Journal of the Marine Biological Association of the United Kingdom, 2012, 92(7), 1595–1601. # Marine Biological Association of the United Kingdom, 2012 doi:10.1017/S0025315412000173 Impact of wave exposure on seasonal morphological and reproductive responses of the intertidal crassa (: Archaegastropoda) jose’ pulgar1, marcos alvarez5, alejandro delgadillo1,4, ines herrera1, samanta benitez1,2, juan pablo morales5, pilar molina3, marcela aldana1,3,6 and victor manuel pulgar7 1Universidad Andres Bello, Departamento de Ecologı´a & Biodiversidad, Repu´blica 470, Santiago Chile, 2Universidad Andres Bello Escuela de Biologı´a Marina, Repu´blica 440, Santiago, Chile, 3Pontificia Universidad Cato´lica de Chile, Alameda 370, Santiago, Chile, 4Escuela de Ingenierı´a en Acuicultura, Universidad Andres Bello, Repu´blica 440, Santiago, Chile, 5Universidad Andres Bello, Facultad de Ciencias Biolo´gicas, Repu´blica 217, Santiago, Chile, 6Escuela de Pedagogı´a en Biologı´a y Ciencias, Facultad de Ciencias de la Educacio´n, Universidad Central de Chile, Santa Isabel 1278, Santiago, 7Center for Research in Obstetrics & Gynecology, Wake Forest School of Medicine and Biomedical Research Infrastructure Center, Winston-Salem State University, Winston-Salem NC, USA

Intertidal organisms have long been considered an ideal system to quantify how physical variations determine differential energy allocations in specimens inhabiting environmental gradients such as exposure to wave action. In with differ- ential intertidal wave exposure (sheltered, Sh; and exposed, E) seasonal gonadal and foot weight variations and their associ- ations with exposure and food availability ( ) were determined in the keyhole limpet Fissurella crassa. Gonadal weight is used as a measure of reproduction allocation whereas foot weight is an indirect indicator of energy allo- cation to survival. RNA:DNA ratio in obtained from Sh and E habitats during the two different seasons was used as an indicator of biosynthetic capability. Our results indicate that algae abundance in E sites was higher in summer and lower in winter compared to Sh sites. In E sites the muscular foot weight of limpet was higher in summer in contrast to Sh sites where F. crassa muscular foot weight of limpet was higher in winter. Gonadal weight in Sh sites was higher in summer and remained constant in winter; whereas in E sites gonadal weight was lower in summer and higher in winter. RNA:DNA ratios indicate that regardless of intertidal wave exposure, F. crassa showed higher biosynthetic capability in summer. Energetic allocation in that inhabit sheltered intertidal habitats would support constant allocation towards reproduction. In contrast, animals that inhabit exposed habitats may favour seasonally reproduction allocation at expense of survival.

Keywords: shell morphology, RNA:DNA ratio, energetic trade-off

Submitted 17 January 2012; accepted 25 January 2012; first published online 28 March 2012

INTRODUCTION Wagner et al., 1998; Dahlhoff, 2004; Pulgar et al., 2011). Physiological constraints are important determinants of the Diversity and variability are key characteristics of life distribution limits of and populations (Gaston & (Spicer & Gaston, 1999). Environmental factors influence an Spicer, 1998; Chown & Gaston, 2000); however, processes animal’s condition at several levels of biological organization, associated with environmental tolerance explaining for including organismal (e.g. feeding rate and metabolic rate: example differential use at the local scale, or species Sanford, 2002) subcellular levels (e.g. protein synthesis and distribution patterns at the geographical scale, remain gene expression: Somero, 2002). To understand the effects poorly understood. of on biological phenomena throughout the Rocky intertidal habitats experience a wide range of phys- biosphere (Hofmann, 2005) is important to evaluate the ical conditions, with daily and seasonal variability including: organism’s responses to environmental variations. As a degree of immersion; isolation; nutrient availability; and result, there has been increasing interest in determining the exposure to different levels of wave action (Newell, 1970; variability in physiological condition and life-history traits Truchot & Duhanel-Jouve, 1980). Organisms that inhabit of organisms in their natural habitats (Colman, 1933; intertidal rocky shores are strongly influenced by a vertical tidal emersion (Denny, 1988; Helmuth & Hofmann, 2001;

