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The effect of salinity on clearance rate in the suspension-feeding estuarine gastropod Crepipatella dilatata under natural and controlled conditions
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Estuarine, Coastal and Shelf Science 76 (2008) 861e868 www.elsevier.com/locate/ecss
The effect of salinity on clearance rate in the suspension-feeding estuarine gastropod Crepipatella dilatata under natural and controlled conditions O.R. Chaparro a,*, Y.A. Montiel a, C.J. Segura a, V.M. Cubillos a, R.J. Thompson b, J.M. Navarro a a
Instituto de Biologı´a Marina ‘‘Dr. Ju¨rgen Winter’’, Universidad Austral de Chile, Casilla 567, Valdivia, Chile b Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada Received 14 December 2005; accepted 22 August 2007 Available online 14 September 2007
Abstract The suspension-feeding gastropod Crepipatella dilatata occurs in estuaries in southern Chile that experience considerable fluctuations in salinity, driven by tidal and atmospheric forces. In the Quempille´n estuary salinities as low as 9 psu may occur after severe rainstorms, and persist for several hours. In this study salinity was the major factor influencing the clearance rate of C. dilatata. At salinities below 20 psu, filtration ceased, whereas at high salinities (>22 psu) mean clearance rate was 0.24 l h1 standard animal1 (S.D. 0.18) for actively filtering individuals. This was confirmed by laboratory experiments under controlled conditions. Endoscopic observations were consistent with measurements of clearance rate, and showed that at salinity 25e30 psu the rate of transport along the gill filaments of particulate material embedded in mucus was 759 mm s1 (S.D. 480), but particle transport ceased at and below salinity 20 psu. Complete or partial isolation of the mantle cavity from the environment may be a mechanism to protect soft tissues and/or incubated egg capsules from osmotic stress. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: clearance rate; salinity; Crepipatella; estuary; suspension-feeding; filtration
1. Introduction Many environmental variables can influence clearance rate (CR) and the feeding process in suspension-feeders, among them seston quality and quantity (Stenton-Dozey and Brown, 1992; Wong and Cheung, 2001; Gardner, 2002), seston volume (Hawkins et al., 2001), chlorophyll a (Hawkins et al., 2001; James et al., 2001), particle concentration and size (Stenton-Dozey and Brown, 1992, 1994), temperature (Wong and Cheung, 2003), and salinity (Navarro, 1988; Hutchinson and Hawkins, 1992; Navarro and Gonzalez, 1998; Spicer and Stro¨mberg, 2003). Most of these investigations were carried out in the laboratory, allowing control of environmental * Corresponding author. E-mail address: [email protected] (O.R. Chaparro). 0272-7714/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2007.08.014
conditions, but some were field studies in which short-term changes in the food supply were demonstrated. However, few attempts have been made to investigate the effects of other potentially important variables on suspension-feeding on short time scales (e.g. phaeopigments, energy content of seston, biochemical composition of seston, feeding indices). In this context estuaries are particularly interesting, owing to the combined effects of tidal cycles and freshwater input (Riaux, 1981; Huang et al., 2003) and their impact on physiological rate functions of estuarine organisms, especially those that determine energy balance (Navarro, 1988). The estuary may experience rapid and profound changes in the physical, chemical and biological environment in the water column associated with tidal forces, wind stress and heavy rainfall (Toro and Winter, 1983; Chaparro et al., 2008). These events also impact the bottom, especially in shallow estuaries,
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such as Quempille´n, southern Chile (Chaparro et al., 2008), leading to resuspension of bottom sediments. The result is a dynamic environment in which the food source for suspension-feeders often varies on short time scales. An important feature of an estuary is the changing salinity that results from the influx and efflux of sea water, which is diluted by the input of fresh water from the river. Salinity has been termed by Kinne (1966) an ‘‘ecological master factor’’ in estuaries, and is an important regulator of many behavioural and physiological processes in estuarine organisms (Navarro, 1988; Hutchinson and Hawkins, 1992; Navarro and Gonzalez, 1998; Kim et al., 2001; Spicer and Stro¨mberg, 2003; Marsden, 2004). The gastropod Crepipatella dilatata is common in the estuaries of southern Chile (Gallardo, 1979), including Quempille´n, where there is a large population. The females of Crepipatella spp., which are all sessile, feed by removing particles from suspension (Newell and Kofoed, 1977a,b; Chaparro et al., 2002). The suspension-feeding mechanism in Crepipatella fecunda has been described by Chaparro et al. (2002). Suspended particles are entrained on a unilamellar gill which is covered by short dorsal and ventral cilia and also possesses long lateral cilia. The particles are trapped in a mucous sheet which covers the surface of the lamella and is moved to the distal region of the branchial filaments, where it forms a mucous string. This particle-laden string is transferred to a muscular canal in the neck, where it forms a compact mucous cord which is moved anteriorly towards the buccal region, where it is seized by the radula and ingested. In a companion paper (Chaparro et al., 2008) we deal with short-term (tidal cycle) and long-term (seasonal) environmental changes in the physical and biochemical characteristics of the Quempille´n estuary, which we found to be very susceptible to wind and rain storms. The purpose of the present study was to investigate the effects of environmental factors on the CR of C. dilatata, specifically how CR responds to short-term changes in the properties of the water column, particularly salinity, during the tidal cycle at different times of the year.
2. Materials and methods 2.1. Water sampling and seston analysis The study was undertaken in the Quempille´n estuary, Chiloe´ Island, southern Chile (41 520 S; 73 460 W). In each season of the year, 2 or 3 tidal cycles of 12 h were investigated. Water was pumped continuously into the laboratory and the following variables measured: temperature, salinity, particle numbers, total particle volume, total particulate matter, particulate organic matter, particulate inorganic matter, chlorophyll a, total phaeopigments, and particulate lipid, protein, carbohydrate and energy. Salinity (practical salinity scale) was monitored continuously with a temperature/conductivity meter (YSI 300). For details of water sampling and methods for measuring environmental variables and properties of seston, see Chaparro et al (2008).
