Prior Exposure Influences the Behavioural Avoidance by An

Estuarine, Coastal and Shelf Science 136 (2014) 82e90

Contents lists available at ScienceDirect

Estuarine, Coastal and Shelf Science

journal homepage: www.elsevier.com/locate/ecss

Prior exposure inﬂuences the behavioural avoidance by an intertidal gastropod, Bembicium auratum, of acidiﬁed waters

Valter Amaral a,b,*, Henrique N. Cabral b, Melanie J. Bishop a a Department of Biological Sciences, Macquarie University, North Ryde, New South Wales 2109, Australia b Centro de Oceanograﬁa, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal article info abstract

Article history: Phenotypic plasticity may be critical to the maintenance of viable populations under future environ- Received 22 April 2013 mental change. Here we examined the role of behavioural avoidance of sub-optimal conditions in Accepted 17 November 2013 enabling the intertidal gastropod, Bembicium auratum, to persist in mangrove forests affected by the low Available online 23 November 2013 pH runoff from acid sulphate soils (ASS). Behaviourally, the gastropod may be able to avoid periods of particularly high acidity by using pneumatophores and/or mangrove trunks to vertically migrate above Keywords: the water line or by retreating into its shell. We hypothesised that (1) B. auratum would display greater adaptation and more rapid vertical migration out of acidified than reference estuarine waters, and (2) responses Bembicium auratum fi crawl-out would be more pronounced in gastropods collected from acidi ed than reference sites. Gastropods from fi fi fi invertebrate acidi ed sites showed signi cantly higher activity in and more rapid migration out of acidi ed waters of microhabitat pH 6.2e7.0, than reference waters or waters of pH < 5.0. Gastropods from reference locations showed a pH significantly weaker response to acidified water than those from acidified waters, and which did not significantly differ from their response to reference water. At extremely low pHs, <5.0, a higher proportion of both acidified and reference gastropods retreated into their shell than at higher pHs. Both the migration of gastropods out of acidified waters and retraction into their shells serves to reduce exposure time to acidified waters and may reduce the impact of this stressor on their populations. The stronger response to acidification of gastropods from populations previously exposed to this stressor suggests that the response may be learned, inherited or induced over multiple exposures. Our study adds to growing evidence that estuarine organisms may exhibit considerable physiological and behaviour adaptive capacity to acidification. The potential for such adaptive capacity should be incorporated into studies seeking to forecast impacts to marine organisms of environmental change. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction organisms to adapt to change may depend on their phenotypic plasticity. Plasticity in life-history, physiology or behaviour may Ecological environments are currently experiencing change at enable populations to persist and maintain viable populations un- unprecedented rates and scales (IPCC, 2007). Where organisms are der a broad range of environmental conditions (Charmantier et al., unable to adapt to this change, restructuring of coastal and estua- 2008; Tuomainen and Candolin, 2011). rine ecosystems (Worm et al., 2006; Richardson and Poloczanska, The acidification of the world’s oceans and estuaries is one 2008), and loss of important ecosystem services (Barbier et al., aspect of environmental change that is presently challenging the 2011) may result. In instances where molecular evolution is con- structure and function of ecosystems. Emissions of CO2 are already strained (Stern and Orgogozo, 2009; Hoffmann et al., 2012), or is producing changes in pH of ecological significance (Fabry et al., insufficiently rapid to keep pace with environmental change (Stern 2008; Hendriks et al., 2010) and are predicted to produce a and Orgogozo, 2009; Lavergne et al., 2013), the capacity of further drop in pH of w0.5 units in the next 100e200 years (Caldeira and Wickett, 2005; IPCC, 2007). Simultaneously, the conversion of wetlands to farmlands is leading to enhanced exposure of acid sulphate soils (ASS; Dent and Pons, 1995), the runoff * Corresponding author. Centro de Oceanografia, Faculdade de Ciências da Uni- from which can reduce the pH of adjacent estuaries to as low as 2e6 versidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal. fi E-mail addresses: [email protected] (V. Amaral), [email protected] (H.N. Cabral), (Sammut et al., 1996; NSW DPI, 2006). The acidi cation of seawater [email protected] (M.J. Bishop). can negatively impact marine organisms by impairing their

