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 limpet Fissurella crassa (Mollusca: 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 habitats 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 (algae abundance) 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 limpets 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 animals 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 animal life distribution limits of species 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 habitat 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 climate change 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 1595 1596 jose’ pulgar et al. 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 phytoplankton (Dortch et al., 1983) and zooplankton 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 cell components. Addition of chloroform followed by centrifugation separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the