2.2. Clearance rate in estuary water Sessile females (shell length 23e33 mm, mean: 27.9 mm) of Crepipatella dilatata were collected from the subtidal zone in the Quempille´n estuary. The individuals in each stack were separated from one another and only those attached directly to the rocky substrate were retained. These were returned to the estuary, where they remained for at least 2e 3 months before being used for measurements of CR. A few days before each set of measurements began, specimens were taken to the laboratory, cleaned of epibionts and placed in 100 L tanks supplied with flowing unfiltered water directly from the estuary 25 m away i.e. under ambient conditions. The limpets were then transferred to the CR measurement apparatus, which consisted of a series of 8 plastic chambers (250 ml) individually supplied with ambient seawater by gravity from a constant head device (Navarro and Thompson, 1995). Six chambers contained individuals of Crepipatella dilatata (one per chamber), whereas 2 chambers contained only flowing water and served as controls. The inflow line to each chamber was fitted with a flow restrictor (a plastic plug drilled lengthwise to produce a hole of the required diameter). The constant head tank was continuously supplied with water pumped from the estuary, the inlet being placed as close as possible to the bottom without removing sediment. The flow through the chambers was adjusted to provide a detectable difference (25e30%) in particle concentration between inflow and outflow. Limpets were left overnight in flowing ambient seawater in the chambers before CR measurements were made. For each measurement of CR, the outflow from each chamber was collected over a measured time period, permitting the calculation of flow rate. Particle numbers were determined with a particle counter (Beckman Coulter Z2) fitted with a 100 mm orifice tube. The size range of the particles counted was nominally 2e40 mm equivalent spherical diameter, which comprised over 95% of the particles in the estuary (Chaparro et al., 2008). Replicate counts were made for each sample. Clearance rate was calculated as CR ¼ FR(Ni No)/Ni where FR ¼ flow rate (l h1), Ni ¼ particles ml1 in inflow (mean of 2 control chambers), No ¼ particles ml1 in outflow from experimental chamber. Measurements were made hourly for 12 h during tidal cycles in November 2002 and January, May, August and November 2003. A fresh set of limpets (6e12 individuals) was used for each series of measurements (for details see Chaparro et al., 2008). The soft tissues of each limpet used were excised, dried to constant mass at 90 C and weighed. CR values were adjusted for a limpet of standard body mass 0.33 g (the mean value for the individuals used). 2.3. Clearance rate under controlled conditions Data from limpets kept in water from the estuary suggested that CR was inhibited at salinities below 20 psu (see Section 3). We therefore carried out a controlled laboratory experiment in August 2004 to investigate this further.
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2.4. Velocity of particle transport along the gill filament Specimens of Crepipatella dilatata were taken from the estuary and immediately prepared for endoscopy. A small hole was drilled through the shell in the ventral region at the mantle margin to permit the insertion of the tip of the rigid tube of the endoscope (Olympus OTV-S4) so that it reached the gill region without damaging the mantle. The endoscope was mounted on a micromanipulator and connected to a xenon light source (Olympus CLV-10), a digital camera, a Sony VHS videorecorder and a Sony monitor. Before being examined by endoscopy, the experimental limpets were held in gently aerated seawater (30 psu, 17 C) for 3e4 days and fed Isochrysis galbana. A series of aquaria containing water of known salinity (10, 15, 20, 25 or 30 psu) was prepared as described above, and 10 experimental limpets placed in each. A food level of 50,000 cells ml1 I. galbana was maintained in each aquarium. Individual specimens were examined in turn by endoscopy over a period of several hours. The rate of particle transport on the gill was measured from video sequences as follows. A clearly identifiable aggregation of particles in mucus was located and its displacement along the filament observed between two identifiable points. The time elapsed was obtained by stepping through the video sequence frame by frame (NTSC format: 30 frames s1, Ward et al., 1991) and the distance traveled estimated by reference to the width of the gill filament (determined by microscopy from measurements of filaments of freshly excised demibranchs from similar specimens).
3. Results 3.1. Relationship between clearance rate and environmental variables Stepwise multiple regression for data combined from all tidal cycles during all months revealed that salinity accounted for 23% of the observed variation in CR of Crepipatella dilatata (ANOVA: F ¼ 209, df ¼ 1, 814, P < 0.0001; R2 ¼ 0.23). Inclusion of other environmental and seston variables (Chaparro et al., 2008) in the model did not increase the proportion of total variation explained. A plot of CR vs. salinity for the pooled data showed that below 15e20 psu there was little or no filtration activity, whereas at higher salinities CR was variable (Fig. 1). Careful inspection of individual tidal cycles confirmed that although there was no consistent overall pattern in CR in relation to salinity, CR responses were observed when salinity either fell below or rose above approximately 20 psu, which occurred in all the cycles examined during winter (August) and spring (November) (Fig. 2). As salinity decreased below approximately 20 psu at low tide, CR declined to zero or to a value close to zero (Fig. 2; C1, C2, C3). Conversely, as salinity rose above approximately 20 psu at high tide, CR increased from zero (Fig. 2; C2, D1, D2). A forward stepwise multiple regression for cases where salinity 20 showed that salinity accounted for only 7.5% of the variation in CR (R2 ¼ 0.075), and that the inclusion of seston variables (number of suspended particles, particulate carbohydrate, energy content of seston, phaeopigments and food index) increased the variation explained to 13% of the total. To investigate further the possibility of a transition from zero or negligible filtration activity at low salinity to high activity at high salinity, the observed CR values for the entire data set were each assigned a binary value for a derived variable CR0 as follows: if CR < 0.05 l h1 individual1 then CR0 ¼ 0, and if CR 0.05 l h1 individual1 then CR0 ¼ 1.
1.4 y = 0.0005x2 -0.0037x -0.0319 1.2
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Specimens of Crepipatella dilatata prepared as described above were kept overnight in estuary water and then placed in 10 L aquaria (one limpet per aquarium). Each aquarium contained water of known salinity (5, 10, 15, 20, 22.5, 25 or 30 psu) prepared by diluting filtered estuarine water (0.45 mm), taken at high tide to obtain the highest salinity possible, with filtered (0.45 mm) fresh water from a well. Six aquaria, i.e. six replicates of C. dilatata, were used for each salinity. Additional aquaria without limpets were also set up as controls. Cultured microalgae (Isochrysis galbana) were added to each aquarium (final concentration 30,000 cells ml1). Circulation was facilitated by gentle bubbling of air through the water. Temperature was maintained at 17 0.5 C (ambient temperature in the estuary). Cell concentration was measured hourly with a particle counter as described above until cell concentration declined to 70% of the initial value. Clearance rate was calculated from the exponential rate of decrease of particle concentration in a closed system (Coughlan, 1969) after correcting for controls. The need to prepare large quantities of water at predetermined salinities precluded the use of a flow-through system to measure CR in the laboratory experiments, but according to Widdows (1985) and Petersen et al. (2004) the two methods (flow-through and static systems) produce similar results. Furthermore, no comparison was required between CR data for limpets in estuary water and limpets in the dilution series.
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The purpose of this transformation was to classify the limpets into two groups, those that were suspension-feeding and those that were not. A graphical representation of the percentage of limpets in each of the two categories at each observed salinity showed that below 20 psu none of the limpets was taking particles from suspension (Fig. 3). At salinity 20e22 psu the percentage of individuals that were feeding increased considerably, and above 25 psu almost all limpets were clearing particles from the water. Close inspection revealed that the presence of a few individuals that were not filtering at a high salinity at any given time did not signify that they were incapable of doing so, but rather that some limpets appeared to close for short periods on occasion.
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Clearance rate was zero at low salinities (5 and 10 psu) and very low at intermediate salinities (15 and 20 psu), but increased substantially at higher salinities (>20 psu; Fig. 4; a ManneWhitney test demonstrated that CR at salinities 22.5 psu or higher was significantly greater than at salinities 20 psu or lower; P < 0001). There was a transition at 20e 25 psu from zero or negligible suspension feeding to active removal of particles. At 22.5 psu, within this transition phase, mean CR for a ‘‘standard animal’’ was 0.52 l h1 individual1 (S.D. 0.36, range 0e0.81). At the highest salinities (data for 25 and 30 psu combined) mean CR was 0.76 l h1 individual1 (S.D. 0.32, range 0.3e1.34).
Fig. 4. Crepipatella dilatata. Relationship between clearance rate and salinity (controlled conditions). Mean S.D. Where no error bar is shown, S.D. is smaller than the symbol.