0272-7714/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecss.2013.11.019 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90 83 physiological regulation, olfactory discrimination, and predator (2) those sourced from sites recurrently exposed to acidic waters avoidance behaviours and/or causing dissolution of the exoskeleton would display stronger responses to this disturbance than those of calcifying organisms (Munday et al., 2009; Ries et al., 2009; from unaffected, reference sites. Melatunan et al., 2013). Among marine organisms, intertidal molluscs (e.g. oysters and gastropods) are among the most vulnerable 2. Materials and methods to acidification (Fabry et al., 2008; Guinotte and Fabry, 2008; Hendriks et al., 2010). They rely on a calcium carbonate exoskel- 2.1. Sampling sites, gastropods and test waters eton to protect them from predation and desiccation stress, they have limited ability to regulate their internal pH and, in the case of To test the hypothesis that Bembicium auratum gastropods species with a sessile life-history stage, cannot escape from acidic previously exposed to runoff from ASS would display stronger waters (Bamber, 1987, 1990; Sammut et al., 1995; Ries et al., 2009). avoidance behaviours to acidified waters than naive gastropods, we Although many molluscs display strong negative responses to reciprocally exposed gastropods from acidified and reference sites acidification, some appear to exhibit considerable capacity to adapt of an estuary to water sourced from acidified and reference sites of to this stressor (Bibby et al., 2007; Ries et al., 2009; Hendriks et al., that same estuary. Water and gastropods were collected from two 2010). For example, the effect of runoff from acid sulphate soils on acidified and two reference sites within each of the Hunter (32.915 the shell strength and density of wild populations of Saccostrea S,151.801 E) and Port Stephens (32.708 S, 152.196 E) estuaries, NSW, glomerata oysters and Bembicium auratum was less than predicted Australia. At each site, B. auratum were found attached to the from experiments exposing naive oysters to this stressor (Amaral pneumatophores of the grey mangrove, Avicennia marina and on et al., 2011a, 2012a). Whereas naive individuals rapidly developed the surface of the sediment. Acidified sites were situated within tissue lesions and experienced shell dissolution that ultimately 900 m of major ASS discharge drains, in areas classified by the NSW resulted in mortality (Dove and Sammut, 2007a,b), wild pop- Government as being of high ASS runoff risk (Naylor et al., 1998; ulations were able to persist in a periodically acidifying environ- NSW DECCW, 2012), and with a history of low pH (NSW DPI, ment (Amaral et al., 2011a, 2012a). Wild populations of molluscs 2006, 2008, 2009). Reference sites were situated at least 2400 m that are recurrently exposed to acidified conditions are likely to away from drains in areas of low ASS runoff risk (Naylor et al., 1998; experience intense selective pressure for resistance to acidification NSW DECCW, 2012) and where we had not observed pH values (Trussell, 1996; Jones and Boulding, 1999; Melatunan et al., 2013). lower than 7.6 (Table 1). Sites within each estuary were of similar Additionally, phenotypically plastic physiological and behavioural water temperature, but those adjacent to drains were on average 10 responses to the conditions may confer some resistance to the times more acidic and of slightly lower (w1 unit) salinity than stressor (Ries et al., 2009; Rodolfo-Metalpa et al., 2011). Oysters can reference sites (Table 1). We have previously documented differ- endure pulses of exposure to sub-optimal conditions by closing ences in the abundance, morphology and growth of molluscs be- their valves and relying on their tough shells, built mainly of calcite, tween these acidified and reference sites (Amaral et al., 2011a, to protect them from predators (Stenzel, 1964; Dove and Sammut, 2012a,b). 2007a; Green and Barnes, 2010). Gastropods, on the other hand, Experiments were repeated on four occasions, during late fall build their shells from the more soluble aragonite and are mobile and early winter of 2012. At low tide of each sampling date, we (Taylor and Reid, 1990), so may benefit from moving to microhab- collected test water from surface waters adjacent to each site, and itats in which they escape acidification or reduce the frequency and Bembicium auratum gastropods of 11e14 mm in shell height from duration of their exposure to it (Marshall et al., 2008). the pneumatophore zone (mean low water þ 0.5e0.7 m). This size Gastropods commonly use behavioural avoidance (i.e. move- of gastropod is numerically dominant at our sampling sites (Amaral ment into protected microhabitats or retraction into the shell) to et al., 2011a) and differs in shell strength (by >60 N) between reduce their susceptibility to predators (Richardson and Brown, acidified and reference sites (Amaral et al., 2012a). Only gastropods 1992; Jacobsen and Stabell, 1999). The role of similar strategies in without shell fouling or visible shell damage were used. Test waters enabling them to persist in acidic water has, however, seldom been were continuously aerated with air stones until experimentation, considered (Bibby et al., 2007), and never using wild organisms that within 1.5 h of collection. Measurements of water quality, with a have been exposed to acidification over multiple generations. We multi-parameter, handheld metre (556 MPS, YSI Incorporated, conducted aquarium experiments to investigate the extent to which Bembicium auratum gastropods are able to behaviourally Table 1 avoid ASS-affected waters. B. auratum is a littorinid that is endemic Mean (SD) temperature (Temp), salinity and pH at the acidified (A1, A2) and to Australasia and common in the intertidal of rocky shores and reference (R1, R2) sites within the Hunter (H) and Port Stephens (P) estuaries, be- mangrove forests, where it is most abundantly found attached to tween 2009 and 2012 (n ¼ 17 sampling dates). Sites were selected on the basis of oyster shells and to trunks and peg roots of mangrove trees (Branch these measurements, previously recorded pH minima (pH min) and acid sulphate risk maps that categorize areas according to the probability of being impacted by and Branch, 1980; Reid, 1988; Underwood and Barrett, 1990). this disturbance (ASS-risk; Naylor et al., 1998; NSW DECCW, 2012). Behavioural plasticity has previously been shown in this species: it is able to quickly adjust its dispersal pattern to the surrounding Site Location ( C) Temp Salinity pH pH min ASS risk microhabitat, responding to extrinsic cues of the new environment HA1 Fullerton Cove 20 (5) 25 (6) 6.90 (0.36) w4* High * (Crowe, 1996; Crowe and Underwood, 1999). Similarly, it may be HA2 Tomago Wetland 19 (3) 25 (5) 6.99 (0.40) w4 High HR1 Southern Ash Island 19 (4) 26 (6) 7.94 (0.27) w7* Low able to avoid periods of particularly high acidity by using pneu- * HR2 Northern Ash Island 20 (4) 26 (3) 7.96 (0.25) w7 Low y z matophores and/or mangrove trunks to vertically migrate above PA1 Fenninghams 19 (4) 27 (7) 6.84 (0.79) <5 , High the water line or by retreating into its shell. Creek (entry) y z We compared the effect of seawater acidification on activity PA2 Fenninghams 19 (4) 28 (7) 6.87 (0.66) <5 , High Creek (middle) patterns and rates of vertical migration above the water line be- z PR1 Stuart’s Island 20 (4) 29 (6) 7.93 (0.42) w6.8 Low z tween populations of gastropods that had been exposed to runoff PR2 4 km North of 19 (4) 29 (7) 7.96 (0.39) w6.8 Low from ASS over many generations, and those that had never before Stuart’s Island been exposed. We predicted that: (1) all gastropods would be more * NSW DPI (2008). y active and display higher rates of movement above the water line NSW DPI (2006). z when exposed to ASS-affected than control (unaffected) waters, but NSW DPI (2009). 84 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90

