 44 plates (n = 11) were arranged within the mid mussel zone, covering a total along-shore stretch of 10 m, while, at Keurboomstrand, 40 plates (n = 10) were arranged, across 15 m of shore, on 6 April 2012. All plates were removed in August 2012. 2.2. Data collection Photographs of each plate were taken every month and new barnacles (C. dentatus) on all settling surfaces were counted, using ImageJ 1.45 software. The dates of sampling at Beacon Isle were: 6 April 2012; 7 May 2012; 7 June 2012; 20 July 2012; 4 August 2012, while at Keurboomstrand, they were: 7 May 2012; 7 June 2012; 20 July 2012; 4 August 2012. Barnacles were also allocated to a size class, so that survival over the months could be estimated. Barnacles were divided into size classes on the basis of the basal diameter as follows: Size class I: visible to the naked eye — 1.00 mm. Size class II: 1.01 mm — 2.50 mm. Size class III: 2.60 mm — 3.50 mm. Size class IV: 3.60 mm — 5.50 mm. Chthamalus fissus reaches maturity at a basal diameter of 2 mm (Hines, 1978). In the absence of data on size at sexual maturity for our species, we took the conservative approach of considering class IV individuals to be adult. Prior to analysis, the counts for the rock mimic plates were adjusted by taking the area of the plastic film attached to the plate and scaling it to correspond to the average area of the other mussel shells used.

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At the end of the study, month six for Beacon Isle and month five for Keurboomstrand, final recruitment on different settling surfaces was estimated and normalised for surface area. Because the mussel shells were similar in size and shell curvature minimal, they were treated as flat surfaces. A total of five observations were available for analysis but only events 1–4 were used as too few plates remained for statistical analysis after observation five. 2.3. Statistical analysis To identify possible substratum preference, initial settlement and adult numbers were analysed separately using a 3-way PERMANOVA. A repeated measure design was used with settling surface (fixed, 4 levels) and site (random, 2 levels), considered orthogonal allowing the detection of interactions between these two factors, while time was considered random, but was excluded from interactions due to a lack of true replication. To represent the adult numbers, the ratio of adults, to total number of settlers (i.e. cumulative number of initial settlers throughout the study) was calculated for each settling surface. These ratios are shown in Fig. 5. Pair-wise tests were performed for significant results relevant to our hypotheses. All statistical analyses were done using PERMANOVA + adds-in to PRIMER (v. 6.1.10; PRIMER-E Ltd.). Normality and homogeneity of data were tested using the Kolmogorov–Smirnov and Levene's tests respectively, and none of the data met these assumptions (p b 0.01 in all cases). Transformation did not improve heterogeneity of data, which were therefore analysed untransformed. This approach was considered robust due to the large size of the dataset (n = 262, Underwood 1997). 3. Results 3.1. Settlers Settlement of C. dentatus size class I individuals (termed settlers hereafter) showed significant differences between sites, among settling surfaces and over time (p b 0.05; Table 1). There was, however, no significant interaction of site and settling surface, and the pattern for both sites was clearly similar, thus sites were pooled (Fig. 1). However, settlement was significantly higher at Beacon Isle across all settling surfaces (not shown). Pairwise tests showed that rock mimic plates had significantly higher settlement rates than the other three settling surfaces (Fig. 1): dead b rock mimic (p b 0.001); replica b rock mimic (p b 0.02) and live b rock mimic (p b 0.001). Replicas also had higher settlement than dead mussels or the live mussel shells (p b 0.001 in both cases). There was no significant difference between the live and dead mussel shells (p N 0.05). We had no hypothesis concerning time, but it was clear that settlement was substantially higher at both sites during the first sampling period (note y-axis scale on Figs. 2 and 3) and relatively consistent thereafter. 3.2. Adult survivors The numbers of barnacles that survived to adulthood (size class IV) were significantly affected by site and time, but not by the factor of

Fig. 1. Overall settlement of Chthamalus dentatus size class one (visible to the naked eye: 1.00 mm), across settling surfaces (N = 78), where “live” is a live mussel, “dead” is a dead mussel (shell only), “replica” is an artificial mussel (replicating the surface texture of a natural mussel shell) and “rock mimic” is a film of hard Safety-Walk™ plastic that resembled natural rock surface. Values are means over all sample periods, error bars indicate standard errors. Sites differed significantly and the letters represent homogenous groups for the term settling surface, identified by pair-wise tests (using PERMANOVA).