3.3. Velocity of particle transport on the gill filament Particles entering the mantle cavity were retained in mucous sheets which covered the dorsal and ventral surfaces of the filaments (see Chaparro et al., 2002 for a detailed description of the feeding mechanism). At high salinities (25e30 psu) mucus-embedded particles were transported towards the distal margin of the filament at a mean velocity of 759 mm s1 (S.D. 480) (Fig. 5). However, at 20 psu or lower, no transport of material embedded in mucus was observed on the gill, and no movement of the mucous sheets was apparent. A ManneWhitney test showed that particle velocity at salinities 25 psu or higher was significantly greater than at salinities 20 psu or lower; P < 0.0001). 4. Discussion
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Salinity Fig. 3. Crepipatella dilatata. Diagrammatic representation of filtration activity in relation to salinity. The variable on the vertical axis is a binary derivative (CR) of clearance rate in which a value of 0 signifies individuals with zero or negligible clearance rate (CR < 0.05 L h1 standard animal1), and a value of 1 signifies individuals that were filtering (CR 0.05 l h1 standard animal1). The area of each circle represents the number of individuals filtering (CR0 ¼ 1) or not filtering (CR0 ¼ 0) expressed as a percentage of the number of individuals for each 1 psu class range for which observations were obtained. Thus the largest symbols represent 100%, i.e. all limpets were filtering (CR0 ¼ 1) or not filtering (CR0 ¼ 0), and smaller symbols represent lesser percentages in proportion to the area of the symbol.
Estuaries are exposed to considerable variation in environmental conditions on both long and short time scales. In particular, tidal cycles (Baird et al., 1987; Navarro et al., 1993; Huang et al., 2003) and stochastic events such as wind and rain storms (Toro and Winter, 1983; Navarro et al., 1993; Huang et al., 2003) can exert a strong influence on the characteristics of the water column. The impact of these factors is enhanced in shallow estuaries, such as Quempille´n (maximum depth 2 m). In addition to increasing the amount of seston through resuspension of bottom sediment and input of allochthonous material, tidal and atmospheric forces can produce rapid and substantial fluctuations in salinity to which estuarine organisms, including the suspension-feeding gastropod Crepipatella dilatata, must respond physiologically. Clearance rate (CR) is a physiological variable that is often strongly influenced by environmental factors, including the quantity and quality of food (Pouvreau et al., 2000; Navarro and Thompson, 1995), although in our study variables related to food availability (such as chlorophyll a, total particulate matter, protein, lipid and carbohydrate) explained very little of the observed variance in CR of Crepipatella dilatata. The
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only significant variable in this context was salinity. Our CR values for C. dilatata in estuary water at salinities above 22.5 psu (grand mean for animals that were filtering: 0.24 l h1 standard animal1; S.D: 0.18; n ¼ 591; range: 0.017e1.33) are similar to those recorded for Crepidula fornicata by Newell and Kofoed (1977a); (approximately 0.25 l1 h1 individual1 for a limpet of shell length about 30 mm). Jørgensen et al. (1984) recorded a CR of 0.66 l h1 animal1 for C. fornicata (dry soft tissue mass 0.132 g) feeding on cultured algae at 15 C and 30 psu, which is consistent with our value of ca. 0.8 l h1 animal1 for C. dilatata (dry mass of soft tissues 0.33 g) under similar conditions (17 C, 30 psu, cultured algae). We found that C. dilatata does not remove particles from the water at salinities below 20 psu. Observations on limpets feeding on algae under controlled salinity conditions demonstrated that between 20 and 25 psu there is a transition from no filtration activity to normal suspension feeding. During periods of low salinity, when no suspension-feeding is taking place, the limpet probably isolates the mantle cavity from the surrounding water by close apposition of the shell and mantle rim to the substrate. Observations with the endoscope revealed that formation of the mucous cord that transports food material from the gill to the mouth ceases, and there is no production of pseudofaeces, which is consistent with the cessation of particle removal from the water. However, although apparently closed the limpet continues to produce faeces, which are retained within the shell margin. The conclusion that suspension feeding ceases at salinities below 20 psu is supported by our endoscopic observations, since no movement of the mucous sheet on the gills was evident at salinity 20 psu or less, whereas at 25 and 30 psu particles trapped in mucus moved distally. Although direct observation of cilia was not possible at this magnification, the cessation of particle transport at low salinity suggests that the ciliary tracts responsible for moving the mucous sheet were inoperative, presumably to conserve energy during a period when food particles were not being drawn into the mantle cavity. The mean velocity with which trapped particles moved along the gill filament of Crepipatella dilatata at 25e30 psu
was 759 mm s1, which is lower than the mean value obtained by Chaparro et al. (2002) for Crepipatella fecunda (mean 1320 mm s1, range 490e2570), although within the same range. As far as we are aware these are the only published data for the rate of particle transport along the gill filament of a suspension-feeding gastropod. Particle transport rate along the gill filament of C. dilatata exceeds that observed in the suspension-feeding bivalve Mytilus edulis (mean 320 mm s1; Ward et al., 1991), in which individual particles or small aggregations are more directly associated with an individual filament and are not bound in a mucous sheet as in Crepipatella spp. (Chaparro et al., 2002). An increase in CR and a faster rate of particle transport may be a response of C. dilatata to the reduction in feeding time imposed by factors like low salinity or exposure to air. No periods of low salinity (<20 psu) were evident in the tidal cycles we studied in the Quempille´n estuary in summer (Chaparro et al., 2008), and all the specimens of Crepipatella dilatata that we examined at that time continued to remove particles from suspension. On the other hand, in winter and during wet days in spring salinity frequently fell below the critical value (20 psu), and these conditions often persisted for several hours. For example, in November (cycle 1) low salinity (<22.5 psu) values were observed for 75% of the 12 h period of a complete tidal cycle and the limpets ceased suspension-feeding, despite an adequate food supply (Chaparro et al., 2008). Hutchinson and Hawkins (1992) observed a reduction in CR of the oyster Ostrea edulis at salinities below 22 psu, and according to Bøhle (1972) the mussel Mytilus edulis exhibits a lower CR and a reduced growth rate when exposed to highly fluctuating salinity conditions. The cessation of feeding by sessile female Crepipatella dilatata during periods of low salinity does not necessarily signify that this also occurs in juveniles and males, which are also able to feed by scraping the substrate with the radula as they move (Chaparro et al., 2005), a process that may or may not require contact with the exterior (i.e. outside the mantle rim). Thus these motile forms may be able to obtain food during low salinity periods by rasping the substrate lying beneath the shell while isolating the mantle cavity from the external environment. This alternative, independent feeding mechanism in motile individuals may permit acquisition of energy during periods of low salinity when suspension-feeding is curtailed, as well as facilitate a greater and more diverse food input during periods of high salinity. The fact that during a complete tidal cycle exhibiting no periods of low salinity most of the limpets are able to clear particles all the time, although at different rates, suggests that there is no pattern of CR (i.e. feeding rate) that follows the tidal cycle in a systematic way. The apparent isolation of the mantle cavity by female Crepipatella dilatata during periods of low salinity is presumably a mechanism for maintaining osmotic pressure and preventing tissue damage. Valve closure at low salinities occurs in many bivalves e.g. Choromytilus chorus (Navarro, 1988), Crassostrea virginica (Hand and Stickle, 1977), Mytilus edulis, Crassostrea gigas, Scrobicularia plana, Cardium edule,