Corresponding author: Somero, 2002) and a horizontal wave exposure gradient J. Pulgar (Jones & Demetropoulos, 1968; Dahlhoff et al., 2002). In inter- Email: [email protected] tidal organisms, biochemical and physiological processes and

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ultimately organismal performance, are modified in response considered an estimation of wave action intensity in each to environmental conditions (Stickle & Bayne, 1987; Pulgar site (Guin˜ez & Pacheco, 1999). et al., 2011). For instance wave action exerts great forces on Individuals of F. crassa were sampled from exposed sessile or less mobile organisms, with the risk of dislodgement (summer E-S, N ¼ 22, winter E-W, N ¼ 22), sheltered being an important selective pressure (e.g. Carrington, 1990; (summer Sh-S, N ¼ 22, winter Sh-W, N ¼ 22) sectors Gaylord et al., 1994; Denny, 1999). Under these conditions during 2008 and winter 2009. Limpets sampled at both animals face an energetic trade-off between survival (ability seasons from each sector were deposited in labelled plastic to attach to the substrate) and reproduction (reproductive bags and then transported to the laboratory. Limpet foot tissue) (Brown & Quinn, 1988; Sibly, 1991). In intertidal mol- weight (g), gonad weight (g), and individual total weight (g) luscs, evidences also indicate that morphological features such were measured using an analytic balance (+/– 0.01 g pre- as shell morphology and body size are associated with differ- cision). We considered gonadal weight as an indirect estima- ences in wave energy (Etter, 1988; Trussell et al., 1993). tor of reproductive tissue investment and foot weight as a Among the biochemical indicators used to determine direct estimator of substrate attaching capability of F. crassa. physiological condition and metabolic activity in situ the Total limpet shell length (cm), shell width (cm) and shell RNA:DNA ratio is widely used as an index to determine con- height (cm) were measured using a digital caliper dition of organisms in the field (Chı´charo & Chı´charo, 2008). (Mitutoyo) (+/– 0.01mm) and analytic balance (0.01 g). This index measures the protein biosynthetic capacity and is Limpet sexual condition was determined by direct observation usually correlated with the nutritional status under a given of gonad colour; male gonads are yellow and female gonads set of environmental conditions (Buckley & Caldarone, are green (Olivares et al., 2009). 1999). Organisms in good condition, therefore tend to have higher RNA:DNA ratios. This index has been used on a F. crassa food availability wide range of marine organisms such as those constituting the (Dortch et al., 1983) and Food availability for F. crassa in low intertidal of both E and (Ikeda et al., 2007), larval fish (Caldarone et al., 2003), juvenile Sh study sectors, was evaluated using 100-m long transects and adult fish (Thorpe et al., 1982), bivalves (Chı´charo et al., parallel to the coast. For each season and degree of exposure 2001), crustaceans (Lemos et al., 2002) and intertidal fish considered, 50 × 50 cm quadrats randomly chosen were sur- (Pulgar et al., 2011). veyed (E-S, N ¼ 12 quadrat; E-W, N ¼ 15, Sh-S, N ¼ 13; and To address physiological responses to wave exposure in a Sh-W, N ¼ 13). In each quadrat, macroalgae cover (as percen- sessile organism we studied morphometric, reproductive and tage of total) of Rodophyta (Mazzaella spp.) and Chlorophyta in situ physiological variables in the intertidal limpet (Ulva spp.) were considered as indirect estimator of food Fissurella crassa (Lamarck, 1882) sampled at exposed and F. crassa availability. sheltered intertidal sites. Fissurella crassa is distributed from Peru to Chile (McLean, 1984; Oliva & Castilla, 1992), inhab- Molecular analyses iting sheltered and exposed