- Split View
-
Views
-
Cite
Cite
Julia C. Marxen, Oleg Prymak, Felix Beckmann, Frank Neues, Matthias Epple, Embryonic shell formation in the snail Biomphalaria glabrata: a comparison between scanning electron microscopy (SEM) and synchrotron radiation micro computer tomography (SRµCT), Journal of Molluscan Studies, Volume 74, Issue 1, February 2008, Pages 19–26, https://doi.org/10.1093/mollus/eym044
- Share Icon Share
Abstract
Embryos of different developmental stages and newly hatched juveniles of the freshwater snail Biomphalaria glabrata were investigated by synchrotron radiation micro computer tomography (SRµCT). Because this method is sensitive for objects with a high X-ray density, it is ideally suited to study mineralized tissues without the need for dissection of the sample, i.e. removal of the soft tissue. This is a clear advantage over scanning electron microscopy (SEM). However, the resolution is inferior to SEM (about 1–2 µm compared to a few nm). After the measurement, computer-processed handling (virtual turning, cutting and measuring) of the object is possible. The development of the calcified shell in embryos before hatching (age 60, 72, 96 and 120 h after oviposition) was investigated and both methods were compared. While it was not possible to find a calcified shell in 60 h old embryos, the shell in 72 h old embryos was almost fully mineralized. By SRµCT, the weight of the calcified shell was estimated to 0.64, 9.59 and 30.3 µg for embryos of 72, 96 and 120 h. All juvenile snails, of 5 days and 4 weeks after hatching, contained concretions in the stomach, mostly consisting of calcium phosphate.
INTRODUCTION
Biomineralized structures such as bones, teeth, mollusc shells or corals are of interest since their mechanical strength is increased compared to pure inorganic minerals. The composite material, consisting of diverse inorganic minerals (Lowenstam & Weiner, 1989; Baeuerlein, 2000, 2004; Mann, 2001) in combination with specific proteins and carbohydrates is constructed in a variety of ultrastructural modes that are determined mainly by the organic part (Albeck et al., 1993; Aizenberg et al., 1996, 1997; Falini et al., 1996; Gotliv et al., 2003). The details of biomineral formation, however, are still unknown. Among biominerals, molluscan shells are of special interest since on the one hand a remarkable number of morphological modifications are seen and on the other hand the shell-forming mantle edge has a clear sequential organization (Timmermanns, 1969; Simkiss & Wilbur, 1989; Addadi et al., 2006).
The shell of the adult snail Biomphalaria glabrata is well investigated with respect to aspects of biochemistry (Marxen & Becker, 1997, 2000; Marxen et al., 1998, 2003b, c) and mineralogy (Hasse et al., 2000; Marxen et al., 2003a; Prymak et al., 2005). The cross-lamellar structured shell consists of calcium carbonate, almost completely in the aragonite phase (Hasse et al., 2000). In embryos, shell building starts 48 h after oviposition with the formation of the periostracum. Six to twelve hours later amorphous calcium carbonate (ACC) is deposited, which transforms to aragonite (Marxen et al., 2003a), in good accord with current models in biomineralization (Weiss et al., 2002; Addadi, Raz & Weiner, 2003; Addadi et al., 2006). Five to six days (120–144 h) after oviposition, the embryos hatch. The early shell formation, the first calcium delivery and the calcium distribution in the embryonic shell are of special interest. This has been reported by us in a study using scanning and transmission electron microscopy (SEM and TEM) in combination with electron spectroscopic imaging (ESI), X-ray diffraction and X-ray absorption spectroscopy (EXAFS) on dissected embryos (Marxen et al., 2003a). However, this always involved a delicate preparation of the embryos which may have caused artefacts, and did not permit a three-dimensional investigation of the developing shell. In a preliminary study, we have applied synchrotron radiation micro computer tomography (SRµCT) to a 120 h old embryo of B. glabrata and have shown that it is possible to derive quantitative structural parameters (like the thickness of the shell at different points) (Prymak et al., 2005). The major advantage of this method is the possibility to distinguish between hard (i.e. mineralized) and soft tissue without the need for preparation (Neues et al., 2006), followed by a three-dimensional analysis of the structures (Vanis et al., 2006). Conventional µCT and SRµCT were applied to a number of biological samples, such as bone from several species, enamel, sea urchin teeth (Nuzzo et al., 2002; Stock et al., 2002, 2003a, b, 2004; Martin-Badosa et al., 2003; Dowker et al., 2004; Jaecques et al., 2004; Kinney et al., 2005), for destruction-free insights into the internal morphology of such biomineralized tissues. In general, µCT is presently gaining increasing application in the study of bone tissues and of biomaterials (Cancedda et al., 2007; Porter et al., 2007; Van Lenthe, 2007). However, it has never been applied to the delicate features of the developing snail shell to gain a picture of its development in the embryonic state.