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Mercenaria mercenaria, Modiolus modiolus (Shumway, 1977) and Anadara senilis (Djangmah et al., 1979). According to Davenport and Fletcher (1978) M. edulis closes its valves to avoid osmotic damage to the frontal cilia at low salinities. In female C. dilatata, which are sessile whether brooding or not, avoidance of low salinity may serve in part to protect the egg capsules, which are attached to the substrate below the mantle cavity. Pechenik (1982, 1983) showed that in some gastropods that do not display parental care the capsule walls reduce the rate at which the osmotic concentration of the capsular fluid falls with decreasing salinity, but they cannot prevent ion exchange with the exterior environment, i.e. the walls provide only partial protection. On the other hand, the capsule walls of C. dilatata are very thin (Ojeda and Chaparro, 2004) and presumably permeable, requiring parental care and isolation from water of low salinity. In this study we did not distinguish between incubating and non-incubating females, but conditions leading to low salinity and subsequent sealing of the mantle cavity and cessation of filtration occurred both in brooding (spring) and non-brooding (winter) seasons. If the observed response to low salinity is a mechanism to protect the capsules, rather than to prevent osmotic damage to the tissues of the adult, we should expect a difference between incubating and non-incubating females in the response to low salinity, a question that remains to be addressed. Acknowledgements We thank M.V. Garrido, A.J. Schmidt and J.A. Videla for their help during field sampling. This study was supported by a grant from Fondecyt-Chile (1020171). A grant for international collaboration (Fondecyt 7020171) allowed R.J.T to travel to Chile during the research. References Baird, D., Winter, P.E.D., Wendt, G., 1987. The flux of particulate material through a well-mixed estuary. Continental Shelf Research 7, 1399e1403. Bøhle, B., 1972. Effects of adaptation to reduced salinity on filtration activity and growth of mussels (Mytilus edulis L.). Journal of Experimental Marine Biology and Ecology 10, 41e47. Chaparro, O.R., Thompson, R.J., Pereda, S.V., 2002. Feeding mechanisms in the gastropod Crepidula fecunda. Marine Ecology Progress Series 234, 171e181. Chaparro, O.R., Montiel, Y.A., Cubillos, V.M., 2005. Direct intracapsular development: implications for feeding mechanisms in early juveniles of the gastropod Crepidula dilatata. Journal of Marine Biology Association U.K 85, 163e169. Chaparro, O.R., Segura, C.J., Montiel, Y.A., Thompson, R.J., Navarro, J.M., 2008. Variations in the quantity and composition of seston from an estuary in southern Chile on different temporal scales. Estuarine. Coastal and Shelf Science 76 (4), 815e830. Coughlan, J., 1969. The estimation of filtering rate from the clearance of suspensions. Marine Biology 2, 356e358. Davenport, J., Fletcher, J.S., 1978. The effects of simulated estuarine mantle cavity conditions upon the activity of the frontal gill cilia of Mytilus edulis. Journal of the Marine Biological Association UK 58, 671e681. Djangmah, J.S., Shumway, S.E., Davenport, J., 1979. Effects of fluctuating salinity on the behaviour of the west African blood clam Anadara senilis and
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on the osmotic pressure and ionic concentrations of the haemolymph. Marine Biology 50, 209e213. Gallardo, C.S., 1979. Especies gemelas del ge´nero Crepidula (Gastropoda, Calyptraeidae) en la costa de Chile; una redescripcio´n de C. dilatata Lamarck y descripcio´n de C. fecunda n.sp. Studies on Neotropical Fauna and Environment 14, 215e226. Gardner, J.P.A., 2002. Effects of seston variability on the clearance rate and absorption efficiency of the mussels Aulacomya maoriana, Mytilus galloprovincialis and Perna canaliculus from New Zealand. Journal of Experimental Marine Biology and Ecology 268, 83e101. Hand, S.C., Stickle, W.B., 1977. Effects of tidal fluctuations of salinity on pericardial fluid composition of the American oyster Crassostrea virginica. Marine Biology 42, 259e271. Huang, S.C., Kreeger, D.A., Newell, R.I.E., 2003. Tidal and seasonal variations in the quantity and composition of seston in a North American, mid-Atlantic saltmarsh. Estuarine, Coastal and Shelf Science 56, 547e560. Hutchinson, S., Hawkins, L.E., 1992. Quantification of the physiological responses of the European flat oyster Ostrea edulis L. to temperature and salinity. Journal of Molluscan Studies 58, 215e226. Hawkins, A.J.S., Fang, J.G., Pascoe, P.L., Zhang, J.H., Zhang, X.L., Zhu, M.Y., 2001. Modelling short-term responsive adjustments in particle clearance rate among bivalve suspension-feeders: separate unimodal effects of seston volume and composition in the scallop Chlamys farreri. Journal of Experimental Marine Biology and Ecology 262, 61e73. James, M.R., Weatherhead, M.A., Ross, A.H., 2001. Size-specific clearance, excretion, and respiration rates, and phytoplankton selectivity for the mussel Perna canaliculus at low levels of natural food. New Zealand Journal of Marine and Freshwater Research 35, 73e86. Jørgensen, C.B., Kiørboe, T., Møhlenberg, F., Riisga˚rd, H.U., 1984. Ciliary and mucus-net filter feeding, with special reference to fluid mechanical characteristics. Marine Ecology Progress Series 15, 283e292. Kim, W.S., Huh, H.T., Huh, S.H., Lee, T.W., 2001. Effects of salinity on endogenous rhythm of Manila clam, Ruditapes philippinarum (Bivalvia: Veneridae). Marine Biology 138, 157e162. Kinne, O., 1966. Physiological aspects of animal life in estuaries with special reference to salinity. Netherlands Journal of Sea Research 3, 222e244. Marsden, I.D., 2004. Effects of reduced salinity and seston availability on growth of the New Zealand little-neck clam Austrovenus stutchburyi. Marine Ecology Progress Series 266, 157e171. Navarro, J.M., 1988. The effects of salinity on the physiological ecology of Choromytilus chorus (Molina, 1782) (Bivalvia: Mytilidae). Journal of Experimental Marine Biology and Ecology 122, 19e33. Navarro, J.M., Gonzalez, C.M., 1998. Physiological responses of the Chilean scallop Argopecten purpuratus to decreasing salinities. Aquaculture 167, 315e327. Navarro, J.M., Thompson, R.J., 1995. Seasonal fluctuations in the size spectra, biochemical composition and nutritive value of the seston available to a suspension-feeding bivalve in a subarctic environment. Marine Ecology Progress Series 125, 95e106. Navarro, J.M., Clasing, E., Urrutia, G., Asencio, G., Stead, R., Herrera, C., 1993. Biochemical composition and nutritive value of suspended particulate matter over a tidal flat of southern Chile. Estuarine, Coastal and Shelf Science 37, 59e73. Newell, R.C., Kofoed, L.H., 1977a. The energetics of suspension-feeding in the gastropod Crepidula fornicata. Journal of the Marine Biological Association U.K 57, 161e180. Newell, R.C., Kofoed, L.H., 1977b. Adjustment of the components of energy balance in the gastropod Crepidula fornicata in response to thermal acclimation. Marine Biology 44, 275e286. Ojeda, J.A., Chaparro, O.R., 2004. Morphological, gravimetric, and biochemical changes in Crepidula fecunda (Gastropoda: Calyptraeidae) egg capsule walls during embryonic development. Marine Biology 144, 263e269. Pechenik, J.A., 1982. Ability of some gastropod egg capsules to protect against low-salinity stress. Journal of Experimental Marine Biology and Ecology 63, 195e208. Pechenik, J.A., 1983. Egg capsules of Nucella lapillus (L.) protect against lowsalinity stress. Journal of Experimental Marine Biology and Ecology 71, 165e179.