Yellow Springs, USA; Table 2), revealed that all test waters were of (Anderson, 2001b). Consequently, unlike ANOVAs, PERMANOVAs similar temperature, but that the salinity of acidified water was on do not have explicit assumptions about the underlying distribu- average slightly lower (on average by w1 unit) than reference tions of data and can use any distance matrix that is appropriate to water. Across all trials, the pH of acidified water ranged from 4.64e the data. Here, PERMANOVAs were used because they allow 7.02, and of reference waters, from 7.91e8.28 (Table 2). interpretation of interaction terms within random factors (Anderson, 2001a). The mixed-model PERMANOVAs had five fac- 2.2. Experimental setup tors: water treatment (2 levels: acidified vs reference, orthogonal, fixed); water site (2 levels; nested within water treatment); Experiments were performed in cylindrical plastic containers, of gastropod treatment (2 levels: acidified vs reference, orthogonal, 10 cm diameter and 10.5 cm height. Each was filled to a depth of fixed); gastropod site (2 levels; nested within gastropod treat- 8 cm with w630 ml of the designated test water, such that the top ment), and trial (4 levels; orthogonal, fixed). The PERMANOVAs 2.5 cm of the container walls were above the water line. The height were run on Rogers-Tanimoto dissimilarity matrices for the bino- of the container walls matched the average length of pneumato- mial variables: (1) moved (yes vs no) and (2) crawled out (yes vs phores at our field sites, and the horizontal distance (10 cm) be- no), and on Euclidean dissimilarity matrices for the numeric vari- tween walls, the typical distance between pneumatophores ables: (3) time to first movement, (4) proportion of time spent (Amaral et al., 2011b). moving and (5) time to crawl-out. Where there was no significant A single gastropod, from the assigned source population, was difference (at P > 0.25; Underwood, 1997) in a variable between the placed in an upright and central position on the bottom of a 2 replicate sites within a water or gastropod treatment (i.e. acidified container of the designated water treatment. For each water and or reference) or in interaction terms involving the factor site, ana- gastropod treatment, there were six replicates per sampling trial. lyses were repeated with data pooled across sites within treat- Each gastropod was left to acclimate in the bottom of its container ments. In the instance that PERMANOVAs detected significant for 10 min before measurements commenced. The activity (moving treatment effects, a posteriori pairwise comparisons were done to vs not moving) and horizontal and vertical displacement along the identify sources of the differences. The analyses were run in Primer container walls of each snail from its starting position was noted v6 (PRIMER-E, Ivybridge, UK). after the first minute, the first 5 min, and then every 5 min there- after, for an hour (n ¼ 13 observations). A 60 min observation 3. Results period was chosen because most Bembicium auratum had crawled- out of each container by this time and this duration represents the For all variables except the time gastropods spent moving, there time taken for pneumatophores to go from emersed to submersed were no significant differences (1) between the two source sites on a rising tide. within a water, or gastropod treatment or (2) in each interaction For each replicate gastropod, we used these observations to term involving sites (at P > 0.25; Underwood, 1997), enabling assess: (1) whether the gastropod had moved from its initial po- pooling of sites within gastropod and water treatments. sition 30 and 60 min after the start of the experiment; (2) the time The response of gastropods to acidified water, in many in- elapsed (to the nearest 5 min) before the gastropod first moved; (3) stances, varied according to whether the gastropods were sourced the proportion of observation points at which the gastropod was from acidified or reference sites (Tables 3 and 4; PERMANOVA, sig. moving, either until it reached the water line or the end of the trial Water treatment [W] Gastropod treatment [G] interactions). (whichever came first); and (4) whether the gastropod had Across all trials [T], and in each of the estuaries, significantly more migrated out of the water by the end of the 60 min trial. For those gastropods from acidified than reference sites had moved following gastropods that did migrate out of the water, we also calculated (5) 30 min of exposure to acidic waters, but in reference waters there the average amount of time (to the nearest 5 min) they took to do was no significant difference in the movement of the two gastropod so. Gastropods and waters were used only once to maintain inde- groups (Fig.1; G(W), a posteriori tests, Moved within 30 min, Table 3). pendence of replicates. After 60 min of exposure to test waters, by which time >80% of gastropods had moved, this pattern of greater movement in acidi- 2.3. Statistical analyses fied waters of gastropods from acidified than reference sites was weaker, though still significant, in the Hunter estuary (Fig. 1; G(W), We tested hypotheses about differing behavioural responses of a posteriori tests, Moved within 60 min, Table 3), but non-significant gastropods from ASS-affected and reference sites to acidified and in Port Stephens where no effect of either water source or reference waters using separate univariate permutational analyses gastropod source was seen (non-sig. effects of W G, G or W; Moved of variance (PERMANOVA; Anderson, 2001a) for each estuary. within 60 min, Table 3). In the Hunter trials, and irrespective of PERMANOVAs apply the traditional ANOVA partitioning procedure gastropod source site, fewer animals moved when exposed to to a distance matrix, but use permutations to obtain P-values acidified than reference waters, but in the Port Stephens trials

Table 2 Temperature, salinity, pH, total dissolved solids (TDS) and oxidation-reduction potential (ORP) of test waters collected at each acidiﬁed (A1, A2) and reference (R1, R2) sites within the Hunter (H) and Port Stephens (P) estuaries, in each of the 4 experimental trials (1e4).

Site Temperature (C) Salinity pH TDS (g L 1) ORP (mV)

12341234123412341234

HA1 17 17 16 15 24 24 23 24 6.75 6.46 4.63 4.76 25.3 25.0 25.8 25.6 186 203 400 393 HA2 17 16 15 15 24 24 23 24 6.55 6.22 4.95 4.41 25.0 25.1 25.4 25.8 184 220 349 369 HR1 18 16 16 16 25 25 25 25 8.17 8.14 7.91 8.07 24.4 24.7 24.4 25.0 160 158 152 160 HR2 17 16 16 17 25 26 24 25 8.18 8.15 8.16 8.12 24.4 25.1 24.8 24.7 169 162 175 167 PA1 16 17 16 16 24 24 24 23 6.31 6.41 6.87 6.67 25.0 25.1 24.8 25.1 197 208 190 192 PA2 16 17 16 16 24 24 24 25 6.34 6.45 7.02 7.01 25.4 25.2 24.8 25.0 193 185 183 172 PR1 17 16 15 15 25 25 25 25 8.15 8.19 8.27 8.28 25.1 24.6 24.4 24.5 159 164 151 165 PR2 16 16 16 16 25 25 25 24 7.94 8.16 8.26 8.23 24.9 24.5 24.8 24.3 158 158 163 146 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90 85

Table 3 Results of three-way PERMANOVAs testing for differences in the behavioural response of Bembicium auratum gastropods sourced from acidified (GA) and reference (GR) locations (Gastropod treatment, G) to exposure to acidified (WA) and reference (WR) waters (Water treatment, W). Four replicate experimental trials (T; 1e4) were run for each of the Hunter and Port Stephens estuaries. Within each estuary, gastropods and waters were sourced from two acidified and two reference sites, but were pooled across these because the factor Site was not significant at a ¼ 0.05. Data were analysed untransformed. Terms significant at a ¼ 0.05 are highlighted in bold. For significant interactions, a posteriori tests were used to identify significant differences among levels of one factor (at a ¼ 0.05; denoted by > and < symbols) within levels of the other (indicated in parentheses).