interest, settling surface. Adult numbers accumulated over the study period, and so were greatest at time 4 at each site. Adult numbers at Beacon Isle were roughly an order of magnitude higher (mean at time 4: 0.75 adult barnacles, across settling surfaces, ± 0.34) than at Keurboomstrand (mean at time 4: 0.09 adult barnacles, across settling surfaces, ± 0.03), reflecting the patterns of settlement. Nevertheless, there was no correlation between total numbers of settlers and adult numbers, even when settlers from time 1 were correlated against adults from time 4 (correlation analysis, P N 0.05). Although settling surface had no significant effect (F ratio: 1.39353; p value: 0.2163), due to high levels of variability, adult numbers were higher on rock mimic and replica surfaces at both sites (Fig. 4, note different x-axis scales). Given the difference in settlement between sites and among surfaces, adults were re-calculated as the ratio of adults to total settlers (Fig. 5). Again, there was no obvious pattern by settling surface, though at time 4, the rock mimic and mussel replica surfaces had the (non-significantly) highest values at Beacon Isle. Likewise, the mussel replica surface, at Keurboomstrand. 4. Discussion Barnacles showed a clear preference for settlement on rock-like substratum (the rock mimic surface) where numbers of settlers were on average double those on replica mussels at both sites. The numbers of settlers on replica surfaces were in turn significantly higher than on

Table 1 PERMANOVA of substratum preference by settlers of Chthamalus dentatus. Significant effects in bold. * indicates p b 0.05, ** p b 0.01, *** p b 0.001.

Site Settling surface Time Site × settling surface Res

Df

SS

MS

pseudo-F

p (perm)

U perms

p (MC)

1 3 3 3 251

19,477 59,382 95,883 6426.8 5.703E5

19,477 19,794 31,961 2142.3 2272.3

8.571 8.711 14.065 1.943

0.0001 0.0001 0.0001 0.462

9936 9925 9932 9931

0.004** 0.0001*** 0.0001*** 0.464

Df = degrees of freedom; SS = sum of squares; MS = mean squares; U = unique permutations; p (MC) = Monte Carlo significant values; p = significance level.

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Fig. 2. Settlement of Chthamalus dentatus size class one, on the different settling surfaces at Beacon Isle (N = 40), showing sampling events (time 1 – time 4). Surfaces are “live”: a live mussel, “dead”: a dead mussel (shell only), “replica”: an artificial mussel (replicating the surface texture of a natural mussel shell) and “rock mimic”: a film of hard, Safety-Walk™ plastic that resembled natural rock surface. Note that the scales of the y axes are different. Values are means and error bars indicate standard errors.

live and dead mussel shell surfaces, implying that there are features of mussel shells that deter barnacle settlement. Inhibition of fouling has been widely studied and may be mechanical, chemical or physical (Scardino et al., 2003), with some species combining these different modes to deter or prevent epibionts (Wahl et al., 1998). The surface roughness of mussel shells has been investigated as a possible physical defence by a number of authors (Crisp and Barnes, 1954; Chabot and Bourget, 1988; Le Tourneux and Bourget, 1988; Raimondi, 1990; Hills

and Thomason, 1996; Walters and Wethey, 1996; Hills and Thomason, 1998; Wahl et al., 1998). We used P. perna individuals as the ‘live’ and ‘dead’ mussels on settlement plates and it has been shown that the properties of the periostracum of P. perna, M. edulis and M. galloprovincialis are all similar (Bers et al., 2006). Reduced fouling has been shown on M. galloprovincialis (Scardino et al., 2003), and different populations of M. edulis (Bers et al., 2006). These antisettlement properties are due to the micro-topography of the shell

Fig. 3. Primary settlement of Chthamalus dentatus on the different settling surfaces at Keurboomstrand (N = 37), showing sampling events (time 1–time 4). Surfaces are “live”: a live mussel, “dead”: a dead mussel (shell only), “replica”: an artificial mussel (replicating the surface texture of a natural mussel shell) and “rock mimic”: a film of hard, Safety-Walk™ plastic that resembled natural rock surface. Values are means and error bars indicate standard errors.