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Petersen, J.K., Bougrier, S., Smaal, A.C., Garen, P., Robert, S., Larsen, J.E.N., Brummelhuis, E., 2004. Intercalibration of mussel Mytilus edulis clearance rate measurements. Marine Ecology Progress Series 267, 187e194. Pouvreau, S., Bodoy, A., Buestel, D., 2000. In situ suspension feeding behaviour of the pearl oyster, Pinctada margaritifera: combined effects of body size and weather-related seston composition. Aquaculture 181, 91e113. Riaux, C., 1981. Tidal variations in phytoplankton biomass and seston in a Northern Brittany estuary: ‘‘ebb-flood’’ asymmetry. Kieler Meeresforsch, Sonderh 5, 274e277. Shumway, S.E., 1977. The effect of fluctuating salinity on the tissue water content of eight species of bivalve molluscs. Journal of Comparative Physiology 116, 269e285. Spicer, J.I., Stro¨mberg, J.-O., 2003. Metabolic responses to low salinity of the shipworm Teredo navalis (L.). Sarsia 88, 302e305. Stenton-Dozey, J.M.E., Brown, A.C., 1992. Clearance and retention efficiency of natural suspended particles by the rock-pool bivalve Venerupis corrugatus in relation to tidal availability. Marine Ecology Progress Series 82, 175e186. Stenton-Dozey, J.M.E., Brown, A.C., 1994. Short-term changes in the energy balance of Venerupis corrugatus (Bivalvia) in relation to tidal availability of natural suspended particles. Marine Ecology Progress Series 103, 57e64.
Toro, J.E., Winter, J.E., 1983. Estudios en la ostricultura Quempille´n, un estuario del sur de Chile. Parte I. La determinacio´n de los factores abio´ticos y la cuantificacio´n del seston como oferta alimenticia y su utilizacio´n por Ostrea chilensis. Memorias de la Asociacio´n Latinoamericana de Acuicultura 5, 129e144. Ward, J.E., Beninger, P.G., MacDonald, B.A., Thompson, R.J., 1991. Direct observations of feeding structures and mechanisms in bivalve molluscs using endoscopic examination and video image analysis. Marine Biology 111, 287e291. Widdows, J., 1985. Physiological procedures. In: Bayne, B.L., Brown, D.A., Burns, K., Dixon, D.R., Ivanovici, A., Livingstone, D.R., Lowe, D.M., Moore, M.N., Stebbing, A.R.D., Widdows, J. (Eds.), The Effects of Stress and Pollution on Marine Animals. Praeger Scientific, pp. 161e178. Wong, W.H., Cheung, S.G., 2001. Feeding rates and scope for growth of green mussels, Perna viridis (L.) and their relationship with food available in Kat O, Hong Kong. Aquaculture 193, 123e137. Wong, W.H., Cheung, S.G., 2003. Seasonal variation in the feeding physiology and scope for growth of green mussels, Perna viridis in estuarine Ma Wan, Hong Kong. Journal of the Marine Biological Association UK 83, 543e552.
Estuarine, Coastal and Shelf Science 76 (2008) 861e868 www.elsevier.com/locate/ecss
The effect of salinity on clearance rate in the suspension-feeding estuarine gastropod Crepipatella dilatata under natural and controlled conditions O.R. Chaparro a,*, Y.A. Montiel a, C.J. Segura a, V.M. Cubillos a, R.J. Thompson b, J.M. Navarro a a
Instituto de Biologı´a Marina ‘‘Dr. Ju¨rgen Winter’’, Universidad Austral de Chile, Casilla 567, Valdivia, Chile b Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada Received 14 December 2005; accepted 22 August 2007 Available online 14 September 2007
Abstract The suspension-feeding gastropod Crepipatella dilatata occurs in estuaries in southern Chile that experience considerable fluctuations in salinity, driven by tidal and atmospheric forces. In the Quempille´n estuary salinities as low as 9 psu may occur after severe rainstorms, and persist for several hours. In this study salinity was the major factor influencing the clearance rate of C. dilatata. At salinities below 20 psu, filtration ceased, whereas at high salinities (>22 psu) mean clearance rate was 0.24 l h1 standard animal1 (S.D. 0.18) for actively filtering individuals. This was confirmed by laboratory experiments under controlled conditions. Endoscopic observations were consistent with measurements of clearance rate, and showed that at salinity 25e30 psu the rate of transport along the gill filaments of particulate material embedded in mucus was 759 mm s1 (S.D. 480), but particle transport ceased at and below salinity 20 psu. Complete or partial isolation of the mantle cavity from the environment may be a mechanism to protect soft tissues and/or incubated egg capsules from osmotic stress. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: clearance rate; salinity; Crepipatella; estuary; suspension-feeding; filtration
1. Introduction Many environmental variables can influence clearance rate (CR) and the feeding process in suspension-feeders, among them seston quality and quantity (Stenton-Dozey and Brown, 1992; Wong and Cheung, 2001; Gardner, 2002), seston volume (Hawkins et al., 2001), chlorophyll a (Hawkins et al., 2001; James et al., 2001), particle concentration and size (Stenton-Dozey and Brown, 1992, 1994), temperature (Wong and Cheung, 2003), and salinity (Navarro, 1988; Hutchinson and Hawkins, 1992; Navarro and Gonzalez, 1998; Spicer and Stro¨mberg, 2003). Most of these investigations were carried out in the laboratory, allowing control of environmental * Corresponding author. E-mail address: [email protected] (O.R. Chaparro). 0272-7714/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2007.08.014
conditions, but some were field studies in which short-term changes in the food supply were demonstrated. However, few attempts have been made to investigate the effects of other potentially important variables on suspension-feeding on short time scales (e.g. phaeopigments, energy content of seston, biochemical composition of seston, feeding indices). In this context estuaries are particularly interesting, owing to the combined effects of tidal cycles and freshwater input (Riaux, 1981; Huang et al., 2003) and their impact on physiological rate functions of estuarine organisms, especially those that determine energy balance (Navarro, 1988). The estuary may experience rapid and profound changes in the physical, chemical and biological environment in the water column associated with tidal forces, wind stress and heavy rainfall (Toro and Winter, 1983; Chaparro et al., 2008). These events also impact the bottom, especially in shallow estuaries,
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such as Quempille´n, southern Chile (Chaparro et al., 2008), leading to resuspension of bottom sediments. The result is a dynamic environment in which the food source for suspension-feeders often varies on short time scales. An important feature of an estuary is the changing salinity that results from the influx and efflux of sea water, which is diluted by the input of fresh water from the river. Salinity has been termed by Kinne (1966) an ‘‘ecological master factor’’ in estuaries, and is an important regulator of many behavioural and physiological processes in estuarine organisms (Navarro, 1988; Hutchinson and Hawkins, 1992; Navarro and Gonzalez, 1998; Kim et al., 2001; Spicer and Stro¨mberg, 2003; Marsden, 2004). The gastropod Crepipatella dilatata is common in the estuaries of southern Chile (Gallardo, 1979), including Quempille´n, where there is a large population. The females of Crepipatella spp., which are all sessile, feed by removing particles from suspension (Newell and Kofoed, 1977a,b; Chaparro et al., 2002). The suspension-feeding mechanism in Crepipatella fecunda has been described by Chaparro et al. (2002). Suspended particles are entrained on a unilamellar gill which is covered by short dorsal and ventral cilia and also possesses long lateral cilia. The particles are trapped in a mucous sheet which covers the surface of the lamella and is moved to the distal region of the branchial filaments, where it forms a mucous string. This particle-laden string is transferred to a muscular canal in the neck, where it forms a compact mucous cord which is moved anteriorly towards the buccal region, where it is seized by the radula and ingested. In a companion paper (Chaparro et al., 2008) we deal with short-term (tidal cycle) and long-term (seasonal) environmental changes in the physical and biochemical characteristics of the Quempille´n estuary, which we found to be very susceptible to wind and rain storms. The purpose of the present study was to investigate the effects of environmental factors on the CR of C. dilatata, specifically how CR responds to short-term changes in the properties of the water column, particularly salinity, during the tidal cycle at different times of the year.