Here we report an investigation of the developing shell of B. glabrata both in the embryonic and in the juvenile state using SRµCT in comparison to classical scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Thus, morphological data (by SEM) are directly compared with three-dimensional structural data (by SRµCT).
MATERIAL AND METHODS
Animals
Adult and juvenile tropical freshwater snails Biomphalaria glabrata (Say, 1818), strain Puerto Rico, were kept in aerated running tap water, preheated to 28±0.5°C, and fed ad libitum with fish food and lettuce. Newly-laid spawning packs were separated and kept under the same conditions. 60, 72, 96 and 120 h after oviposition the eggs were cooled to 8°C and the embryos were carefully removed from the egg capsules for further treatment. Furthermore, juvenile snails between 5 days and 4 weeks after hatching were investigated. Note that 5 days after hatching corresponds to about 240 h after oviposition.
Scanning electron microscopy (SEM) and energy-dispersiveX-ray spectroscopy (EDX)
Whole intact embryos of different ages, and juvenile snails between 5 days and 4 weeks after hatching with a shell diameter of 1–3 mm were rinsed with 10 mM tris–maleate buffer (pH 7.4) and fixed for 2 h at 20°C in 2% glutaraldehyde (E.M. grade, Agar Scientific) in the same buffer. Thereafter the specimens were washed three times with 10 mM tris–maleate buffer at 4°C, dehydrated in ascending series of ethanol, transferred to amylacetate/ethanol (3:1=v:v) and critical-point dried. The embryos (about 20 of each age) were attached with double-sided tape on specimen holder stubs, sputtered either with gold (SEM) or graphite (EDX) (sputter coater GEA 004 S), and investigated on a scanning electron microscope (Leo 1525 Gemini) equipped with an EDAX Analysis System Falcon HX 0291 for elemental analysis.
Synchrotron radiation micro computer tomography (SRμCT)
After fixation, rinsing and dehydration as described above between six and eight embryos and juveniles of each age were embedded in Spurr's medium (Spurr, 1969) in plastic dishes of suitable shape. The edge length of the Spurr blocks was 1.8–2 mm for the 60, 72, 96 and 120 h old embryos and 4–5 mm for the juvenile snails. Microtomography was carried out at beamline BW2 at HASYLAB/DESY (Hamburg, Germany). The synchrotron radiation was monochromatized to 17 keV photon energy for the juvenile snails (2–3 mm shell diameter), to 12 keV photon energy for the 72 h embryos and to 10 keV photon energy for all other samples. The 72 h embryos and the juvenile snails were measured at higher photon energy because of the higher absorption of the samples (increasing degree of mineralization). The measurements were typically carried out with one animal per age; in some cases two were studied. Due to the limited beamtime at the synchrotron, it was not possible to investigate more animals.
