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Journal of Experimental Marine Biology and Ecology

Examining the physiological plasticity of particle capture by the blue mussel, Mytilus edulis (L.): Confounding factors and potential artifacts with studies utilizing natural seston

ABSTRACT

Historically, particle capture efficiency (CE) in suspension-feeding bivalve molluscs has been shown to be strongly dependent on particle size, increasing asymptotically to a maximum of about 100% for particles ca. ≥  4 μm in diameter. Recent advances in the analysis of the particulate matter of seston have allowed for more precise studies of bivalve feeding under natural conditions. Some studies have reported that the mechanisms associated with particle capture exhibit physiological plasticity, and under certain conditions smaller cells and particles are captured in preference to larger ones. For bivalves, however, there is no mechanistic explanation that would account for such fine-scale control of CE based on size. The current study experimentally assessed the seasonal control of CE by the blue mussel, Mytilus edulis , employing a flow-through system to examine particle capture of natural seston. The natural particle field was analyzed using two different types of particle analyzers, the LISST-100X and the Coulter Multisizer IIe. Mussels were simultaneously delivered synthetic microspheres of defined diameter (2–45 μm) to control for the effects of seasonal differences in the size and shape of natural particles. The capture of microspheres was quantified by means of flow cytometry (FCM), and results cross-checked with the Multisizer. Additionally, gene expression of a mucosal lectin (MeML) associated with the feeding organs of mussels was examined as a biomarker for physiological response to seasonal changes in the particle-food supply. Results demonstrated that for microspheres ≥  4   μm CE of mussels was always near 100%, and did not change seasonally. In contrast, there was an apparent seasonal shift in CE of natural particles, with particles 17-to-35   μm in equivalent spherical diameter (ESD) occasionally being captured at lower efficiencies than particles 4-to-15 μm in ESD (e.g., during September and December). No relationship between MeML expression and seasonal CE was found. These findings call into question the physiological plasticity of CE in mussels and alternative hypotheses are presented. We suggest that the purported changes in CE are not a consequence of behavioral or physiological responses of mussels, but rather a result of one or more of the following confounding factors; 1) instrument artifacts that can arise as a result of the way in which laser and electronic particle counters calculate ESD to estimate particle size; 2) disaggregation of flocculent material collected from control chambers; 3) post-capture escape of highly motile phytoplankton cells from the infrabranchial camber; 4) qualitative factors of the particles that could affect capture; or 5) mathematical happenstance of calculating CE on particle-size classes that contain widely different numbers of particles.

Introduction

Suspension-feeding bivalve molluscs are one of the most important groups of animals in coastal ecosystems, often dominating the macrobenthos (Dame, 1996). Bivalves are exposed to large amounts of suspended matter that include both nutritious and non-nutritious particles (Newell, 1965, Owen, 1966, Newell et al., 1989). As a way to process efficiently the complex mixture of material that they encounter, bivalves have evolved capabilities for selective feeding that allow them to reject some of the captured material in pseudofeces (Loosanoff, 1949, Foster-Smith, 1975, Shumway et al., 1985, Bacon et al., 1998). Selective feeding can be based on physical factors such as particle size and shape (Bayne et al., 1977, Shumway et al., 1985, Cognie et al., 2003, Mafra et al., 2009), as well as physicochemical interactions between the particles and the feeding organs (see reviews by Jørgensen, 1996, Ward and Shumway, 2004). Rosa et al. (2013) demonstrated that the eastern oyster, Crassostrea virginica, and the blue mussel, Mytilus edulis, can discriminate between particles of the same size based upon the surface charge and wettability of particles. More specific chemical interaction between lectins in the mucus of pallial organs and carbohydrates present on the surfaces of microalgal cells has also been demonstrated. In several studies, workers confirmed that carbohydrate-lectin interactions are involved in mediating particle sorting in both C. virginica and M. edulis (Pales Espinosa et al., 2009, Pales Espinosa et al., 2010a). A mucosal C-type lectin (dubbed MeML) in the blue mussel, M. edulis, whose expression could be involved in particle sorting was also identified (Pales Espinosa et al., 2010b). Most recently, Pales Espinosa and Allam (2013) demonstrated that the expression of MeML is regulated in response to the quality and quantity of food offered, further suggesting a physiological basis for qualitative particle selection.

