The influence of season and the tidal cycle on division of labour by the radula during feeding in the estuarine brooding gastropod Crepipatella dilatata (Calyptraeidae)

Many marine organisms are constantly exposed to fluctuations in environmental factors such as temperature, salinity, hydrostatic pressure, light intensity and food availability over various time scales [1–3]. This is most evident in shallow coastal zones, especially estuaries, where environmental variation can change rapidly owing to tidal cycles and stochastic atmospheric forces (wind, precipitation) that have a major impact on the water column as a result of the relatively small volumes of water in the system [4]. Temporal variation in factors such as water temperature, salinity, suspended particulate matter and oxygen concentration in bays and estuaries is well known [1, 5–9] and generally results in physiological and behavioral responses by organisms living in these environments [10–13]. Salinity is one of the most important environmental variables in estuarine systems [14–16] and may be a significant source of stress for estuarine invertebrates and other organisms [11, 13], particularly sessile forms that are unable to move to more favorable locations [17].

Physiological functions in suspension-feeding estuarine invertebrates are influenced by factors such as temperature, salinity, quality and quantity of the seston and exposure to air, most of which are entirely or partially determined by the tidal cycle [1, 7, 8, 11, 13, 18]. The concentration and quality of suspended particulate matter and their role in regulating feeding and ingestion rates has been studied extensively [19–21].

In suspension-feeding marine gastropods such as calyptraeids, the radula is used as a supplementary feeding structure which collects food by rasping the substrate [22–24], a process which is relatively more important in motile than sessile individuals (Crepidula fecunda; [25]). The radula, however, also plays a role in suspension-feeding because it is responsible for transferring the mucous cords containing sequestered particles from the gill margin to the mouth via a food canal in the neck [23, 24, 26, 27]. A third feeding function of the calyptraeid radula is the removal of the mucous balls formed in the food pouch, a depression located in the frontal region of the mantle [28–30], either to the mouth for ingestion or to the mantle rim for rejection as pseudofaeces [23, 27, 30]. The material in these mucous balls is acquired by suspension-feeding and originates either on the branchial filaments closest to the head of the animal or from the lateral canal of the mantle, which moves particulate material from the inhalent area of the mantle cavity [23, 24, 27].

Calyptraeid gastropods brood their young within capsules [23, 31, 32] which are affixed to the substrate by the sessile female, ventral to the neck, and are irrigated by the inhalent water current [33]. The protandric hermaphroditic calyptraeid Crepipatella dilatata is common in the subtidal and intertidal zones of some estuaries in southern Chile; individuals < 18 mm shell length are male and also motile, whereas those > 18 mm long are female and sessile [34]. The female incubates the capsules until the juveniles are released (direct development; [32, 35]). This species lives in an environment in which variation in salinity and seston is high, owing to high winds and rainfall in winter and spring [7]. In order to minimize osmotic stress to the soft tissues and to the capsules, the female seals the shell to the substrate when the salinity falls below 22.5, preventing entry of water to the mantle cavity and thereby curtailing suspension-feeding [8, 17]. There is little information on the action of the radula, particularly in relation to environmental variables, although it is more associated with motile individuals (all males and juveniles) than with sessile individuals (females), in which the scraping action of the radula is reduced and suspension-feeding is the principal means of food acquisition [23, 25]. Males also isolate the mantle cavity from the exterior environment to prevent osmotic stress to the soft tissues at low salinity, although the critical salinity value [19] is lower than for females. Although isolation of the mantle cavity prevents suspension-feeding, it is not clear whether the radula continues to scrape the substrate, either in males or females.

The three modes of action of the radula in feeding by calyptraeid species (manipulation of the mucous cord, removal of mucous balls from the food pouch and scraping the substrate) vary in importance according to the stage of development [23, 24, 27] and to environmental conditions [36]. In C. dilatata suspension-feeding continues uninterrupted at salinities above 22.5 [8, 17], and the radula plays a role by transferring the mucous cord to the mouth. Factors other than salinity may also be important, however, such as the characteristics of the water column, mobility or sessility of the individual, sex and, for females, reproductive condition (incubating or non-incubating). The objective of this study was to determine the roles played by the radula in feeding in females (brooding and non-brooding) and males during various stages of the tidal cycle in different seasons of the year. In particular, the study contributes to an understanding of the impact of temporal changes in the water column on the radular feeding behaviour of an organism that is sessile as female and therefore unable to escape adverse conditions. Furthermore, the paper explores the dependence of feeding responses on the developmental stage of the individual and, for females, the brooding status (brooding or non-brooding).