The projection images were recorded in the angular range of 0°–180° with incremental steps of 0.25°. The detector was a KX2 instrument (Apogee Instruments; 14-bit digitization at 1.25 MHz, 1,536·1,024 pixel2; each 9·9 µm2). For normalization of the recorded data, flat field images of the beam were recorded every eight projections. Reconstruction was performed using a filtered backprojection algorithm (Donath et al., 2004). Qualitative three-dimensional reconstruction work was done at HASYLAB (Donath et al., 2004). Three-dimensional renderings were created with the program VG Studio MAX 1.2. Using the known voxel side lengths, the characteristic lengths (e.g. the diameters) were calculated (see also Table 1).
Age . | Magnification . | Binning factor . | Edge length of voxel edge/µm . | Volume per voxel/µm3 . | Number of voxels in the shell . | Total shell volume/µm3 . | Shell weight/µg . |
---|---|---|---|---|---|---|---|
72 h after oviposition | 6.0 | 1 | 1.49 | 3.32 | 6.54·104 | 2.17·105 | 0.64 |
96 h after oviposition | 4.7 | 1 | 1.91 | 7.02 | 4.63·105 | 3.25·106 | 9.59 |
120 h after oviposition | 6.0 | 1 | 1.51 | 3.41 | 3.02·106 | 1.03·107 | 30.3 |
240 h after oviposition (5 days after hatching) | 4.7 | 2 | 3.83 | 56.2 | 1.77·106 | 9.92·107 | 293 |
4 weeks after hatching | 2.9 | 2 | 6.18 | 235.9 | 2.93·106 | 6.92·108 | 2,041 |
Age . | Magnification . | Binning factor . | Edge length of voxel edge/µm . | Volume per voxel/µm3 . | Number of voxels in the shell . | Total shell volume/µm3 . | Shell weight/µg . |
---|---|---|---|---|---|---|---|
72 h after oviposition | 6.0 | 1 | 1.49 | 3.32 | 6.54·104 | 2.17·105 | 0.64 |
96 h after oviposition | 4.7 | 1 | 1.91 | 7.02 | 4.63·105 | 3.25·106 | 9.59 |
120 h after oviposition | 6.0 | 1 | 1.51 | 3.41 | 3.02·106 | 1.03·107 | 30.3 |
240 h after oviposition (5 days after hatching) | 4.7 | 2 | 3.83 | 56.2 | 1.77·106 | 9.92·107 | 293 |
4 weeks after hatching | 2.9 | 2 | 6.18 | 235.9 | 2.93·106 | 6.92·108 | 2,041 |
The length of the voxel edge = 9 (µm) B M−1 (9 µm=length of pixel edge of the CCD camera; B, binning factor; M, magnification). One animal was studied for each age.
Age . | Magnification . | Binning factor . | Edge length of voxel edge/µm . | Volume per voxel/µm3 . | Number of voxels in the shell . | Total shell volume/µm3 . | Shell weight/µg . |
---|---|---|---|---|---|---|---|
72 h after oviposition | 6.0 | 1 | 1.49 | 3.32 | 6.54·104 | 2.17·105 | 0.64 |
96 h after oviposition | 4.7 | 1 | 1.91 | 7.02 | 4.63·105 | 3.25·106 | 9.59 |
120 h after oviposition | 6.0 | 1 | 1.51 | 3.41 | 3.02·106 | 1.03·107 | 30.3 |
240 h after oviposition (5 days after hatching) | 4.7 | 2 | 3.83 | 56.2 | 1.77·106 | 9.92·107 | 293 |
4 weeks after hatching | 2.9 | 2 | 6.18 | 235.9 | 2.93·106 | 6.92·108 | 2,041 |
Age . | Magnification . | Binning factor . | Edge length of voxel edge/µm . | Volume per voxel/µm3 . | Number of voxels in the shell . | Total shell volume/µm3 . | Shell weight/µg . |
---|---|---|---|---|---|---|---|
72 h after oviposition | 6.0 | 1 | 1.49 | 3.32 | 6.54·104 | 2.17·105 | 0.64 |
96 h after oviposition | 4.7 | 1 | 1.91 | 7.02 | 4.63·105 | 3.25·106 | 9.59 |
120 h after oviposition | 6.0 | 1 | 1.51 | 3.41 | 3.02·106 | 1.03·107 | 30.3 |
240 h after oviposition (5 days after hatching) | 4.7 | 2 | 3.83 | 56.2 | 1.77·106 | 9.92·107 | 293 |
4 weeks after hatching | 2.9 | 2 | 6.18 | 235.9 | 2.93·106 | 6.92·108 | 2,041 |
The length of the voxel edge = 9 (µm) B M−1 (9 µm=length of pixel edge of the CCD camera; B, binning factor; M, magnification). One animal was studied for each age.