Although post-capture selection has been well studied, much less is known about selective retention during particle capture. Particle capture is the first step in suspension feeding and is a consequence of two interrelated processes: particle encounter and particle retention (Ward et al., 1998). Encounter efficiency relates to the proportion of particles that come into contact with the capture unit, in this case the gill filaments, whereas retention efficiency is the proportion of encountered particles that are actually retained (see Shimeta and Jumars, 1991). Although previous workers have used the term "retention efficiency" to describe particle capture efficiency in bivalves (Riisgård, 1988, MacDonald and Ward, 1994, Cranford and Hill, 1999, Strøhmeier et al., 2012), unless in vivo techniques are employed to differentiate the number of particles that are encounter from those that are actually retained (Ward et al., 1998), retention efficiency cannot be determined. Therefore, in this study we use the term capture efficiency (CE) to accurately describe the process being measured. Future studies using in situ techniques such as those described herein should use CE, which is the more exact term, in place of retention efficiency.

In general, capture efficiency has been reported to increase non-linearly with increasing particle diameter to a maximum of about 100%, with some species of bivalves being more efficient at capturing small particles than other species (see Ward and Shumway, 2004). If particles were differentially captured at the larger size threshold, this would be a form of particle selection. Preferential retention of particles could thus be an important discriminatory mechanism that alters the composition of material subjected to post-capture selection and ingestion. Although previous studies have demonstrated that many feeding processes of bivalves are under physiological control and respond to changing environmental conditions (=   physiological plasticity; see Bayne, 2004 for review), uncertainty exists regarding the physiological plasticity of CE. Some studies suggest that different bivalve species can shift their maximum capture efficiency as a response to changes in the particle size distribution of the seston (reviewed in Ward and Shumway, 2004). This includes shifts in CE to coincide with larger particles containing a higher organic content by the rock-tide bivalve Venerupis corrugatus (Stenton-Dozey and Brown, 1992), and lower capture of smaller particles as the concentration of inorganic particles and overall particle loads increase by the scallop Placopecten magellanicus, and the oyster Crassostrea gigas (e.g., clay, Cranford and Gordon, 1992, Barillé et al., 1993, respectively). Recently, Strøhmeier et al. (2012) reported a seasonal variation in capture efficiency in the blue mussel M. edulis. These workers used a flow-through system to calculate CE and clearance rate, and compared these to the size distribution and concentration of particles in the natural seston. They reported that CE increased and reached a maximum for larger particles (30 to 35   μm) in early summer. Later in the season, when smaller particles dominated the seston, 7-to-15   μm particles were retained at higher efficiencies over the larger particles (30 to 35   μm), indicating a seasonal, size-dependent shift in particle capture. Overall, these studies suggest shifts in CE as a result of changing environmental conditions (e.g., tide, season).

In a few cases, qualitative factors have also been shown to affect CE. The European oyster, Ostrea edulis, for example, was found to capture the dinoflagellate Prorocentrum minimum preferentially over two other algal species of the same size (Shumway et al., 1985). Other studies have reported similar results for mussels and scallops (Newell et al., 1989, Shumway et al., 1997), and the authors suggested that capture was based on cell properties other than size. Hernroth et al. (2000) examined the effects of surface properties on particle uptake, and found that changing the electrostatic charge of bacteria (~   1   μm) affected their capture by M. edulis. Similarly, Yahel et al. (2009) reported size-independent capture of particles in the tropical bivalve Lithophaga simplex. Using flow cytometry to examine the types of particles entering the inhalant siphon and comparing these with particles exiting the exhalent siphon, the authors determined that some algal species were captured at higher proportions than others, despite the overlap in mean cell size distribution.