intertidal zones (Pino et al., 1994). These limpets are dioecious without external sexual For molecular analyses, limpets were collected both in winter dimorphism, bearing a single gonad. Individuals may release (E sectors N ¼ 8 limpets, Sh, N ¼ 9) and summer (E, N ¼ 10 gametes during two major spawning events (Bretos et al., and Sh, N ¼ 10). The extraction of RNA and DNA was per- 1983, 1988; Bretos & Chihuailaf, 1993). Fissurella crassa formed using TRIZOLw Reagent for the isolation of total shows seasonal differences in growth rate: in late summer RNA from cells and tissues (Chomczynski & Sacchi, 1987). and autumn, there is an accelerated growth, which declines We extracted 200 mg of foot tissue from each individual. again in late autumn and winter. This decrease in growth During the homogenization of the sample previously rate may coincide with the spawning period (Mclean, 1984; extracted, TRIZOLw Reagent maintains the integrity of the Oliva & Castilla, 1986). RNA, while disrupting cells and dissolving components. Addition of chloroform followed by centrifugation separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase. After transfer MATERIALS AND METHODS of the aqueous phase, the RNA is recovered by precipitation with isopropyl alcohol. After removal of the aqueous phase, Quantification of wave action and collection the DNA in the supernatant can be recovered by sequential of F. crassa specimens precipitation (Chomczynski, 1993). After extracting, the RNA and DNA were reconstituted in 50 and 900 mlof Wave action was measured in winter 2009 in intertidal shel- nuclease-free water respectively. Both RNA and DNA were tered (Sh) and exposed (E) sectors in central Chile (Quintay quantified spectrophotometrically to 260/280 nm (Perkin (33811′S71841′W)), using the methodology described by Elmer Lambda Bio L7110184) and expressed as mg/ml, cor- Doty (1971) and Gerard & Mann (1979). This method rected for body and sample size. involves the measurement of the rate at which plaster shapes dissolve assuming their diffusion rate is proportional Statistical analyses to the mass flow of water in motion (Guin˜ez & Pacheco, 1999). For these measurements ten wear units were used; One-way analysis of variance (ANOVA) (general linear these units were dried to constant weight, and during a com- models) was used to compare loss of weight of waste units plete tidal cycle the units were removed and dried at 408C among sheltered and exposed sectors. Two-way ANOVA until achieving constant weight. The decrease in weight of (general linear models) was used to compare seasonal vari- the units (measured as the difference in weight, D)is ations of the morphological and reproductive characteristics morphological and reproductiveresponseoff. crassa 1597 of F. crassa, and algae richness and abundance between exposed and sheltered intertidal sampled sites. Season and wave exposure represent extreme intertidal ecological con- ditions and were considered fixed factors. The Tukey a-posteriori test was used to assess specific differences between factor levels. Residual analysis was used to evaluate the effect of season and wave exposure, on foot and gonad weight; results were expressed with respect to total individual weight. Residual analysis was also used to compare RNA:DNA ratios between exposed limpets and sheltered shore limpets, in relation to Fig. 1. Habitat variability: wave action and food availability. (A) Wave energy fresh body limpet weight. A significance level of P , 0.05 exposition measured as the decrease in weight of wear units (D) in sheltered (Sh) and exposed (E) sectors: (B) seasonal algae cover (%) in Sh and E was selected for rejection of a null hypothesis of no significant ∗ sampled sites. Vertical bars indicate + 1 SEM, P , 0.05. differences (Zar, 1996).