Estuarine systems experience variations in environmental conditions in the water column as a result of stochastic atmospheric forcing [2, 37, 38] and tidal cycles [2, 8, 39]. In our study significant tidal and seasonal variation was observed in the concentration of seston (total particulate matter, TPM) in the Quempillén estuary, values being generally highest in summer (January), which is consistent with previous studies [37, 40, 41] and is typical of the seasonality characteristic of estuarine and shallow coastal systems in mid- to high latitudes [38, 41, 42]. The high TPM values we observed are associated with high standing stocks of phytoplankton in this estuary [7]. TPM values were low in winter (August), despite resuspension of sediment produced by strong wind events and precipitation [43]. As in nearby coastal areas [42], seston levels at Quempillén are largely determined by phytoplankton dynamics rather than sediment resuspension [7]. Similar patterns of variation (tidal superimposed on seasonal) in seston have also been recorded in coastal habitats, including estuaries, in other latitudes [2, 44, 39].

On several occasions we observed high variation in salinity associated with the tidal cycle in the Quempillén estuary, especially in winter and spring, when precipitation is high (http://164.77.222.61/climatologia/) and salinity can fall rapidly to values very close to zero at low tide during periods of heavy rainfall. Occasionally heavy rain can also occur in summer, but we did not encounter rainstorms during our January sampling. Salinity variation, particularly low salinity, is an important stress factor in estuarine systems [15, 16] and a significant regulator of physiological and behavioral processes in estuarine organisms [11, 13, 17]. The feeding behaviour of marine invertebrate suspension-feeders can be strongly impacted by exposure to low salinity, which frequently results in cessation of feeding or at least a reduction in clearance rate [1, 8, 11, 13, 17]. In many cases the individual also responds by partially or completely isolating itself from the surrounding environment to minimise osmatic stress [17]. Critical values of salinity have been identified below which calyptraeid gastropods adhere closely to the substrate, isolating the mantle cavity from the exterior, with the result that suspension-feeding ceases (e.g. salinity 22–24 for C. peruviana, 52; salinity 23–24 for C. dilatata, 17).

In our study, the degree to which the mantle cavity of C. dilatata was isolated from the ambient water, and the use of the radula in its various feeding modes, depended largely on environmental conditions, particularly salinity, and on the sex and reproductive status of the individual. In general, non-brooding females exhibited higher rates of radular extrusion for ingesting the mucous cord and for scraping the substrate than did brooders. In some suspension-feeding brooding mollusc species the incubated embryos, whether free or encapsulated, can remain for days or weeks within the mantle cavity of the female [23, 45, 46]. Their presence can physically interfere with the capture of food particles by the gill, thereby modifying clearance rate [47, 48]. In calyptraeids, particles captured on the gills are bound in mucous cords on the distal margins of the gill filaments and moved towards the mouth [23, 30]. Our observation that radular activity related to ingestion of the mucous cord is lower in brooding female C. dilatata than in non-brooders is consistent with a lower clearance rate. In some species, e.g. C. peruviana, the frequency of formation of mucous cords depends on the concentration of particles in the water column [36], as long as salinity exceeds the threshold level for isolation. Nevertheless, our data show that under the low salinity regimes prevailing in many of the tidal cycles we studied, salinity was a determinant of the amount of mucous cord material produced.

According to Navarro and Chaparro [25], removal of particles by the gill and their transfer to the mouth within a mucous cord located in a muscular canal on the right side of the neck is the principal means of food acquisition by female calyptraeids, which are sessile. In our study of C. dilatata the rate of radular extrusion for ingesting the mucous cord varied seasonally and between brooding and non-brooding females. The lowest activity was recorded in the winter during periods in the tidal cycle when salinity decreased below 23. Under these conditions the mantle cavity is sealed from the external environment, suspension-feeding ceases and there is no production of a mucous cord. This behaviour has been recorded previously for C. dilatata by Montory et al. [49] and for C. peruviana and the oyster Ostrea chilensis by Chaparro et al. [17]. The difference in the critical salinity between females [23] and males [19] may be attributable to the fact that larval stages of marine invertebrates are often more sensitive than adults to low salinity [50–53].