Some studies have also reported that bivalves can capture smaller particles at a higher efficiency than larger particles (e.g., Strøhmeier et al., 2012). Lesser et al. (1991), for example, reported the clearance of the toxic dinoflagellate Alexandrium tamarensis (30–45   μm) by scallops at significantly lower rates than smaller (~   16   μm diameter) phytoplankton species. Using a Coulter counter to examine particle selection in M. edulis, Bayne et al. (1977) reported that cells of Phaeodactylum tricornotum (~   6   μm) were captured in higher quantities than larger inorganic particles, a shift the authors suggested may be a result of the coulter counter using spherical equivalents to calculate diameter. Using flow cytometry, Pile and Young (1999) reported that the cold-seep mussel Bathymodiolus childressi captured bacteria at significantly higher proportions than larger protozoans. Although the above reports suggest that particle capture in bivalves may be physiologically plastic and responsive to changes in the particle food supply, controls for the effects of seasonal changes in the relative abundance of different shaped particles, such as using microspheres of defined geometries, were not usually included in these studies. Apparent changes in CE could result from changes in the proportion of particles with different geometries, or other effects unrelated to physiological plasticity. To date, no mechanism has been proposed for bivalves that would account for the capture of smaller particles in preference to larger sized particles. Resolving results from field studies of CE with current models of particle capture mechanisms is important not only for a deeper knowledge of particle capture and selection processes, but also for a better understanding of how selective grazing by bivalves could affect phytoplankton species composition and impact food web dynamics in near-shore waters (see Dame, 1996).

This project was designed to assess experimentally the seasonal shifts in particle CE of M. edulis, and to compare CE of natural particles to that of microspheres with defined size and shape in order to specifically examine the physiological plasticity of particle capture. Changes in gene expression of a mucosal lectin (MeML), previously shown to be involved in particle feeding in mussels (Pales Espinosa et al., 2010b), were also investigated. A correlation between seasonal changes in CE and expression levels of MeML would further demonstrate a physiological response to changing particle fields.

Section snippets

Study animals

Blue mussels, M. edulis, were collected locally from wild populations at the Avery Point Campus of the University of Connecticut in Groton, CT, USA. Animals were maintained in lantern nets hung from a dock adjacent to the intake line of the flow-through experimental system (see below). Mussels were acclimated to these conditions for at least 2   weeks prior to start of the feeding experiments.

Feeding experiments

Studies were carried out seasonally on two separate days for a period of 1   year (March, May, July,

Environmental and seston parameters

As expected, environmental conditions varied seasonally during the one-year study (Table 1). Water temperature ranged from 3.5 to 24.5   °C, and salinity ranged from 30 to 35. The total particulate matter (TPM) ranged from 4.04   mg/L in December to 7.50   mg/L in March 2014. There were no significant differences in TPM between each of the seasons sampled. The particulate organic matter (POM) fraction was significantly higher during March 2013 (3.09   mg/L) and significantly lower in December 2013 (0.55

Discussion

Results reported here demonstrate several important points regarding the capture of particles by mussels and point out potential errors associated with use of different instruments. First, the capture of the spherical particles ≥   4   μm in diameter was consistently high across all sampling months, with only the 2-μm particles being captured at a lower efficiency than particles of greater size. These data are consistent with current understanding of hydrosol filtration mechanisms employed by

Acknowledgments

Funding for this project was provided by a National Science Foundation grant to JEW and SES (IOS-1147122), and EPE and BA (IOS-1050596 and IOS-1146920). Additional support was provided by student research grants from the University of Connecticut, Department of Marine Sciences and the American Malacological Society to MR. The authors would like to thank the Dierssen COLORS laboratory for use of their LISST-100X, and B. Russell, K. Bostrom, and K. Randolph for logistical and MATLAB assistance.

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