Limpet morphology: soft structures RESULTS Limpets from the Sh sector showed greater gonadal weight in summer than in winter. In contrast, individuals from the E Data describing limpet morphological variables such as sector showed higher gonadal weight in winter (Figure 3A; gonadal weight, foot weight and limpet size, are indicated in Table 5: Tukey a-posteriori test P , 0.05). Residual of foot Table 1. weight of F. crassa was higher in winter in the Sh sector whereas in animals from the E sector the foot weight was higher in summer (Figure 3B; Table 4: P , 0.05). Habitat variability: wave action and food availability Molecular analysis Wear units showed a greater weight loss in E than Sh sectors Residual of the relationship between RNA:DNA ratio to foot (Figure 1A; Table 2), indicating that the former sectors are and gonadal weight indicates that regardless of exposure, subjected to greater wave energy. Algae cover was significantly animals present higher RNA:DNA ratios in summer com- greater in winter in Sh, and in summer in E sites (Figure 1B; pared to winter (Figure 4; Table 6). Table 3), indicating differences in the amount of food avail- ability to F. crassa depending on the season and wave exposure. DISCUSSION

Our results indicate that, in the intertidal limpets F. crassa, traits associated with survival and reproduction, as well as bio- Limpet morphology: shell structures synthetic capabilities present environmental and seasonal Analyses of shell morphometric characteristics of F. crassa variation. Whereas hard structures were thinner in winter, indicate that independent of wave exposure length, width soft structures showed both exposition and season-related and height shell s were thinner in winter than summer variations. Gonadal tissue weight was similar between (Figure 2; Table 4). seasons and foot weight was greater in winter in sheltered sectors. In the exposed sectors, gonadal weight was higher in winter and foot weight higher in summer. At the molecular level, a greater RNA:DNA ratio was observed in summer Table 1. Basic morphological description of keyhole limpet in sampled regardless of intertidal exposure. sectors (sheltered and exposed) during both seasons studied (summer, s; The balance between energy acquisition and expenditure is winter, w). Results are expressed as mean + 1 SEM. E/s, exposed/ critical for animal survival and reproductive success (Sibly, summer; E/w, exposed/winter; Sh/s, sheltered/summer; Sh/w, sheltered/ winter. 1991). This balance depends on the interplay between food intake, digestion, and the allocation of energy to various func- Sector/season Mean SEM tions such as growth and reproduction (Karasov, 1986; Wiener, 1992). In animals inhabiting an environment with high phys- Gonadal weight (g) E/s 4.30 0.62 E/w 9.10 0.91 ical variability, such as the intertidal system, traits related to Sh/s 7.60 0.76 energy allocation dealing with survival (foot development), Sh/w 9.2 0.89 Foot weight (g) E/s 21.82 1.6 E/w 21.26 2.50 Table 2. General linear model (analysis of variance) results comparing Sh/s 18.94 1.96 the decrease in mean mass of the waste units from sheltered and Sh/w 26.40 2.32 exposed zones. df, degrees of freedom; MS, mean square; F, F value; Limpet size (cm) E/s 6.5 1.8 P, probability value. E/w 1.9 2.8 Effect df MS F P Sh/s 6.2 2.3 Sh/w 6.5 2.7 Wave exposition 1 47.95 14.22 0.0011 1598 jose’ pulgar et al.

Table 3. General linear model (analysis of variance) results comparing Table 4. General linear model (analysis of variance) results comparing algae abundance of sheltered and exposed zones during summer and Fissurella crassa shell length (cm), shell width (cm), shell height (cm) of winter. df, degrees of freedom; MS, mean square; F, F value; P, probability sheltered and exposed zones during summer and winter. df, degrees of value. freedom; MS, mean square; F, F value; P, probability value.