Source Hunter Pseudo-FP Port Stephens Pseudo-FP

df MS df MS

Moved within 30 min W 1 57,526 37.6 <0.001 1 <1 <0.1 1.000 G 1 16,276 10.6 <0.002 1 5104 4.6 0.027 T 3 33,568 21.9 <0.001 3 1424 1.3 0.295 W G 1 11,484 7.5 <0.003 1 3750 3.4 0.042 W T 3 37,179 24.3 <0.001 3 1181 1.1 0.369 G T 3 3012 2.0 0.108 3 1007 0.9 0.419 W G T 3 1971 1.3 0.296 3 1458 1.3 0.251 Residual 368 1530 368 1116 A posteriori tests W G W G G(W); [WA]:GA > GR; [WR]:GA ¼ GR G(W); [WA]:GA > GR; [WR]:GA ¼ GR W(G); [GA]:WA < WR; [GR]:WA < WR W(G); [GA]:WA¼WR; [GR]:WA¼WR W T T(W); [WA]:(1 ¼ 2)>(3 ¼ 4); [WR]:1 ¼ 2 ¼ 3 ¼ 4 W(T); [1]: WA > WR; [2]: WA ¼ WR; [3,4]: WA < WR Moved within 60 min W 1 62,526 46.1 <0.001 1 1667 2.5 0.105 G 1 9401 6.9 <0.009 1 938 1.4 0.201 T 3 44,540 32.8 <0.001 3 903 1.3 0.226 W G 1 5859 4.3 0.042 1 938 1.4 0.215 W T 3 34,679 25.5 <0.001 3 1319 2.0 0.110 G T 3 3359 2.5 0.055 3 313 0.5 0.699 W G T 3 1207 0.9 0.469 3 590 0.9 0.450 Residual 368 1358 368 670 A posteriori tests W G G(W); [WA]:GA > GR; [WR]:GA ¼ GR W(G); [GA]:WA < WR; [GR]:WA < WR W T T(W); [WA]:(1 ¼ 2)>(3 ¼ 4) [WR]:1 ¼ 2 ¼ 3 ¼ 4 W(T); [1,2]: WA¼WR; [3,4]: WA < WR Time to start moving W 1 905 9.9 <0.004 1 1396 13.4 <0.001 G 1 4284 46.8 <0.001 1 3555 34.2 <0.001 T 3 215 2.4 0.075 3 89 0.9 0.462 W G 1 3942 43.1 <0.001 1 2617 25.2 <0.001 W T 3 322 3.5 <0.024 3 14 0.1 0.95 G T 3 93 1.0 0.401 3 47 0.4 0.696 W G T 3 142 1.6 0.189 3 67 0.7 0.571 Residual 247 91 340 104 A posteriori tests W G W G G(W); [WA]:GA < GR; [WR]:GA ¼ GR G(W); [WA]:GA < GR; [WR]:GA ¼ GR W(G); [GA]:WA < WR; [GR]:WA > WR W(G); [GA]:WA < WR; [GR]:WA¼WR W T T(W): [WA]:(1 ¼ 2 ¼ 3)<4; [WR]:1 ¼ 2 ¼ 3 ¼ 4 W(T); [1,2]: WA¼WR; [3,4]: WA > WR Crawled out W 126<1 0.848 1 2604 1.1 0.245 G 1 13,776 8.2 <0.004 1 33,750 14.3 <0.001 T 3 41,762 24.9 <0.001 3 2569 1.1 0.378 W G 1 3151 1.9 0.2 1 17,604 7.5 <0.008 W T 3 32,387 19.3 <0.001 3 4757 2.0 0.12 G T 3 2248 1.3 0.24 3 3264 1.4 0.255 W G T 3 2040 1.2 0.279 3 1840 0.8 0.512 Residual 368 1677 368 2360 A posteriori tests W T W G T(W): [WA]:(1 ¼ 2)>(3 ¼ 4) G(W); [WA]:GA > GR; [WR]:GA ¼ GR [WR]:1 ¼ 2 ¼ 3 ¼ 4 W(G); [GA]:WA > WR; [GR]:WA¼WR W(T); [1,2]: WA > WR; [3,4]: WA < WR Time to crawl-out W 1 307 2.0 0.19 1 6 3.3E 2 0.844 G 1 4997 32.1 <0.001 1 9743 58.3 <0.001 T 3 20 0.1 0.961 3 33 0.2 0.912 W G 1 2087 13.4 <0.002 1 3879 23.2 <0.001 (continued on next page) 86 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90

Table 3 (continued )

Source Hunter Pseudo-FP Port Stephens Pseudo-FP

df MS df MS

W T 2 116 0.7 0.492 3 99 0.6 0.629 G T 3 106 0.7 0.55 3 335 2.0 0.108 W G T 1 405 2.6 0.115 3 224 1.3 0.241 Residual 120 156 180 167 A posteriori tests W G W G G(W); [WA]:GA < GR; [WR]:GA ¼ GR G(W); [WA]:GA < GR; [WR]:GA ¼ GR W(G); [GA]:WA¼WR; [GR]:WA > WR W(G); [GA]:WA < WR; [GR]:WA > WR neither gastropod group displayed a significant difference in the Table 3). Gastropods exposed to the extremely acidified waters of proportion of individuals that moved between the two water the Hunter trials 3 and 4, took significantly longer to start moving treatments (Fig. 1; W(G), a posteriori tests, Moved within 30 min, than those exposed to reference waters (Fig 2; W(T), a posteriori 60 min, Table 3). tests, Time to start moving, Table 3), such that a smaller proportion In acidified waters, gastropods from acidified sites took on in acidified waters crawled out (W(T), a posteriori tests, Crawled out, average 4 times less time to start moving (Fig 2; G(W), a posteriori Table 3). Indeed, only one gastropod was able to crawl-out of acidic tests, Time to start moving, Table 3) and spent proportionally twice test waters of the Hunter trials 3 and 4 (Fig 1). Overall, gastropods as much time moving than gastropods from reference sites (Fig 3; exposed to the acidified treatment of Hunter trials 3 and 4 spent G(W), a posteriori tests, Time spent moving, Table 4). Furthermore, about half the amount of time moving as their counterparts those gastropods also crawled out of acidic waters in significantly exposed to the acidified treatment in trials 1 and 2 (Fig 3; a pos- higher numbers (Fig 1; only significant in Port Stephens; G(W), a teriori tests, T(W); Time spent moving, Table 4). posteriori tests, Crawled out, Table 3) and in half the time (Fig 4; G(W), a posteriori tests, Time to crawl out, Table 3) as gastropods 4. Discussion sourced from reference sites within the same estuary. By contrast, no difference in any of the considered behavioural responses were Here, we have experimentally demonstrated that the gastropod found between gastropods from acidified and reference sites tested Bembicium auratum modifies its behaviour in response to acidifi- in waters sourced from areas unaffected by ASS discharge (G(W), a cation, and that the magnitude of this response, which acts to posteriori tests, Tables 3 and 4). Consequently, gastropods from minimize exposure to the stressor, is dependent on prior exposure. acidified sites took less time to start moving in acidified than When exposed to waters within the normal pH range of healthy reference waters (Fig. 2), and consequently crawled out of acidified estuaries, gastropods sourced from sites with a history of recurrent waters at an equal (Hunter) or greater (Port Stephens) rate and in exposure to acid sulphate soil (ASS) did not differ in behaviour from equal (Hunter) or greater (Port Stephens) numbers than in refer- gastropods sourced from reference sites that had not previously ence waters (Fig. 4; W(G), a posteriori tests, Table 3). Gastropods been impacted by this stressor. When exposed to waters of pH 5 to from reference sites, by contrast, took an equal (Port Stephens) or 7, however, a greater proportion of gastropods from the acidified greater (Hunter) time to start moving and greater time to crawl out than reference sites moved and sought refuge from the acidified of acidified than reference waters (Figs. 2 and 4; W(G), a posteriori waters by moving above the water line. The proportion of gastro- tests, Table 3). pods from acidified sites that moved out of waters of pH 6.2 to 7.0 The behavioural response of gastropods to acidified water did was greater than for reference waters. When exposed to a pH < 5, not differ among the four trials using waters and gastropods however, significantly more gastropods remained inside their sourced from Port Stephens (PERMANOVAs: non-sig. W G T, shells and with their opercula closed than in reference water. The G T and W T interactions; non-sig. main effect of T; Tables 3 and weaker response to acidification of gastropods from reference than 4). By contrast among the four trials using water and gastropods acidified sites may result from behavioural responses to this from the Hunter, the effect of water source differed (PERMANOVAs: stressor being learned. Alternatively, the alteration of behaviour sig. W T interactions; Tables 3 and 4). Whereas gastropods dis- under acidified conditions may represent an inducible response, played a statistically indistinguishable behavioural response to activated only after repeated exposure to the stressor or be the reference waters across the four Hunter trials, their behavioural outcome of genetic selection over multiple generations of exposure responses to acidified waters in many instances differed between to the stressor. Our study, which used each gastropod only once, trials 1 and 2, as compared to trials 3 and 4 (a posteriori tests, T(W), was unable to experimentally test these potential mechanisms. a posteriori tests, sig. W T interaction, Tables 3 and 4). Not only did more gastropods from acidified than reference During Hunter trials 1 and 2, in which the pH of acidified waters sites move out of acidic waters, but the gastropods from acidified ranged between 6.2 and 7.0, more gastropods in acidified than sites did so at greater speed. Ninety seven percent of gastropods reference waters crawled out (Fig.1; W(T), a posteriori tests, Crawled from acidified sites initiated movement within the first 10 min of out, Table 3) during the 60 min study, although the number of exposure to acidified water, and these gastropods from acidified gastropods that moved (a posteriori tests, W(T); Moved within locations typically migrated above the water line (8 cm height) 60 min, Table 3), the time they took to commence movement (Fig. 2; within 20 min for Port Stephens and 25 min for Hunter gastro- W(T), a posteriori tests, Time to crawl out, Table 3) and the time they pods. By comparison, gastropods from reference sites in Port spent moving (Fig. 3; W(T), a posteriori tests, Time spent moving, Stephens took >40 min, and in the Hunter >50 min, to reach the Table 4) did not differ between the water treatments. During trials 3 water line of acidified treatments. In their natural environment, and 4, in which the pH of acidified waters from the Hunter was Bembicium auratum have access to intertidal pneumatophores of below 5.0, fewer gastropods exposed to acidified than reference 10e14 cm height. These are completely submerged by rising tides waters moved from their initial position either within 30 (Fig. 1; within 1.5e2 h, and remain at least partially submerged for w7h. W(T), a posteriori tests, Moved within 30 min, Table 3) or 60 min of Consequently, if B. auratum vertically migrate up these pneu- exposure (Fig. 1; W(T), a posteriori tests, Moved within 30 min, matophores with the rising tide to avoid inundation by acidified V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90 87

