Preparation of large bivalve specimens for scanning electron microscopy using hexamethyldisilazane (HMDS).

ABSTRACT Treatment with hexamethyldisilazane (HMDS) for drying specimens for Scanning Electron Microscopy (SEM) is described for relatively large (>20 mm shell height, SH) individuals of two scallop species, Placopecten magellanicus and Argopecten irradians. The traditionally used method of critical point drying (CPD) has been applied successfully for smaller specimens (~350 [micro]m up to 15 mm SH); however, occasional rupture of the mantle occurred in these specimens. Furthermore, size limitation of the CPD chamber did not permit preparation of larger individuals. An alternative experimental protocol using HMDS was tested, which successfully preserves the morphology of the bivalve feeding organs, mantle surface and the cilia of the two scallop species with negligible shrinkage and few artifacts. Another benefit of this method is that the number of specimens and sample size is not restricted.

KEY WORDS: bivalve, scanning electron microscopy, method, hexamethyldisilazane (HMDS), ciliation, shrinkage

INTRODUCTION

Scanning electron microscopy (SEM) is a very useful technique to study morphology and surface microstructure of various biological specimens in three dimensions. The drying process is one of the critical steps in specimen preparation for SEM (Laforsch & Tollrian 2000). However, removing water from specimens, particularly hydrated organisms normally causes distortion and collapse because of the effects of surface tension. The two principal methods used to reduce distortion during specimen drying for SEM are critical point drying (CPD) and freeze drying or lyophilization (Maugel et al. 1980, Boyde 1980). Air drying after preparation in organic compounds with low surface tension such as Peldri II (Brown 1990), hexamethyldisilazane (HMDS) (Nation 1983), tetramethylsilane (TMS) (Dey et al. 1989) and dimethoxypropane (DMP) (Muller & Jacks 1975) has also been used.

Critical point drying was first proposed by Anderson (1951) and is one of the techniques more commonly used to dry biological specimens for SEM (Maugel et al. 1980, Boyde 1980). Although we have used CPD for smaller bivalve specimens, namely sea scallop postlarvae, Placopecten magellanicus, ~350 [micro]m to 15 mm in shell height (SH), with excellent results (Barre 2001, Veniot et al. 2003), intermittent rupture of the fragile mantle occurs with this method. Additionally, size limitations of the CPD chamber do not permit the use of this method for larger animals (>20 mm). In this study larger bivalve specimens were dried using a HMDS protocol that minimizes shrinkage and preserves the cilia and fragile mantle of the organisms.

The principle of the technique relies on replacement of an ethanol dehydration medium with HMDS, which is subsequently evaporated. HMDS has a lower surface tension than water and is believed to provide structural support by cross-linking proteins (Nation 1983, Braet et al. 1997). The suitability of HMDS for drying biological specimens has been shown on insects (Nation 1983, Heraty & Hawks 1998), aquatic organisms (shrimp eggs, daphnids, gastrotrichs) (Moraes & Bouzon 1995, Hochberg & Litvaitis 2000, Laforsh & Tollrian 2000), organic microstructures on bivalve shells (Schone & Bentley 2002), pollen (Chissoe et al. 1994), dinoflagellates (Botes et al. 2002), anaerobic biofilms and granular sludge (Araujo et al. 2003), and cultured rat hepatic endothelial cells (Braet et al. 1997). Most of the studies have examined insects and small specimens, typically <10 mm.

Here we use SEM to show the morphology of feeding organs (gill, foot, labial palps and arborescent lips), and ultrastructural characteristics such as ciliation in large specimens of two bivalve species, the sea scallop, Placopecten magellanicus, and bay scallop, Argopecten irradians. An experimental protocol has been used that successfully preserves the organ morphology and cilia of large bivalves (>20 mm SH). Our observations will contribute to understanding the feeding mechanisms of these animals during development.

MATERIALS AND METHODS

Large specimens (>20 mm SH) of the two scallop species P. magellanicus and A. irradians were collected for examination by SEM. Scallops were sampled from field sites and reared at the Institute for Marine Biosciences' Marine Research Station (MRS), Ketch Harbour, Nova Scotia, Canada.

Animals were refrigerated at 4 [degrees]C for one hour prior to anesthetization to decrease their metabolism. A gradual addition of MS-222 (Tricaine Methanesulfonate) and KCI (5% potassium chloride) allowed for both muscle tissue relaxation (gaping valves and non contracted tissues) and limitation of mucus production. During this process, sample vials (250-mL glass jars) were not agitated and kept at 4 [degrees]C. Scallops were fixed in IG4F (1% glutaraldehyde, 4% formaldehyde, in 0.2M P[O.sub.4], pH = 7.5) for a minimum of 48 h at 4 [degrees]C.