Effect df MS F P Effect df MS F P

Wave exposure (WE) 1 1598.90 2.72 0.10 Shell length Season (S) 1 1109.90 1.89 0.17 Wave exposure (WE) 1 67.1 0.49 0.48 WE∗S 1 7305.7 12.48 0.00004 Season (S) 1 60720 443.84 0.0001 Error 194 WE∗S 1 24.2 0.17 0.67 Error 84 136.8 Shell width and reproduction (reproductive tissue), are under strong selec- Wave exposure (WE) 1 14.34 0.28 0.59 tive pressure (Sibly & Calow, 1986; Stearns, 1992; Weiner, Season (S) 1 19065.93 377.78 0.0002 WE∗S 1 8.17 0.16 0.68 1992). Intertidal invertebrates inhabit a wide range of variation in a number of physical–chemical variables (Moore & Seed, Error 84 50.47 1986; Raffaelli & Hawkins, 1996). In this heterogeneous Shell height habitat (e.g. substrate characteristics and physical–chemical Wave exposure (WE) 1 0.14 0.018 0.89 Season (S) 1 2564.89 323.55 0.0003 variables), wave action can affect energy intake, the time for ∗ WE S 1 2.76 0.34 0.55 or success in feeding, and some predator species may benefit Error 84 7.29 from wave prey dislodgement (Sebens, 2002). We characterized two sectors as sheltered and exposed depending upon the energy of the wave action observed (Figure 1A). Our results indicated seasonal variability in algae abundance in these mobility and attachment to the substrate, could be associated two sites, with the sheltered sector displaying higher abun- with individual survival (Serra et al., 2001). Our results indi- dance in winter and exposed sites displaying higher algae cate that gonadal weight as well as foot weight development abundance in summer (Figure 1B). To understand how the showed a seasonal response to environmental variability variable environment in these two sites may affect energy allo- (Figure 3). Limpet gonadal weight in sheltered habitats did cations, we determined a number of presumably related mor- not show seasonal changes whereas in exposed habitats phological parameters in F. crassa. We observed thinner gonadal tissue weight increased from summer to winter keyhole shell in winter (Figure 2), probably associated with (Figure 3A). On the other hand, foot weight in sheltered habi- wave action, which reportedly affects animal shell mineral tats increased from summer to winter, whereas in the exposed deposition (Moore & Seed, 1986; Raffaelli & Hawkins, 1996). sector foot weight decreased from summer to winter The effect of wave exposure has been described as a modulator (Figure 3B). The increased foot weight in winter would of body shape, corporal position, movements and thread pro- enable individuals to remain associated with substrate and duction in intertidal organisms (Denny & Blanchette, 2000; the decrease in foot weight in exposed sites would evidence Astorga et al., 2002; Moeser et al., 2006). The foot weight the costs of inhabiting a physically stressful habitat in com- and F. crassa shell variability found in Sh animals and E sites parison to animals from the most benign sheltered habitat. may be interpreted as wave exposure action on our sampled We interpret the opposite variation of gonadal weight and population (Figure 2). foot tissue weight, i.e. increase in winter in exposed sites The most important energetic trade-off reported occurs and in summer in sheltered sites (Figure 3), as a strategy to between reproduction and growth energy allocation, where differentially allocate energy resources associated with differ- compensatory responses to habitat variability reveal the ent intertidal wave exposure. The change in allocated energy action of natural selection on reproduction and survival to growth or reproduction processes is the principal trade-off (Warner, 1984; Spicer & Gaston, 1999; Zera & Harshman, described for animals that inhabit environmental gradients 2001). In limpets, gonadal weight is associated with reproduc- (Chown & Gaston, 1999; Smith et al., 2008). tive potential whereas muscular foot weight, responsible for Table 5. General linear model (analysis of variance) results comparing Fissurella crassa gonadal and foot weight of sheltered and exposed zones during summer and winter. df, degrees of freedom; MS, mean square; F, F value; P, probability value.

Effect df MS F P

Gonadal weight Wave exposure (WE) 1 4.37 0.79 0.37 Season (S) 1 172.34 31.39 0.0001 WE∗S 1 108.35 19.74 0.0002 Error 84 5.48 Foot weight Wave exposure (WE) 1 0.43 0.08 0,44 Season (S) 1 29.60 5.83 0.002 WE∗S 1 157.02 30.94 0.0001 Fig. 2. Limpet morphology: hard structures. Shell morphometric variability in Error 84 5.07 summer and winter. Vertical bars indicate + 1 SEM, ∗P , 0.05. morphological and reproductiveresponseoff. crassa 1599

Table 6. General linear model (analysis of variance) results comparing Fissurella crassa RNA:DNA ratio from sheltered and exposed zones during summer and winter. df, degrees of freedom; MS, mean square; F, F value; P, probability value.