Table 4 Results of five-way PERMANOVAs testing for differences in the time Bembicium auratum gastropods sourced from acidified (GA) and reference (GR) locations (Gastropod treatment, G) spent moving when exposed to acidified (WA) and reference (WR) waters (Water treatment, W). Four experimental trials (T, 1e4) were run, within each of the Hunter and Port Stephens estuaries. Within each estuary, gastropods and waters were sourced from two acidified and two reference sites (gastropod sites, GS: G1, G2; water sites, WS: W1, W2). Terms significant at a ¼ 0.05 are in highlighted in bold. All data were analysed untransformed. For significant interactions, a posteriori tests were used to identify significant differences among levels of one factor (at a ¼ 0.05; denoted by > and < symbols) within levels of the other (indicated in parentheses).

Source Hunter Pseudo-FP Port Stephens Pseudo-FP

df MS df MS

Time spent moving W 1 497 0.8 0.378 1 11,759 21.1 <0.001 G 1 9899 16.6 <0.001 1 34,019 61.0 <0.001 T 3 2685 4.5 <0.006 3 493 0.9 0.441 WS(W) 1 1684 2.8 0.109 2 344 0.6 0.531 GS(G) 1 221 0.4 0.547 2 860 1.5 0.238 W G 1 3179 5.3 <0.033 1 22,632 40.6 <0.001 W T 3 1567 2.6 <0.047 3 1020 1.8 0.157 G T 3 424 0.7 0.543 3 16 3E 2 0.994 W GS(G) 1 527 0.9 0.368 2 80 0.1 0.872 WS(W) G 1 113 0.2 0.669 2 12 2E 2 0.986 WS(W) T 5 435 0.7 0.598 6 325 0.6 0.74 GS(G) T 5 324 0.5 0.735 6 32 5E 2 1 W G T 3 386 0.6 0.593 3 179 0.3 0.808 WS(W) GS(G) 4 182 0.3 0.867 4 758 1.4 0.283 W GS(G) T 5 318 0.5 0.766 6 660 1.2 0.345 WS(W) G T 5 114 0.2 0.967 6 353 0.6 0.715 WS(W) GS(G) T 10 575 1.0 0.455 12 318 0.6 0.864 Residual 202 596 292 558 A posteriori tests W G W G G(W); [WA]:GA > GR; [WR]:GA ¼ GR G(W); [WA]:GA > GR; [WR]:GA ¼ GR W(G); [GA]:WA¼WR; [GR]:WA < WR W(G); [GA]:WA > WR; [GR]:WA¼WR W T T(W); [WA]:(1 ¼ 2)>(3 ¼ 4) [WR]:1 ¼ 2 ¼ 3 ¼ 4 W(T); [1,2,3,4]: WA¼WR waters, gastropods from acidified sites may reduce exposure When exposed to waters of pH < 5 (trials 3 and 4 within the times to acidified waters by as much as 1/2, and those from Hunter), most gastropods remained inside their shells and with reference sites by over 1/3, as compared to gastropods remaining their opercula closed, and only one gastropod crawled above the on the ground. water line. Remaining within the shell minimizes contact of soft tissues with the highly acidic waters. Hence, the behavioural Hunter Gastropod treatment: acidified reference response of Bembicium auratum to acidification may be dependent 24 on the magnitude of the stressor, with the crawl-out response the 20 optimal solution at more moderate levels of acidification. Consis- 16 tent with this hypothesis, a freshwater gastropod, Physella gyrina, 12 displays crawl-out behaviour that varies according to the intensity of predator cues (Klose, 2011). Alternatively, severe acidification 8 may impede the locomotion of gastropods. Acidified water is 4 known to reduce the metabolic rates and even to cause narcotic/ 0 anaesthetic effects in many animal groups (Ellis and Morris, 1995; Port Stephens Portner et al., 2004; Hendriks et al., 2010). Several accounts of 24 extensive fish kills have been associated with ASS-affected waters 20 of pH < 5(Brown et al., 1983; Sammut et al., 1995), and degener- 16 ative lesion on the mantle and gill soft tissue of Saccostrea glom-

