Volume 48

Combining Confocal Laser Scanning Microscopy with SEM to investigate the nature, function and ontogeny of the mantle ciliation of bivalve larvae

Sam Stanton

Institute of Marine Sciences, School of Biological Sciences, University of Portsmouth, Ferry Road, Portsmouth P04 9LY, UK

A combination of EM and fluorescence microscopy is being used to reveal the development of ciliation on the larval mantle and the velum of Crassostrea gigas larvae. These observations are providing insights into the functional importance of cilia-bearing mantle sense organs in, for example, settlement behaviour and of the velar cells in feeding mechanisms.

The catecholamines
Catecholamines are a group of neurotransmitters which have previously been located within the larvae of Crassostrea gigas (Croll et al. 1997; Croll and Dickinson 2004). They have been found to act as morphogens in early development, as controllers of ciliary activity and as inducers of settlement in the larva. Previous studies have used methods such as liquid chromatography, or exposure of larvae to varying catecholamine concentrations to demonstrate catecholamine presence and/or effect. In the current study, the distribution of catecholamines has been investigated using aldehyde-induced fluorescence. This information is then compared with images from bright field microscopy and scanning electron microscopy (SEM) to determine where catecholamines occur within the larval anatomy.
Catecholamine-positive cells have been found around the ventral rim of the mantle and near the gill bud in larvae that are ready to settle (pediveligers). The mantle signal occurs within the twin tracts of cilia previously identified using the SEM. Figure 1 illustrates discrete patches of staining, perhaps individual cells, whilst Figure 2 shows the same region using SEM.

Figure 1 UV image of catecholamine fluorescence in C.gigas pediveliger larva.	Figure 2 SEM of C.gigas pediveliger showing cilia on mantle rim and gill bud

The gill bud appears as the point of origin for the inner mantle rim ciliation in the veliger. As the larva develops from free-swimming veliger to the swimming-crawling pediveliger ciliation increases. By late-stage pediveliger mantle ciliation is dense in the region of the gill bud/mantle rim join. It is especially interesting to note the presence of catecholamines in the ventral region adjacent to the foot exiting between the valves (Figure 1). This region is held close to the substrate when seeking a settlement site.
In addition to the ventral mantle/gill bud, catecholamine-positive cells have also been localised in the posterior-dorsal region (Figure 3c). This location matches that of a group of stereocilia (Figures 3b & d) that are associated with a shell feature known as the posteriodorsal notch. The notch is a feature which leaves a visible 'track' in the shell during growth (Figures 3a & b), resulting in a small gap between the shell valves even when they are closed (Figure 3a). As a result, this group of stereocilia will always have some degree of contact with the water surrounding the animal.

Figure 3a posteriodorsal notch	Figure 3b stereocilia under notch
Figure 3c Catecholamine signal (C) and notch region (N)	Figure 3d stereocilia under notch

Serotonin
Like the catecholamines, serotonin has been implicated in the control of ciliary activity of invertebrate larvae, but has also been identified as having a more fluid role in other larval organisms. For example, in the larvae of the sea star Patiriella regularis serotonin has been identified in the ciliated feeding bands of the early larva, suggesting a role in control of ciliary activity. However as the larvae develop, serotonin redistributes within the larva, suggesting the activity of serotonin is being redirected towards settlement (Chee and Byrne 1999).
Results from this study suggest a similar process may be occurring in the larva of Crassostrea gigas.

Figure 4 (a) LM image of a mid term veliger stage larva (b) Optical sections from CLSM showing serotonin sinal in the velum (arrows). Note that there is no signal from the rest of the larva.

Figures 4a & b above show a mid-term veliger stage larva in which the antibody-labelled serotonin is restricted to the velum (the swimming/feeding organ of this planktonic larva).
In contrast to the veliger, the late-stage pediveliger features serotonin signals in the region of the mantle and gill bud (figure 5 & 6).

	Figure 5 Serotonic image and contrasting SEM. Note that the signal corresponds with the gill bud and the beginnings of cilia tracts
	Fig. 6 Serotonin signal from the mantle musculature region of C.gigas pediveliger

Additional CLSM work
In addition to the serotonin and catecholamine work this study also aims to identify the cells bearing the large pre-oral cilia of the larval velum. The nuclear stain Topro-3 is ideal for this work, enabling the staining of the large nuclei of these cells. Initial results are encouraging and suggest enumeration of nuclei of these cells is feasible (figure 7).

Figure 7. Topro-3 nuclei staining of the velum (V), and the corresponding LM image of the veliger.

Summary
Initial results from this study suggest an association of both serotonin and catecholamines with ciliated structures seen with SEM. Catecholamines have been localised in the posteriodorsal region, an area associated with stereocilia. Results suggest that serotonin signal in the veliger is localised in the velum, with little or no signal being detected from the mantle. Both catecholamines and serotonin have been identified in the mantle and the gill bud of the pediveliger, specifically in those areas bearing the significant and organised ciliation in the form of cilia tracts. This is suggestive of the serotonin function changing through development, altering from ciliary control of the feeding/swimming cilia bands of the velum to a role within the mantle in settlement.

References

Acknowledgements
I would like to thank the Malacological Society of London for the centenary grant that has made this piece of work possible and for the conference opportunities to present my research to a wider audience. Finally, Dr Simon Cragg for his continued support and input, and Matthew Harris for his assistance with my larval cultures.