Effect df MS F P

Wave exposure (WE) 1 84.137 0.41 0.52 Season (S) 1 4695.73 22.96 0.00001 WE∗S 1 399.46 1.65 0.20 Error 41

among competing energy functions (Stearns, 1989; Ricklefs & Wikelski, 2002). Energetic allocation for the herbivorous F. crassa, is depen- dent on season, intertidal wave exposure and food availability. The greater food availability in sheltered sites in winter as well as the greater food availability in exposed sites in summer is associated with increased limpet foot weight in both habitats indicating that the energy budget is mostly allocated to Fig. 3. Limpet morphology: soft structures. Seasonal Fissurella crassa gonadal enhance survival (Figures 1 & 3). (A) and foot (B) weight residual in sheltered (Sh) and exposed (E) sectors. Understanding the mechanisms by which environmental Vertical bars indicate + 1 SEM. variability modifies physiological performance of organisms in nature is of great interest when considering the foundations of dynamics (Parmesan & Yohe, 2003; Dahlhoff, Environmental variability associated with the sheltered 2004). The RNA:DNA ratio is considered as an in situ indi- habitat is related to foot weight increase from summer to cator of the physiological status, because of its association winter, without affecting reproduction investment with the nutritional condition and growth in several marine (Figure 3B). However, in exposed habitats energetic restric- organisms (Buckley & Caldarone, 1999; Chı´charo & tions are evident, and thus drastic decline in foot weight Chı´charo, 2008). We observed a higher RNA:DNA ratio in from summer to winter is necessary to offset the summer to summer animals, regardless of their intertidal location winter increase in gonadal tissue weight (Figure 3A). (Figure 4); in this context summer limpets would be increas- Our results indicate that in sheltered habitats, limpets may ingly well-off nutritionally (Palumbi, 2003). have low maintenance costs compared with limpets inhabiting A higher RNA:DNA ratio in F. crassa in summer suggests exposed sites, and this fact would allow the former to experi- greater protein synthetic activity producing greater foot ence an increase in foot weight during winter with no effects weight gain in exposed sites in summer, and greater gonadal on reproduction. In opposition, animals from stressful habi- weight gain in sheltered habitats. At the molecular level tats may show physiological compensation (Hernandez our evidences indicate a dynamic seasonal change in the et al., 2002; Tomanek & Helmuth, 2002), that would result F. crassa biosynthetic capability (Figure 4), and no effect of in allocation of resources in limpets to reproduction at the intertidal exposure. A higher summer limpet RNA:DNA expense of survival (Stearns, 1992; Roff, 2002). Our evidences ratio may be associated with an accelerated growth indicate that animals in exposed habitats reallocate energy (McLean, 1984; Oliva & Castilla, 1986) and greater gonadal tissue weight in summer than in winter (Bretos et al., 1988); this evidence may help to understand dynamic reproductive cycles in an important intertidal . To our knowledge this represents the first evidence of adjustments in the rates of energy acquisition and/or energy expenditure in the commer- cially important herbivorous mollusc F. crassa. This species is also an important component in the control of the algal com- munity (Aguilera, 2011). These allocations are thought to be ultimately responsible for positive energy budgets in animals (Hammond & Wunder, 1991; Piersma & Lindstrom, 1997) associated with organism ‘decisions’ regarding energy allo- cation into maintenance, growth and reproduction (Wiener, 1992).

ACKNOWLEDGEMENTS

Fig. 4. Molecular analysis: RNA:DNA ratio. Seasonal RNA:DNA ratio in This study was funded by grants DI0508 and DI 17-10/R and limpets from sheltered (Sh) and exposed (E) sectors. Vertical bars DI-16-12/R to J.P., Universidad Andres Bello. We thank the indicate + 1 SEM. Molecular Biology Laboratory staff of Universidad Andres 1600 jose’ pulgar et al.

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Departamento de Ecologı´a & Biodiversidad Serra G., Chelazzi G. and Castilla J.C. (2001) Temporal and spatial Repu´blica 470, Santiago Chile activity of the key-hole limpet Fissurella crassa (Mollusca: email: [email protected]