Number of gastropods erata oysters, associated with abnormal valve movements and 12 altered filtration rates have been reported after only 6h of exposure 8 to acid sulphate discharge of similar pH (Dove and Sammut, 2007a). 4 Behaviours that minimize exposure to acid sulphate discharge have previously been observed among both sessile and mobile 0 ASS REF ASS REF ASS REF ASS REF animals. Juvenile and adult oysters keep their shells closed to avoid 1 2 3 4 direct contact of soft tissues with affected waters (Dove and Water treatment / Trial Sammut, 2007a; Green and Barnes, 2010). When given a choice between control (pH 7.3e8.1) and acidified waters (pH 5.0e6.9), Fig. 1. Number (out of 24) of Bembicium auratum gastropods from acidified (white e fi bars) and reference (black bars) sites that moved within 30 min (solid bars) or crawled 80 100% of the individuals of four sh and one prawn species out within 60 min (crossed bars) of exposure to acidified (ASS) or reference (REF) moved away from acidic waters (Kroon, 2005). Our results clearly waters. Four replicate trials (numbered 1e4) were run using water and gastropods show that intertidal mangrove gastropods are able to reduce their from each of the Hunter and Port Stephens estuaries. Within each trial, gastropods and exposure to acidified water by actively crawling out, or by retract- test waters were sourced from two acidified and two reference sites of each estuary, but were pooled across sites within treatments of an estuary (acidified vs reference) ing into their shells when in extreme acidity, and that this response due to an absence of significant differences (at a ¼ 0.25) between these. is dependent on prior exposure to the stressor. 88 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90

Hunter Gastropod treatment: acidified reference Gastropod treatment: acidified reference 60 60 50 50 40 40 30 30 20 20 Time (min) 10 10 0 0 Port Stephens ASS REF ASS REF 60 Hunter Port Stephens Water treatment / Estuary 50 40 Fig. 4. Mean (þS.D.) amount of time taken for Bembicium auratum gastropods from acidified (white bars) and reference (black bars) sites to crawl-out of acidified (ASS) 30 and reference (REF) test waters. Data were pooled across replicate sampling sites Time of first movement (min) ¼ fi 20 (n 2) within gastropod and water treatments (acidi ed or reference), and across four experimental trials within an estuary, because they were statistically indistinguishable 10 (at a ¼ 0.25). 0 ASS REF ASS REF ASS REF ASS REF 1 2 3 4 increased mortality among Sydney rock oysters, particularly those Water treatment / Trial under 2 years of age (>85% mortality; Dove and Sammut, 2007b). We have recently shown that sustained exposure to acid sulphate þ Fig. 2. Mean ( S.D.) amount of time taken for Bembicium auratum gastropods from runoff reduces the abundance of gastropods and oysters and acidified (white bars) and reference (black bars) sites to start moving in acidified (ASS) and reference (REF) test waters. Four replicate trials (numbered 1e4) were run using weakens the strength of their exoskeletons (Amaral et al., 2011a, water and gastropods from each of the Hunter and Port Stephens estuaries. Within 2012a) to a lesser extent than predicted from experiments utiliz- each trial, gastropods and test waters were sourced from two acidified and two ing organisms naive to acidification (Bamber, 1987, 1990; Dove and reference sites of each estuary, but were pooled across sites within treatments of an Sammut, 2007a,b). The explanation may be that animals are able to estuary (acidified vs reference) due to an absence of significant differences (at modify their behaviour to minimize their exposure to this stressor. a ¼ 0.25) between these. Behavioural responses of organisms to stressors are likely to involve trade-offs (e.g. DeWitt et al., 1998). In the present study, Although this study did not follow the consequences of this gastropods that minimized contact with acidic waters by moving reduced exposure to acidification through to survival and fitness of above the water line generally spent w50% more of their time the gastropods, we have reason to believe that it may translate to moving than gastropods that did not exhibit this response. Hence, greater survivorship under these conditions. Prolonged exposure to the avoidance of acidified waters is likely to reduce the amount of acidification weakens mollusc shells, and this in turn renders them energy available for other activities. In previous studies, similar more susceptible to predation (Amaral et al., 2012a). Prolonged avoidance responses of gastropods to predators or predator cues exposure to acid sulphate acidification causes reduced growth and reduced the time and energy available for foraging, feeding, growth and reproduction (Richardson and Brown, 1992; Jones and Boulding, 1999; Dove and Sammut, 2007a; Dalesman et al., 2009). Hunter Gastropod treatment: acidified reference 100 Bembicium auratum are smaller at acidified than reference sites (Amaral et al., 2011a). Hence, the trade-off for behaviourally mini- 80 mizing contact with acidified waters might be a reduction in 60 growth rate. Gastropods experimentally exposed to CO2-enriched waters also show reduced growth rates, although the magnitude of 40 responses may not vary significantly between moderate and 20 extreme values of pCO2 (Hendriks et al., 2010). B. auratum gastropods from our acidified sites only displayed high activity and crawl- 0 out responses when exposed to reference waters, suggesting that Port Stephens 100 they may be able to determine when it will be beneficial to engage in energy-demanding behavioural responses. 80 Our study adds to growing evidence that aquarium studies that fi 60 assess biological impacts of acidi cation on naive organisms, from family lines never before exposed to this stressor, might over- 40 estimate impacts. Our study has demonstrated that the behavioural

Porportion of time spent moving (%) response of gastropods to acidification is dependent on previous 20 exposure to this stressor. Similarly, physiological adaptation of 0 oysters to acidification has been observed following multiple gen- ASS REF ASS REF ASS REF ASS REF 1 2 3 4 erations of exposure to this stressor (Parker et al., 2012). Our study Water treatment / Trial has also reinforced the importance of microhabitats in potentially providing refugia to organisms from the effects of ongoing envi- Fig. 3. Mean (þS.D.) percentage of time that Bembicium auratum gastropods from ronmental change. Future studies examining responses of aquatic acidified (white bars) and reference (black bars) sites spent moving when exposed to organisms to ocean acidification need to consider the potential for acidified (ASS) and reference (REF) test waters. Within each of the four trials adaptation of organisms across multiple generations, and that it is (numbered 1e4), gastropods and test waters were sourced from two acidified and two reference sites, but were pooled across sites within treatments (i.e. acidified vs refer- the nature of microhabitats, not average site conditions, which ence) due to an absence of significant differences (at a ¼ 0.25) between these. determine environmental exposure of organisms to stressors. V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90 89