During subsequent processing, samples were gently agitated in a fume hood at room temperature. Specimens were rinsed twice for one hour in a 0.2 M P[O.sub.4] buffer solution (pH = 7.2) and dehydrated in ascending ethanol washes (twice at each concentration for one hour in 50, 70, 80, 95 and finally 100% ethanol). Transitional steps of 100% ethanol (30 min, 30 min, 1h) and then HMDS: ethanol (1:1) (30 min, 30 min, 1 h) were followed by two baths (1 h each) of 100% HMDS. Finally the jars were placed in a dessicator with silica gel with just enough HMDS to cover the specimens.

The gross morphology of the scallops was observed using a Wild M400 Photomakroskop and a Nikon Coolpix 4500 digital camera. For SEM observation, specimens were mounted on aluminum stubs with double-sided carbon tape and the right valve was removed. Samples were then sputter-coated with gold-palladium (Model SC7620 Polaron) and examined using a Hitachi Model S-3000N SEM at 20kV.

RESULTS AND DISCUSSION

Specimens of P. magellanicus dried after HMDS treatment showed well-preserved surfaces with negligible shrinkage of the tissues and few artifacts (Plate 1, Fig. 1, 2, 3). The extended foot and intact gill (Plate 1) were relaxed and showed good preservation. The fragile mantle surface was well preserved with no shrinkage or tears (Plate 1, Fig. 2). The radial muscles were visible and well preserved. Feeding organs (lips, labial palps and foot) were intact and the arborescent lips (described and drawn by Beninger et al. 1990) were photographically recorded for the first time (Plate 1, Fig. 3). The labial palps--gill connection (type III according to Satsek 1963) was also shown (Plate 1, Fig. 3).

[FIGURES 1-3 OMITTED]

HMDS drying also provided excellent results in the preservation of microstructural characteristics such as ciliation of scallop feeding organs, as illustrated in the gills and labial palps of A. irradians specimens (Plate 1, Fig. 4, 5, 6). There were no visible distortions of the cilia as shown on the frontal cilia of the gill (Plate 1, Fig. 4) or the cilia of the ridges of labial palps (Plate 1, Fig. 6). Tufts of cilia on dorsal expansions of the gill were not shrunken and cilia at the base of the tufts were not clumped (Plate 1, Fig. 5).

[FIGURES 4-6 OMITTED]

HMDS treatment has been used for a number of different specimen types, generally less than 10 mm and was shown to require less time and expertise than critical point drying (Hochberg & Litvaitis 2000, Laforsch & Tollrian 2000, Schone & Bentley 2002). The method is used in this study to prepare large specimens of scallops P. magellanicus and A. irradians for observation of feeding organs and cilia development. Oshel (1997) showed that HMDS use was effective for preparing soft and highly hydrated specimens. Also, HMDS drying preserved excellent surface details in insect tissues (Nation 1983, Heraty & Hawks 1998) and hepatic endothelial cells (Braet et al. 1997). This work shows that large specimens (20-30 mm SH) of marine bivalves can be preserved and dried for SEM without major distortion or collapse and microstructural details are retained.

Intermittently, when using the CPD method for smaller specimens (scallops <15 mm SH) the mantle surface was partially or totally destroyed. All of the samples in this study consistently had intact mantle surfaces. Another difficulty encountered in the preparation of ciliated epithelia for SEM is the tendency for the cilia to stick together and for foreign particles to remain on the cilia. The ciliated surfaces of the feeding organs in A. irradians dried using HMDS showed nonclumped, relatively particle-free tufts (Plate 1, Fig. 4, 5, 6). Drying from HMDS seemed to prevent shrinkage, whereas the CPD method appeared to have caused some distortions (Barre 2001). Another advantage of the HMDS method is that the number of specimens and sample size are not restricted. Thus we conclude that the quality of the results obtained and ease of use of the HMDS drying process make it preferable to the CPD method for processing large bivalve specimens.

ACKNOWLEDGMENTS

The authors thank Steve McKenna and Scott MacQuarrie of the National Research Council, Institute for Marine Biosciences for helping rear the animals at MRS; Cindy Leggiadro of the Microscopy Facility for her expertise; Anne Veniot, University of Moncton, New Brunswick, Canada, for assistance in training and C. Barre in SEM methodology, during early stages of her research. This research was supported by an NCE AquaNet grant to V. M. Bricelj (IMB) and C. Parrish and R. Thompson from the Ocean Sciences Centre, Memorial University of Newfoundland.

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CELINE BARRE, (1,2) DAVID O'NEIL (2) AND V. MONICA BRICEL J (2)

(1) Department of Biology Dalhousie University, Halifax, Nova Scotia, B3H 4J1 Canada; (2) Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax Nova Scotia, B3H 3Z1 Canada

Corresponding author. E-mail: David.O'[e-mail]