2. Materials and methods 2.1. Water sampling and seston analysis The study was undertaken in the Quempille´n estuary, Chiloe´ Island, southern Chile (41 520 S; 73 460 W). In each season of the year, 2 or 3 tidal cycles of 12 h were investigated. Water was pumped continuously into the laboratory and the following variables measured: temperature, salinity, particle numbers, total particle volume, total particulate matter, particulate organic matter, particulate inorganic matter, chlorophyll a, total phaeopigments, and particulate lipid, protein, carbohydrate and energy. Salinity (practical salinity scale) was monitored continuously with a temperature/conductivity meter (YSI 300). For details of water sampling and methods for measuring environmental variables and properties of seston, see Chaparro et al (2008).
2.2. Clearance rate in estuary water Sessile females (shell length 23e33 mm, mean: 27.9 mm) of Crepipatella dilatata were collected from the subtidal zone in the Quempille´n estuary. The individuals in each stack were separated from one another and only those attached directly to the rocky substrate were retained. These were returned to the estuary, where they remained for at least 2e 3 months before being used for measurements of CR. A few days before each set of measurements began, specimens were taken to the laboratory, cleaned of epibionts and placed in 100 L tanks supplied with flowing unfiltered water directly from the estuary 25 m away i.e. under ambient conditions. The limpets were then transferred to the CR measurement apparatus, which consisted of a series of 8 plastic chambers (250 ml) individually supplied with ambient seawater by gravity from a constant head device (Navarro and Thompson, 1995). Six chambers contained individuals of Crepipatella dilatata (one per chamber), whereas 2 chambers contained only flowing water and served as controls. The inflow line to each chamber was fitted with a flow restrictor (a plastic plug drilled lengthwise to produce a hole of the required diameter). The constant head tank was continuously supplied with water pumped from the estuary, the inlet being placed as close as possible to the bottom without removing sediment. The flow through the chambers was adjusted to provide a detectable difference (25e30%) in particle concentration between inflow and outflow. Limpets were left overnight in flowing ambient seawater in the chambers before CR measurements were made. For each measurement of CR, the outflow from each chamber was collected over a measured time period, permitting the calculation of flow rate. Particle numbers were determined with a particle counter (Beckman Coulter Z2) fitted with a 100 mm orifice tube. The size range of the particles counted was nominally 2e40 mm equivalent spherical diameter, which comprised over 95% of the particles in the estuary (Chaparro et al., 2008). Replicate counts were made for each sample. Clearance rate was calculated as CR ¼ FR(Ni No)/Ni where FR ¼ flow rate (l h1), Ni ¼ particles ml1 in inflow (mean of 2 control chambers), No ¼ particles ml1 in outflow from experimental chamber. Measurements were made hourly for 12 h during tidal cycles in November 2002 and January, May, August and November 2003. A fresh set of limpets (6e12 individuals) was used for each series of measurements (for details see Chaparro et al., 2008). The soft tissues of each limpet used were excised, dried to constant mass at 90 C and weighed. CR values were adjusted for a limpet of standard body mass 0.33 g (the mean value for the individuals used). 2.3. Clearance rate under controlled conditions Data from limpets kept in water from the estuary suggested that CR was inhibited at salinities below 20 psu (see Section 3). We therefore carried out a controlled laboratory experiment in August 2004 to investigate this further.
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2.4. Velocity of particle transport along the gill filament Specimens of Crepipatella dilatata were taken from the estuary and immediately prepared for endoscopy. A small hole was drilled through the shell in the ventral region at the mantle margin to permit the insertion of the tip of the rigid tube of the endoscope (Olympus OTV-S4) so that it reached the gill region without damaging the mantle. The endoscope was mounted on a micromanipulator and connected to a xenon light source (Olympus CLV-10), a digital camera, a Sony VHS videorecorder and a Sony monitor. Before being examined by endoscopy, the experimental limpets were held in gently aerated seawater (30 psu, 17 C) for 3e4 days and fed Isochrysis galbana. A series of aquaria containing water of known salinity (10, 15, 20, 25 or 30 psu) was prepared as described above, and 10 experimental limpets placed in each. A food level of 50,000 cells ml1 I. galbana was maintained in each aquarium. Individual specimens were examined in turn by endoscopy over a period of several hours. The rate of particle transport on the gill was measured from video sequences as follows. A clearly identifiable aggregation of particles in mucus was located and its displacement along the filament observed between two identifiable points. The time elapsed was obtained by stepping through the video sequence frame by frame (NTSC format: 30 frames s1, Ward et al., 1991) and the distance traveled estimated by reference to the width of the gill filament (determined by microscopy from measurements of filaments of freshly excised demibranchs from similar specimens).
3. Results 3.1. Relationship between clearance rate and environmental variables Stepwise multiple regression for data combined from all tidal cycles during all months revealed that salinity accounted for 23% of the observed variation in CR of Crepipatella dilatata (ANOVA: F ¼ 209, df ¼ 1, 814, P < 0.0001; R2 ¼ 0.23). Inclusion of other environmental and seston variables (Chaparro et al., 2008) in the model did not increase the proportion of total variation explained. A plot of CR vs. salinity for the pooled data showed that below 15e20 psu there was little or no filtration activity, whereas at higher salinities CR was variable (Fig. 1). Careful inspection of individual tidal cycles confirmed that although there was no consistent overall pattern in CR in relation to salinity, CR responses were observed when salinity either fell below or rose above approximately 20 psu, which occurred in all the cycles examined during winter (August) and spring (November) (Fig. 2). As salinity decreased below approximately 20 psu at low tide, CR declined to zero or to a value close to zero (Fig. 2; C1, C2, C3). Conversely, as salinity rose above approximately 20 psu at high tide, CR increased from zero (Fig. 2; C2, D1, D2). A forward stepwise multiple regression for cases where salinity 20 showed that salinity accounted for only 7.5% of the variation in CR (R2 ¼ 0.075), and that the inclusion of seston variables (number of suspended particles, particulate carbohydrate, energy content of seston, phaeopigments and food index) increased the variation explained to 13% of the total. To investigate further the possibility of a transition from zero or negligible filtration activity at low salinity to high activity at high salinity, the observed CR values for the entire data set were each assigned a binary value for a derived variable CR0 as follows: if CR < 0.05 l h1 individual1 then CR0 ¼ 0, and if CR 0.05 l h1 individual1 then CR0 ¼ 1.