Acknowledgements Hoffmann, A.A., Chown, S.L., Clusella-Trullas, S., 2012. Upper thermal limits in terrestrial ectotherms: how constrained are they? Funct. Ecol. http://dx.doi.org/ 10.1111/j.1365-2435.2012.02036.x. Published online: 16 JUL 2012. We are in debt to J. Rowland for help in the field. We acknowledge IPCC, 2007. Climate Change 2007: Synthesis Report. An Assessment of the Inter- funding from Fundação para a Ciência e a Tecnologia, Portugal governmental Panel on Climate Change (IPCC). Geneva, Switzerland. Available ¼ ¼ ¼ ¼ ¼ ¼ (project PEst-OE/MAR/UI0199/2011, and postdoctoral grant SFRH/ at: http://www.google.pt/url?sa t&rct j&q &esrc s&source web&cd 3&ved¼0CEgQFjAC&url¼http%3A%2F%2Fwww.ipcc.ch%2Fpdf%2Fassessment- BPD/44566/2008 to V. Amaral). A NSW Environmental Trust seeding report%2Far4%2Fsyr%2Far4_syr.pdf&ei¼54bSUc8pi5DsBqXAgIgL&usg¼ grant (to M. J. Bishop) helped support the cost of fieldwork. We thank AFQjCNEbpKeh3EYdLi3VcEy972ibw7jb1Q&bvm¼bv.48572450,d.ZGU&cad¼rja. A. Underwood, I. Valiela and one anonymous reviewer for comments Jacobsen, H.P., Stabell, O.B., 1999. Predator-induced alarm responses in the common periwinkle, Littorina littorea: dependence on season, light conditions, and that improved the quality of this manuscript. All work complied with chemical labelling of predators. Mar. Biol. 134, 551e557. New South Wales and Australian government laws. Jones, K.M.M., Boulding, E.G., 1999. State-dependent habitat selection by an intertidal snail: the costs of selecting a physically stressful microhabitat. J. Exp. Mar. Biol. Ecol. 242, 149e177. Klose, K., 2011. Snail responses to cues produced by an invasive decapod predator. References Invertebr. Biol. 130, 226e235. Kroon, F.J., 2005. Behavioural avoidance of acidified water by juveniles of four Amaral, V., Cabral, H.N., Bishop, M.J., 2011a. Resistance among wild invertebrate commercial fish and prawn species with migratory life stages. Mar. Ecol. Prog. populations to recurrent estuarine acidification. Estuar. Coast. Shelf Sci. 93, Ser. 285, 193e204. 460e467. Lavergne, S., Evans, M.E.K., Burfield, I.J., Jiguet, F., Thuiller, W., 2013. Are species’ Amaral, V., Cabral, H.N., Bishop, M.J., 2011b. Effect of runoff from acid-sulfate soils responses to global change predicted by past niche evolution? Phil. Trans. R. on pneumatophores of the grey mangrove, Avicennia marina. Mar. Freshw. Res. Soc. B Biol. Sci. 368. 62, 974e979. Marshall, D.J., Santos, J.H., Leung, K.M.Y., Chak, W.H., 2008. Correlations between Amaral, V., Cabral, H.N., Bishop, M.J., 2012a. Effects of estuarine acidification on gastropod shell dissolution and water chemical properties in a tropical estuary. predator-prey interactions. Mar. Ecol. Prog. Ser. 445, 117e127. Mar. Environ. Res. 66, 422e429. Amaral, V., Cabral, H.N., Bishop, M.J., 2012b. Moderate acidification affects growth Melatunan, S., Calosi, P., Rundle, S.D., Widdicombe, S., Moody, A.J., 2013. Effects of but not survival of 6-month-old oysters. Aqua. Ecol. 46, 119e127. ocean acidification and elevated temperature on shell plasticity and its ener- Anderson, M.J., 2001a. A new method for non-parametric multivariate analysis of getic basis in an intertidal gastropod. Mar. Ecol. Prog. Ser. 472, 155e168. variance. Aust. Ecol. 26, 32e46. Munday, P.L., Dixson, D.L., Donelson, J.M., Jones, G.P., Pratchett, M.S., Devitsina, G.V., Anderson, M.J., 2001b. Permutation tests for univariate or multivariate analysis of Doving, K.B., 2009. Ocean acidification impairs olfactory discrimination and variance and regression. Can. J. Fish. Aquat. Sci. 58, 626e639. homing ability of a marine fish. Proc. Natl. Acad. Sci. U S A. 106, 1848e1852. Bamber, R.N., 1987. The effects of acidic sea water on young carpet-shell clams Ven- Naylor, S.D., Chapman, G.A., Atkinson, G., Murphy, C.L., Tulau, M.J., Flewin, T.C., erupis decussata (L) (Mollusca, Veneracea). J. Exp. Mar. Biol. Ecol. 108, 241e260. Milford, H.B., Morand, D.T., 1998. Guidelines for the Use of Acid Sulfate Soils Risk Bamber, R.N., 1990. The effects of acidic seawater on three species of lamellibranch Maps, second ed. NSW Department of Land and Water Conservation, Sydney. mollusk. J. Exp. Mar. Biol. Ecol. 143, 181e191. Available at: http://www.environment.nsw.gov.au/resources/acidsulfatesoil/ Barbier, E.B., Hacker, S.D., Kennedy, C., Koch, E.W., Stier, A.C., Silliman, B.R., 2011. The assmapsguide.pdf. (accessed 24.01.13.). value of estuarine and coastal ecosystem services. Ecol. Monogr. 81, 169e193. NSW DECCW, 2012. Acid Sulfate Soil Risk Maps. NSW Department of Environment, Bibby, R., Cleall-Harding, P., Rundle, S., Widdicombe, S., Spicer, J., 2007. Ocean Climate Change and Water. http://www.environment.nsw.gov.au/ acidification disrupts induced defences in the intertidal gastropod Littorina acidsulfatesoil/riskmaps.htm (accessed 01.04.13.). littorea. Biol. Lett. 3, 699e701. NSW DPI, 2006. Assay: a Newsletter about Acid Sulfate Soils. No. 40 Oct 2006. NSW Branch, G.M., Branch, M.L., 1980. Competition in Bembicium auratum (Gastropoda) Department of Primary Industries, Wollongbar. Available at: http://www.dpi. and its effect on microalgal standing stock in mangrove muds. Oecologia 46, nsw.gov.au/__data/assets/pdf_file/0005/168557/assay-40.pdf (accessed 106e114. 01.04.13.). Brown, T.E., Morley, A.W., Sanderson, N.T., Tait, R.D., 1983. Report of a large fish kill NSW DPI, 2008. Acid Sulfate Soils Priority Investigations for the Lower Hunter River resulting from natural acid water conditions in Australia. J. Fish. Biol. 22, 335e Estuary. Report to the Department of Environment, Water, Heritage and the 350. Arts. NSW Department of Primary Industries (Aquatic Habitat Rehabilitation), Caldeira, K., Wickett, M.E., 2005. Ocean model predictions of chemistry changes Port Stephens. Available at: http://www.dpi.nsw.gov.au/fisheries/habitat/ from carbon dioxide emissions to the atmosphere and ocean. J. Geophys. Res.