The rate of radular extrusions for removing the mucous ball from the food pouch, however, did not differ between brooding and non-brooding females in our study. The origin of the material in the food pouch is not clear, although some comes from the anterior region of the gill and some from the mantle wall [30]. The mucous ball plays a much smaller role in feeding than the mucous cord, which carries far more material to the mouth; furthermore, material from the food pouch can be rejected as pseudofaeces as well as ingested, at least in C. peruviana [36]. Thus it is possible that the process of moving material to and from the food pouch can continue in brooding females despite the apparently reduced feeding activity.

In both brooding and non-brooding females the activity of the radula in removing the mucous ball from the food pouch was strongly influenced by the tidal cycle during winter, reaching minimum values at low tide. Under these conditions salinity can decrease well below the critical value of 22–23 at which the female responds by isolating the mantle cavity from the external environment, thereby protecting the soft tissues and the embryos (in the case of brooders) from osmotic stress [17, 49, 50, 54]. Suspension-feeding ceases, and the very low rates of exclusion of the radula for removal of material from the food pouch may represent the arrival in the food pouch of residual particles in the mantle water and/or particles in the mantle tracts, or the use of material accumulated in the food pouch before isolation of the mantle cavity. In males there was no evidence that the tidal cycle affected radular activity in removing the mucous ball at any time of the year.

We observed differences among seasons and within tidal cycles in radular activity associated with scraping of the substrate in C. dilatata, as well as differences among males, brooding females and non-brooding females. Incubating females displayed much less scraping than non-brooders throughout the year. In C. peruviana the incubated capsules are affixed to the substrate and occupy as much as 87% of the area within the mantle cavity [55], restricting access to the substrate by the radula. Assuming that C. dilatata is similar in this respect, the substrate surface accessible for grazing is limited to the small area beneath the shell margin, reducing the availability of biofilm material for grazing. The fact that radular activity is reduced when the area available for grazing is limited may be a response by the individual to increase its efficiency of use of the food resource. Males exhibit a higher rate of radular activity in scraping throughout the year, including periods of low salinity, than in removing the mucous ball or ingesting the mucous cord. This does not necessarily imply that more energy is obtained through substrate grazing than suspension-feeding, since the relationship between the frequency of radular extrusion and food ingestion must vary among the different forms of feeding and according to environmental conditions. The data for rates of radular exclusion indicate the “division of labour” for the radula among three separate tasks associated with feeding, and inferences cannot be drawn about the nutritional significance of each type of radular activity under any given set of conditions.

In C. dilatata, juveniles and males are motile [35] and therefore have much more opportunity to complement suspension-feeding with substrate grazing [28]. Similar behaviour has been observed in motile individuals of C. convexa [56] and C. adunca [57]. During periods when the salinity is below the critical value of 19 and the C. dilatata male isolates the mantle cavity, preventing suspension-feeding, substrate rasping can continue, as demonstrated by a high rate of radular extrusion. When salinity increases and the mantle is re-irrigated, suspension-feeding resumes and the radula both scrapes the substrate and pulls the mucous cord into the mouth.

Throughout the year, about 65% of radular extrusions in the brooding female C. dilatata are for the purpose of ingesting the mucous cord, 18% for rasping the substrate and 15% for removing the mucous ball from the food pouch. In non-brooding females, mucous cord ingestion remains the principal activity of the radula (46% of total extrusions), but substrate rasping is relatively greater (40%) than in brooders and mucous ball removal accounts for a similar proportion (14%). Most of the radular activity in males (55%) was associated with substrate grazing, 12% with removing the mucous ball (similar to females) and 33% with manipulating the mucous cord. Thus, unlike males, females use the radula more for ingesting the mucous cord (i.e. suspension-feeding) than for substrate grazing. The feeding behaviour of both males and females is modulated by salinity, independently of the quality and quantity of seston in the estuary, but the principal determinants of radular activity are the mode of reproduction (brooding in females) and the ability to move (males). Calyptraeid gastropods are unusual in having two discrete feeding mechanisms, rasping the substrate with the radula and suspension-feeding with the gill.

In estuarine invertebrates, especially sessile suspension-feeders, salinity is the principal environmental factor to which feeding activity responds [8, 15, 17]. Our data for C. dilatata demonstrate differential responses in feeding behaviour in a sequential hermaphroditic species according not only to salinity but also to the degree of mobility of the individual (sessile vs. non-sessile), which is dependent on its sex and reproductive status. Incubation of embryos in calyptraeids results in modification of the feeding behaviour of the female, including restriction of the area available for rasping, as in C. dilatata, and changes in availability of suspended particles owing to changes in the inhalent flow associated with the presence of embryos, as in C. fecunda [33, 55].