1.4 y = 0.0005x2 -0.0037x -0.0319 1.2
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Specimens of Crepipatella dilatata prepared as described above were kept overnight in estuary water and then placed in 10 L aquaria (one limpet per aquarium). Each aquarium contained water of known salinity (5, 10, 15, 20, 22.5, 25 or 30 psu) prepared by diluting filtered estuarine water (0.45 mm), taken at high tide to obtain the highest salinity possible, with filtered (0.45 mm) fresh water from a well. Six aquaria, i.e. six replicates of C. dilatata, were used for each salinity. Additional aquaria without limpets were also set up as controls. Cultured microalgae (Isochrysis galbana) were added to each aquarium (final concentration 30,000 cells ml1). Circulation was facilitated by gentle bubbling of air through the water. Temperature was maintained at 17 0.5 C (ambient temperature in the estuary). Cell concentration was measured hourly with a particle counter as described above until cell concentration declined to 70% of the initial value. Clearance rate was calculated from the exponential rate of decrease of particle concentration in a closed system (Coughlan, 1969) after correcting for controls. The need to prepare large quantities of water at predetermined salinities precluded the use of a flow-through system to measure CR in the laboratory experiments, but according to Widdows (1985) and Petersen et al. (2004) the two methods (flow-through and static systems) produce similar results. Furthermore, no comparison was required between CR data for limpets in estuary water and limpets in the dilution series.
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The purpose of this transformation was to classify the limpets into two groups, those that were suspension-feeding and those that were not. A graphical representation of the percentage of limpets in each of the two categories at each observed salinity showed that below 20 psu none of the limpets was taking particles from suspension (Fig. 3). At salinity 20e22 psu the percentage of individuals that were feeding increased considerably, and above 25 psu almost all limpets were clearing particles from the water. Close inspection revealed that the presence of a few individuals that were not filtering at a high salinity at any given time did not signify that they were incapable of doing so, but rather that some limpets appeared to close for short periods on occasion.
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Clearance rate was zero at low salinities (5 and 10 psu) and very low at intermediate salinities (15 and 20 psu), but increased substantially at higher salinities (>20 psu; Fig. 4; a ManneWhitney test demonstrated that CR at salinities 22.5 psu or higher was significantly greater than at salinities 20 psu or lower; P < 0001). There was a transition at 20e 25 psu from zero or negligible suspension feeding to active removal of particles. At 22.5 psu, within this transition phase, mean CR for a ‘‘standard animal’’ was 0.52 l h1 individual1 (S.D. 0.36, range 0e0.81). At the highest salinities (data for 25 and 30 psu combined) mean CR was 0.76 l h1 individual1 (S.D. 0.32, range 0.3e1.34).
Fig. 4. Crepipatella dilatata. Relationship between clearance rate and salinity (controlled conditions). Mean S.D. Where no error bar is shown, S.D. is smaller than the symbol.
3.3. Velocity of particle transport on the gill filament Particles entering the mantle cavity were retained in mucous sheets which covered the dorsal and ventral surfaces of the filaments (see Chaparro et al., 2002 for a detailed description of the feeding mechanism). At high salinities (25e30 psu) mucus-embedded particles were transported towards the distal margin of the filament at a mean velocity of 759 mm s1 (S.D. 480) (Fig. 5). However, at 20 psu or lower, no transport of material embedded in mucus was observed on the gill, and no movement of the mucous sheets was apparent. A ManneWhitney test showed that particle velocity at salinities 25 psu or higher was significantly greater than at salinities 20 psu or lower; P < 0.0001). 4. Discussion
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Salinity Fig. 3. Crepipatella dilatata. Diagrammatic representation of filtration activity in relation to salinity. The variable on the vertical axis is a binary derivative (CR) of clearance rate in which a value of 0 signifies individuals with zero or negligible clearance rate (CR < 0.05 L h1 standard animal1), and a value of 1 signifies individuals that were filtering (CR 0.05 l h1 standard animal1). The area of each circle represents the number of individuals filtering (CR0 ¼ 1) or not filtering (CR0 ¼ 0) expressed as a percentage of the number of individuals for each 1 psu class range for which observations were obtained. Thus the largest symbols represent 100%, i.e. all limpets were filtering (CR0 ¼ 1) or not filtering (CR0 ¼ 0), and smaller symbols represent lesser percentages in proportion to the area of the symbol.
Estuaries are exposed to considerable variation in environmental conditions on both long and short time scales. In particular, tidal cycles (Baird et al., 1987; Navarro et al., 1993; Huang et al., 2003) and stochastic events such as wind and rain storms (Toro and Winter, 1983; Navarro et al., 1993; Huang et al., 2003) can exert a strong influence on the characteristics of the water column. The impact of these factors is enhanced in shallow estuaries, such as Quempille´n (maximum depth 2 m). In addition to increasing the amount of seston through resuspension of bottom sediment and input of allochthonous material, tidal and atmospheric forces can produce rapid and substantial fluctuations in salinity to which estuarine organisms, including the suspension-feeding gastropod Crepipatella dilatata, must respond physiologically. Clearance rate (CR) is a physiological variable that is often strongly influenced by environmental factors, including the quantity and quality of food (Pouvreau et al., 2000; Navarro and Thompson, 1995), although in our study variables related to food availability (such as chlorophyll a, total particulate matter, protein, lipid and carbohydrate) explained very little of the observed variance in CR of Crepipatella dilatata. The
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866
Transport velocity (µm sec-1)
1400 1200 1000 800 600 400 200 0
0
5
10
15
20
25
30
35
Salinity Fig. 5. Crepipatella dilatata. Effect of salinity on the velocity of particle transport along the gill filament. Mean S.D. Where no error bar is shown, S.D. is smaller than the symbol.