- publications/threats/acid-sulphate-soils (accessed 01.04.13.). Oceans 110, C09S04. NSW DPI, 2009. Tilligerry Creek. Floodgate Assessment. Report to the Hunter- Charmantier, A., McCleery, R.H., Cole, L.R., Perrins, C., Kruuk, L.E.B., Sheldon, B.C., Central Rivers, Catchment Management Authority, Project number HCR07_ 2008. Adaptive phenotypic plasticity in response to climate change in a wild 106. NSW Department of Primary Industries, Orange. Available at: http://www. bird population. Science 320, 800e803. dpi.nsw.gov.au/__data/assets/pdf_file/0005/280922/Tilligerry-creek-floodgate- Crowe, T., 1996. Different effects of microhabitat fragmentation on patterns of assessment-report.pdf (accessed 24.01.13.). dispersal of an intertidal gastropod in two habitats. J. Exp. Mar. Biol. Ecol. 206, Parker, L.M., Ross, P.M., O’Connor, W.A., Borysko, L., Raftos, D.A., Portner, H.O., 2012. 83e107. Adult exposure influences offspring response to ocean acidification in oysters. Crowe, T.P., Underwood, A.J., 1999. Differences in dispersal of an intertidal Glob. Change Biol. 18, 82e92. gastropod in two habitats: the need for and design of repeated experimental Portner, H.O., Langenbuch, M., Reipschlager, A., 2004. Biological impact of elevated transplantation. J. Exp. Mar. Biol. Ecol. 237, 31e60. ocean CO2 concentrations: lessons from animal physiology and earth history. Dalesman, S., Rundle, S.D., Cotton, P.A., 2009. Crawl-out behaviour in response to J. Oceanogr. 60, 705e718. predation cues in an aquatic gastropod: insights from artificial selection. Evol. Reid, D.G., 1988. The genera Bembicium and Risellopsis (Gastropoda: Littorinidae) in Ecol. 23, 907e918. Australia and New Zealand. Rec. Aust. Mus. 40, 91e150. Dent, D.L., Pons, L.J., 1995. A world perspective on acid sulfate soils. Geoderma 67, Richardson, A.J., Poloczanska, E.S., 2008. Under-resourced, under threat. Science 263e276. 320, 1294e1295. DeWitt, T.J., Sih, A., Wilson, D.S., 1998. Costs and limits of phenotypic plasticity. Richardson, T.D., Brown, K.M., 1992. Predation risk and feeding in an intertidal Trends Ecol. Evol. 13, 77e81. predatory snail. J. Exp. Mar. Biol. Ecol. 163, 169e182. Dove, M.C., Sammut, J., 2007a. Histological and feeding response of Sydney rock Ries, J.B., Cohen, A.L., McCorkle, D.C., 2009. Marine calcifiers exhibit mixed re- oysters, Saccostrea glomerata, to acid sulfate soil outflows. J. Shellfish Res. 26, sponses to CO2-induced ocean acidification. Geology 37, 1131e1134. 509e518. Rodolfo-Metalpa, R., Houlbreque, F., Tambutte, E., Boisson, F., Baggini, C., Patti, F.P., Dove, M.C., Sammut, J., 2007b. Impacts of estuarine acidification on survival and Jeffree, R., Fine, M., Foggo, A., Gattuso, J.P., Hall-Spencer, J.M., 2011. Coral and growth of Sydney rock oysters Saccostrea Glomerata (Gould 1850). J. Shellfish mollusc resistance to ocean acidification adversely affected by warming. Nat. Res. 26, 519e527. Clim. Change 1, 308e312. Ellis, B.A., Morris, S., 1995. Effects of extreme pH on the physiology of the Australian Sammut, J., Melville, M.D., Callinan, R.B., Fraser, G.C., 1995. Estuarine acidification: ‘yabby’ Cherax destructor: acute and chronic changes in haemolymph carbon impacts on aquatic biota of draining acid sulphate soils. Aus. Geograph. Stud. dioxide, acid-base and ionic status. J. Exp. Biol. 198, 409e418. 33, 89e100. Fabry, V.J., Seibel, B.A., Feely, R.A., Orr, J.C., 2008. Impacts of ocean acidification on Sammut, J., White, I., Melville, M.D., 1996. Acidification of an estuarine tributary marine fauna and ecosystem processes. Ices J. Mar. Sci. 65, 414e432. in eastern Australia due to drainage of acid sulfate soils. Mar. Freshw. Res. 47, Green, T.J., Barnes, A.C., 2010. Reduced salinity, but not estuarine acidification, is a 669e684. cause of immune-suppression in the Sydney rock oyster Saccostrea glomerata. Stenzel, H.B., 1964. Oysters: composition of larval shell. Science 145, 155e156. Mar. Ecol. Prog. Ser. 402, 161e170. Stern, D.L., Orgogozo, V., 2009. Is genetic evolution predictable? Science 323, Guinotte, J.M., Fabry, V.J., 2008. Ocean acidification and its potential effects on 746e751. marine ecosystems. In: Ostfeld, R.S., Schlesinger, W.H. (Eds.), Year in Ecology Taylor, J.D., Reid, D.G., 1990. Shell microstructure and mineralogy of the Littorinidae: and Conservation Biology 2008. Wiley-Blackwell, Malden, pp. 320e342. ecological and evolutionary significance. Hydrobiologia 193, 199e215. Hendriks, I.E., Duarte, C.M., Alvarez, M., 2010. Vulnerability of marine biodiversity to Trussell, G.C., 1996. Phenotypic plasticity in an intertidal snail: the role of a common ocean acidification: a meta-analysis. Estuar. Coast. Shelf Sci. 86, 157e164. crab predator. Evolution 50, 448e454. 90 V. Amaral et al. / Estuarine, Coastal and Shelf Science 136 (2014) 82e90

Tuomainen, U., Candolin, U., 2011. Behavioural responses to human-induced envi- mangrove swamp in New South Wales, Australia. J. Exp. Mar. Biol. Ecol. 137, ronmental change. Biolog. Rev. 86, 640e657. 25e45. Underwood, A.J., 1997. Experiments in Ecology: Their Logical Design and Interpre- Worm, B., Barbier, E.B., Beaumont, N., Duffy, J.E., Folke, C., Halpern, B.S., tation Using Analysis of Variance. Cambridge University Press, Cambridge, Jackson, J.B.C., Lotze, H.K., Micheli, F., Palumbi, S.R., Sala, E., Selkoe, K.A., p. 504. Stachowicz, J.J., Watson, R., 2006. Impacts of biodiversity loss on ocean Underwood, A.J., Barrett, G., 1990. Experiments on the inﬂuence of oysters on the ecosystem services. Science 314, 787e790. distribution, abundance and sizes of the gastropod Bembicium auratum in a