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Fig. 4. Adult numbers of Chthamalus dentatus (size class IV: 3.60 mm – 5.50 mm) on the different Settling surfaces at Beacon Isle (N = 41) and Keurboomstrand (N = 37), showing sampling events (time 1–time 4). Surfaces are “live”: a live mussel, “dead”: a dead mussel (shell only), “replica”: an artificial mussel (replicating the surface texture of a natural mussel shell) and “rock mimic”: a film of hard, Safety-Walk™ plastic that resembled natural rock surface. Error bars indicate standard errors. Note that the scales of the y axes are different.

rather than the thickness of the periostracum, which differs among species (Harper, 1997). Micro-texture is another aspect of the periostracum that differs from surface roughness in that it is the regular, defined surface structure (Andersson et al., 1999; Köhler et al., 1999; Berntsson et al., 2000a; Callow et al., 2002). Investigations into micro-texture have often used artificial micro-topographies and these include the scales of 50–100 μm (Andersson et al., 1999) and 30–45 μm (Berntsson et al., 2000b). With micro-texture at these scales, settlement of barnacle larvae was reduced. Wethey (1986) proposed that surface

contour is the most important factor affecting recruitment of S. balanoides. Our results showed that C. dentatus cyprids avoid live mussels as well as dead mussels that still have an intact periostracum, but not replicas with the same surface texture as the live and dead mussel shell surfaces, suggesting that there must be antifouling cues given off by the periostracum itself. Scardino et al. (2003) came to similar conclusions, suggesting a possible species-specific surface bound chemical that repels larvae, but they acknowledged that this anti-fouling mechanism requires further investigation. Our results showed a hierarchy of defensive

Fig. 5. Adults of Chthamalus dentatus as a proportion of total numbers of settlers on the different settling surfaces at Beacon Isle and Keurboomstrand (N = 78), showing sampling events (time 1–time 4). Surfaces are “live”: a live mussel, “dead”: a dead mussel (shell only), “replica”: an artificial mussel (replicating the surface texture of a natural mussel shell) and “rock mimic”: a film of hard Safety-Walk™ plastic that resembled natural rock surface. Error bars indicate standard errors. Note that the scales of the y axes are different.

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mechanism, with settlers preferentially settling on rock-like substratum, then replica surfaces, followed by live and dead mussel shell surfaces. This suggests that chemical cues are a primary deterrent, followed by micro-topography, with settlers showing a strong preference for the rock-like substratum. Given that shell areas were considered flat, shell curvature may also have been important, but this would not affect the hierarchy among shell/shell-type settling surfaces, as all were similar shapes. There is such a broad range of settlement cues for marine invertebrate larvae (Berntsson et al., 2004) that inferring acceptance or rejection of different cues from final settlement on plates or panels in the field is the most effective means of studying substratum preference. There are multiple aspects to consider, such as differences in settlement and/or recruitment between sites (Raimondi, 1990), which were evident in the significant effect of site for settlers as well as adults. Additionally, factors that have been shown to influence settlement of cyprids include biofilm that has formed on the substratum (Maki et al., 1988; Wieczorek and Todd, 1998), physio-chemical properties of the substratum (Crisp and Barnes, 1954; Becker, 1993), macro- and micro-scale hydrodynamics (Crisp, 1955; Shanks, 1986; Bertness et al., 1996), predation and competition (Connell, 1961a; Dayton, 1971; Wethey, 1984; Berlow and Navarrete, 1997), the age of the cyprid (Satuito et al., 1996; Jarrett, 1997) and the presence of other barnacles (Knight-Jones, 1953). It is possible that the micro-hydrodynamics associated with the complex structure of mussel beds influences settlement, but in our design experimental substrata were set up as isolated individuals and there was a clear effect of settling surface. There was, however, no interaction between site and settling surface, indicating a clear effect of substratum on the choice of settlement site by cyprids. When considering the effect of site, settlement plates were deployed a month apart (in March and April, 2012) but within the same reproductive season in the area of Plettenberg Bay (Bownes and McQuaid, 2009), so this difference is considered minimal. Gregarious behaviour is widespread among barnacles and chemical cues from conspecific adults have been found to encourage settlement in the laboratory (Knight-Jones, 1953; Crisp and Meadows, 1962; Rittschof et al., 1984) and in the field (Raimondi, 1988; Jarrett, 1997). Biochemical cues from other invertebrate species (Raimondi, 1988), biofilms (Maki et al.,1988) and macroalgae (Le Tourneux and Bourget, 1988) also affect barnacle settlement and many of our settlement plates were overgrown with algae (pers. Obs.). A further consideration is the availability of space and recruitment rates. Numbers of settlers were much higher on the first sampling occasion than the later occasions. This could simply be a result of variation in larval supply, but Raimondi (1990) found similar patterns in the field for Chthamalus anisopoma, with recently settled barnacles seeming to inhibit further settlement. S. balanoides also displayed a positive relationship between settlement rate and recruit density until approximately 30% of the free space was covered, after which settlement rates decreased (Chabot and Bourget, 1988). Thus, there appears to be a threshold level of available free space, below which cyprids settle less readily (Minchinton and Scheibling, 1993). Given the high initial settlement on some of our settling surfaces, this may have affected our results. Gaines and Roughgarden (1985), however, regarded settlement of barnacles as a simple function of the availability of free space, adjusted for larval supply, time of site immersion and average bulk flow over the substratum. They found that settlement plays as important a role as post-settlement processes such as predation and competition through physical dislodgement. Higher settlement will result in higher recruitment rates, unless there is a major disturbance. Large, denser populations will be more stable over time, but if settlement is low, the population will be recruitment-limited and sensitive to fluctuations in settlement (Underwood et al., 1983). Larval supply is particularly important when unoccupied space becomes rare. Adult abundances showed no significant effect of settling surface and, although initial settlement is an indication of preference, ultimately how many individuals