For the determination of the weight of the shells, the voxels associated with mineralized shell tissue were counted and the total volume was calculated using the known (cubic) voxel size. The density of aragonite was taken as 2.95 g cm−3.
RESULTS
Embryos of Biomphalaria glabrata, 60, 72, 96 and 120 h after oviposition and juveniles between 5 days and 4 weeks after hatching were investigated by SEM and micro computer tomography. By SEM, the shells of 60 h old embryos were small cap-like structures with a diameter of 120 µm on the caudal side (not shown), but no definite X-ray dense structure could be found by SRµCT. However, by electron-spectroscopic imaging (ESI), calcium was found in the shell of 60 h old embryos in a layer of about 200 nm thickness (Marxen et al., 2003a).
Seventy-two hours after oviposition, the shell had increased in size, but still did not cover the whole animal (Fig. 1A). The corresponding SRµCT image (Fig. 1B) is oriented here in the same direction as in Figure 1A, which means that the shell is seen from inside and has a convex curvature. The shell had an almost oval shape and was about 350 µm wide and about 450 µm high with respect to the curvature. Even in its thickest part in the centre the thickness of the shell did not exceed 3 µm. While in Figure 1A the start of the coiling was hidden behind the mantle rim on the left side of the animal, in Figure 1B the spindle was clearly visible (arrow). To see a video of the shell in rotation go to the web address: http://www.biologie.uni-hamburg.de/zim/physiologie/freel-cycle.avi.
In the 96 h old embryos the shell almost fully covered the animal and the coiling over its left side is visible in SEM (Fig. 1C). SRµCT confirmed that the shell was well mineralized. The inner spindle which is the very first coil can be clearly seen on the left (Fig. 1D).
The shell of 120 h old embryos (Fig. 1E) showed all characteristics of the shell of an adult snail, and the embryo was then ready to leave the egg. At the shell edge the periostracum was soft, demonstrating that this youngest part of the shell was mineralized only to a small degree. By SRµCT an imaginary sagittal section (Fig. 1F) confirmed that the mineralized part in fact ended further proximally and led to a very slightly mineralized periostracum that was almost invisible in SRµCT.
Counting the voxels of the SRµCT image permitted the estimation of the volume and the weight of the embryonic shell as a function of the age of the animal (Table 1, Fig. 2).
Juvenile snails, i.e. 5 days after hatching (240 h after oviposition) with a shell diameter of about 1 mm, are shown in Figure 3. This demonstrates that once the reconstruction of the primary data has been done, it is possible to turn the object freely and to perform imaginary sections in all planes. Similarly, Figure 4 shows juvenile snails four weeks after hatching with a shell diameter of 3 mm in three different views: the surface (Fig. 4A), a transparent view (Fig. 4B) and a section in the sagittal plane (Fig. 4C).
In all cases the juvenile snails contained irregular concretions in the penultimate whorl, which is around the region of the stomach. To examine more closely these granules by SEM and EDX, newly hatched juvenile snails with a shell diameter from one to three mm were sectioned in the sagittal plane. The concretions were indeed found in the stomach (Fig. 5A). EDX measurements of different stones identified them either as calcium phosphate stones (Fig. 5B, C, and spectrum C) or as silicate stones (silicon, aluminium, iron, calcium, magnesium and oxygen; Fig. 5D, spectrum D).