only significant variable in this context was salinity. Our CR values for C. dilatata in estuary water at salinities above 22.5 psu (grand mean for animals that were filtering: 0.24 l h1 standard animal1; S.D: 0.18; n ¼ 591; range: 0.017e1.33) are similar to those recorded for Crepidula fornicata by Newell and Kofoed (1977a); (approximately 0.25 l1 h1 individual1 for a limpet of shell length about 30 mm). Jørgensen et al. (1984) recorded a CR of 0.66 l h1 animal1 for C. fornicata (dry soft tissue mass 0.132 g) feeding on cultured algae at 15 C and 30 psu, which is consistent with our value of ca. 0.8 l h1 animal1 for C. dilatata (dry mass of soft tissues 0.33 g) under similar conditions (17 C, 30 psu, cultured algae). We found that C. dilatata does not remove particles from the water at salinities below 20 psu. Observations on limpets feeding on algae under controlled salinity conditions demonstrated that between 20 and 25 psu there is a transition from no filtration activity to normal suspension feeding. During periods of low salinity, when no suspension-feeding is taking place, the limpet probably isolates the mantle cavity from the surrounding water by close apposition of the shell and mantle rim to the substrate. Observations with the endoscope revealed that formation of the mucous cord that transports food material from the gill to the mouth ceases, and there is no production of pseudofaeces, which is consistent with the cessation of particle removal from the water. However, although apparently closed the limpet continues to produce faeces, which are retained within the shell margin. The conclusion that suspension feeding ceases at salinities below 20 psu is supported by our endoscopic observations, since no movement of the mucous sheet on the gills was evident at salinity 20 psu or less, whereas at 25 and 30 psu particles trapped in mucus moved distally. Although direct observation of cilia was not possible at this magnification, the cessation of particle transport at low salinity suggests that the ciliary tracts responsible for moving the mucous sheet were inoperative, presumably to conserve energy during a period when food particles were not being drawn into the mantle cavity. The mean velocity with which trapped particles moved along the gill filament of Crepipatella dilatata at 25e30 psu
was 759 mm s1, which is lower than the mean value obtained by Chaparro et al. (2002) for Crepipatella fecunda (mean 1320 mm s1, range 490e2570), although within the same range. As far as we are aware these are the only published data for the rate of particle transport along the gill filament of a suspension-feeding gastropod. Particle transport rate along the gill filament of C. dilatata exceeds that observed in the suspension-feeding bivalve Mytilus edulis (mean 320 mm s1; Ward et al., 1991), in which individual particles or small aggregations are more directly associated with an individual filament and are not bound in a mucous sheet as in Crepipatella spp. (Chaparro et al., 2002). An increase in CR and a faster rate of particle transport may be a response of C. dilatata to the reduction in feeding time imposed by factors like low salinity or exposure to air. No periods of low salinity (<20 psu) were evident in the tidal cycles we studied in the Quempille´n estuary in summer (Chaparro et al., 2008), and all the specimens of Crepipatella dilatata that we examined at that time continued to remove particles from suspension. On the other hand, in winter and during wet days in spring salinity frequently fell below the critical value (20 psu), and these conditions often persisted for several hours. For example, in November (cycle 1) low salinity (<22.5 psu) values were observed for 75% of the 12 h period of a complete tidal cycle and the limpets ceased suspension-feeding, despite an adequate food supply (Chaparro et al., 2008). Hutchinson and Hawkins (1992) observed a reduction in CR of the oyster Ostrea edulis at salinities below 22 psu, and according to Bøhle (1972) the mussel Mytilus edulis exhibits a lower CR and a reduced growth rate when exposed to highly fluctuating salinity conditions. The cessation of feeding by sessile female Crepipatella dilatata during periods of low salinity does not necessarily signify that this also occurs in juveniles and males, which are also able to feed by scraping the substrate with the radula as they move (Chaparro et al., 2005), a process that may or may not require contact with the exterior (i.e. outside the mantle rim). Thus these motile forms may be able to obtain food during low salinity periods by rasping the substrate lying beneath the shell while isolating the mantle cavity from the external environment. This alternative, independent feeding mechanism in motile individuals may permit acquisition of energy during periods of low salinity when suspension-feeding is curtailed, as well as facilitate a greater and more diverse food input during periods of high salinity. The fact that during a complete tidal cycle exhibiting no periods of low salinity most of the limpets are able to clear particles all the time, although at different rates, suggests that there is no pattern of CR (i.e. feeding rate) that follows the tidal cycle in a systematic way. The apparent isolation of the mantle cavity by female Crepipatella dilatata during periods of low salinity is presumably a mechanism for maintaining osmotic pressure and preventing tissue damage. Valve closure at low salinities occurs in many bivalves e.g. Choromytilus chorus (Navarro, 1988), Crassostrea virginica (Hand and Stickle, 1977), Mytilus edulis, Crassostrea gigas, Scrobicularia plana, Cardium edule,
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Mercenaria mercenaria, Modiolus modiolus (Shumway, 1977) and Anadara senilis (Djangmah et al., 1979). According to Davenport and Fletcher (1978) M. edulis closes its valves to avoid osmotic damage to the frontal cilia at low salinities. In female C. dilatata, which are sessile whether brooding or not, avoidance of low salinity may serve in part to protect the egg capsules, which are attached to the substrate below the mantle cavity. Pechenik (1982, 1983) showed that in some gastropods that do not display parental care the capsule walls reduce the rate at which the osmotic concentration of the capsular fluid falls with decreasing salinity, but they cannot prevent ion exchange with the exterior environment, i.e. the walls provide only partial protection. On the other hand, the capsule walls of C. dilatata are very thin (Ojeda and Chaparro, 2004) and presumably permeable, requiring parental care and isolation from water of low salinity. In this study we did not distinguish between incubating and non-incubating females, but conditions leading to low salinity and subsequent sealing of the mantle cavity and cessation of filtration occurred both in brooding (spring) and non-brooding (winter) seasons. If the observed response to low salinity is a mechanism to protect the capsules, rather than to prevent osmotic damage to the tissues of the adult, we should expect a difference between incubating and non-incubating females in the response to low salinity, a question that remains to be addressed. Acknowledgements We thank M.V. Garrido, A.J. Schmidt and J.A. Videla for their help during field sampling. This study was supported by a grant from Fondecyt-Chile (1020171). A grant for international collaboration (Fondecyt 7020171) allowed R.J.T to travel to Chile during the research. References Baird, D., Winter, P.E.D., Wendt, G., 1987. The flux of particulate material through a well-mixed estuary. Continental Shelf Research 7, 1399e1403. Bøhle, B., 1972. Effects of adaptation to reduced salinity on filtration activity and growth of mussels (Mytilus edulis L.). Journal of Experimental Marine Biology and Ecology 10, 41e47. Chaparro, O.R., Thompson, R.J., Pereda, S.V., 2002. Feeding mechanisms in the gastropod Crepidula fecunda. Marine Ecology Progress Series 234, 171e181. Chaparro, O.R., Montiel, Y.A., Cubillos, V.M., 2005. Direct intracapsular development: implications for feeding mechanisms in early juveniles of the gastropod Crepidula dilatata. Journal of Marine Biology Association U.K 85, 163e169. Chaparro, O.R., Segura, C.J., Montiel, Y.A., Thompson, R.J., Navarro, J.M., 2008. Variations in the quantity and composition of seston from an estuary in southern Chile on different temporal scales. Estuarine. Coastal and Shelf Science 76 (4), 815e830. Coughlan, J., 1969. The estimation of filtering rate from the clearance of suspensions. Marine Biology 2, 356e358. Davenport, J., Fletcher, J.S., 1978. The effects of simulated estuarine mantle cavity conditions upon the activity of the frontal gill cilia of Mytilus edulis. Journal of the Marine Biological Association UK 58, 671e681. Djangmah, J.S., Shumway, S.E., Davenport, J., 1979. Effects of fluctuating salinity on the behaviour of the west African blood clam Anadara senilis and
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