survive to reproduce is fundamental, both ecologically and evolutionarily (Pineda et al., 2009). It has been shown that high settler densities reliably predict adult barnacle densities (Menge, 2000), but in this study settlers did not consistently determine the numbers of adults. In our study, “new” settlers were only counted once a month and any post-settlement mortality that occurred between observations was not taken into account. In particular, the hard, Safety-Walk™ plastic that made up the “rock mimic” surface may have experienced higher temperatures than expected, as was the case in a study by Shanks (2009). This limitation can be remedied by daily observations but this was not logistically possible. It is well known that marine invertebrates have extremely high fecundities, with very few individuals surviving to reproduce (Pineda et al., 2009). This is due to extremely high pre- and post-settlement mortality (Gosselin and Qian, 1996; Hunt and Scheibling, 1997). Although there was no significant difference among settling surfaces for adult numbers (i.e. barnacles in size class IV), various other processes shape adult numbers. Survival rates are continuously influenced by biotic factors such as predation (Connell, 1961a; Dayton, 1971; Wethey, 1984) and abiotic effects including extreme weather events (Connell, 1961b; de Wolf, 1973). These effects vary in space and time, potentially obscuring the effect of settling surface on adult numbers. The fact that there was no correlation between settler and adult numbers suggests that in this case adult populations are determined by factors other than settlement rates, but, at a larger scale, Beacon Isle had both higher settlement and adult survival than Keurboomstrand indicating that differences in larval supply could persist from settlement to adulthood with considerable effects on these barnacle populations. 5. Conclusion There was a clear hierarchy of larval preference at both sites that strongly favoured a rock-like substratum over mussel shells or mimic shells, suggesting active avoidance of chemical properties of the periostracum of mussel shells, with micro-topography being a less critical element of site selection. Importantly, although settlers appeared to make active choices among substrata, preferred substrata did not improve survival to adulthood. The fact that mussels at our study sites experience high levels of barnacle epibiosis, suggests limited availability of substratum and thus barnacles are obliged to settle onto the shells of live mussels. Clearly, mussel defences against epibionts discourage settlement, but cannot eliminate it, and barnacle preferences for avoiding certain substrata are indeed just preferences, that can be overcome when the situation demands. Therefore, substratum choice presumably reflects selective pressures acting on life-time fitness and is shaped at local scales by biotic and abiotic processes. Acknowledgments This work is based upon research supported by the South African Research Chairs Initiative of the Department of Science and Technology and the National Research Foundation [SS]. References Andersson, M., Berntsson, K., Jonsson, P., Gatenholm, P., 1999. Microtextured surfaces: towards macrofouling resistant coatings. Biofouling 14, 167–178. Bayne, B.L., 1964. Primary and secondary settlement in Mytilus edulis L. (Mollusca). J. Anim Ecol. 33, 513–523. Becker, K., 1993. Attachment strength and colonization patterns of two macrofouling species on substrata with different surface tension (in situ studies). Mar. Biol. 117, 301–309. Berlow, E.L., Navarrete, S.A., 1997. Spatial and temporal variation in rocky intertidal community organization: lessons from repeating field experiments. J. Exp. Mar. Biol. Ecol. 214, 195–229. Berntsson, K.M., Andreasson, H., Jonsson, P.R., Larsson, L., Ring, K., Petronis, S., Gatenholm, P., 2000a. Reduction of barnacle recruitment on micro-textured surfaces: analysis of

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