DISCUSSION
Comparing the possibilities and limitations of SEM and SRµCT with respect to the investigation of snail embryos we can state the following: SEM is an ideal method to investigate surfaces, either of the hard material (biomineral) or the soft tissue. It shows all fine structures of the surface morphology, e.g. growth patterns of the shell, at a high resolution, i.e. down to a few nm. SEM is still irreplaceable to get a fast morphological impression of an organism or a structure, or for the study of fine details. However, it is only sensitive to the surface of a sample, and the acquisition of information on internal features necessitates suitable cutting or dissection. When using SEM, equipped with an EDX system, it is possible to perform an elemental analysis without much additional effort.
The SRµCT is a more modern method that can be used in addition to SEM investigations. The X-ray absorption of soft tissue at these high energies is much lower than of mineralized tissue; therefore, the soft tissue does not show up in SRµCT. This high contrast between mineralized tissue and soft tissue makes the method particularly advantageous in the study of biomineralization so that SRµCT is well suited to analyse the mineralization in developing snail embryos. From SEM it can only be guessed which parts of the shell are already mineralized and which are not. The picture of the shell of the 72 h old embryo (Fig. 1B) is a good example of a delicate structure of which the degree of mineralization was not shown before. These quantitative measurements allow the calculation of the mineral content as shown in Table 1. We used the density of aragonite (2.95 g cm−3) i.e. the material of the final shell, to convert the volume into weight. We estimate the experimental error associated with this computation to be at least ±10%, mainly due to the approximation by cubic voxels with a limited resolution and the unknown density of the shell. However, this is probably smaller than the biological variation between different animals of the same age.
Another advantage of SRµCT is the possibility to investigate the objects without the need for physical dissection that can affect the appearance and location of the structure in question. In principle, it is also possible to carry out experiments in water, i.e. even living organisms can be studied (provided that their movement is sufficiently restricted). It is difficult to distinguish between two mineralized tissues, but dentine and enamel were well distinguishable in a human tooth (Prymak et al., 2005). It is not sensitive to the elemental composition of a sample, apart from the variation in X-ray absorption which is related to the atomic weight of the constituting elements. The main advantage of SRµCT is the possibility for virtual sections that allow insights which would be very time-consuming in classical histological or electron microscopic studies. We can say that SRµCT develops its full abilities and a perfect three-dimensional impression when presented as a video of an object in motion or of a series of slices.
A clear disadvantage with respect to electron microscopy is the limited resolution, of a few µm at best (depending on the size range of the sample and the photon energy). We note that the spatial resolution is in the order of about 10 µm for laboratory equipment as applied to biomineralized tissues (Stock et al., 2002, 2003a, b, 2004;), i.e. the application of synchrotron radiation has clear advantages for the study of such small objects. A good example for the potential of the method was the detection of small stones in the stomach of the juvenile snails, a biologically not surprising fact, but definitely the discovery of objects that would otherwise have escaped our attention. The active ingestion of mineral particles by molluscs has been known for a long time (Storey, 1970), and microcapsules are under debate for the specific administration of molluscicides controlling Biomphalaria glabrata, the intermediate host of Schistosoma mansoni (Thomas, 2001).
Finally, it should be noted that the time needed for the sample preparation is about the same for both methods, but with SRµCT the measurement itself as well as the final processing of the files is rather delicate and time consuming. In this case, the additional application of SRµCT to embryos of B. glabrata revealed the ingestion of stones immediately after hatching and permitted a quantitative calculation of the mineral content of the growing shell.
Supplementary material
Supplementary material is available at Journal of Molluscan Studies online.
ACKNOWLEDGEMENTS
We thank the Deutsche Forschungsgemeinschaft for financial support (Priority Program ‘Principles of Biomineralisation’, SPP 1117) and HASYLAB at DESY (Hamburg, Germany) for generous allocation of beamtime.