Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________ Microscopic features of the tunic in young Styela canopus (Tunicata, Styelidae) M. A. Di Bella and G. De Leo Department of Biopatologia e Biotecnologie Mediche e Forensi, Sezione di Biologia e Genetica, University of Palermo, Via Divisi 83, 90133, Palermo, Italy The tunic is a unique tissue in metazoans covering the epidermis of ascidians and other tunicates. The tunic is a multifunctional tissue that provides support, mechanical rigidity, and protection against infection and predation. The outermost surface of it is covered by a thin continuous layer called tunic cuticle. The tunic cuticle density and integrity must be maintained during the growth of the animal after metamorphosis, as it represents the primary body surface barrier preventing the mechanical damage. Using transmission electronic microscopy we attempted to characterize the cuticle morphology of young Styela canopus, a solitary ascidian (Styelidae), in comparison with the cuticle architecture of adult animals. Keywords: Styela; ascidians; tunic; ultrastructure 1. Introduction The tunic is a peculiar tissue unique in metazoans. It is an integument covering the epidermis of tunicates, ascidians and thaliaceans. Its main components are cellulose, protein and carbohydrates, acid mucopolysaccharides that form a fibrous material arranged in a matrix where free cells are randomly embedded. The tunic among the various ascidian species can be rigid or flexible, transparent or opaque, and its consistency ranges from soft to cartilaginous on account of variations in the fibrous constituents [1-6]. Styela canopus is a solitary ascidian belonging to the Stolidobranchia, Styelidae; its tunic is a leathery matrix and can be structurally distinguished into two areas, the cuticle and the ground substance. The cuticle is the layer that represents the external limit of the body and appears as an electron-dense sheet. The ground substance provides mechanical rigidity and support to the animal body as well as owing to its cellular component it serves various functions such as storage, protection, immune response. The S. canopus ground substance is a thick layer consisting of fibrous structures in an amorphous firm matrix. Elementary fibrils are usually associated to form regular bundles, closely interwoven each other [1, 7]. Here we report on some features of the tunic in young S. canopus specimens as its architecture appears a bit different in comparison with that of adult specimens. 2. Material and Methods S. canopus specimens about 1 cm long were collected from the coast lake of Ganzirri, Messina (Italy). They were carried in plastic containers with natural seawater and kept in aerated aquaria at 15–18 ◦C until used. Animals free of encrusting marine matter were used. Cubes of the tunic, 1 to 3 mm long, were fixed using the following procedure. They were placed for 1 hr at 4 °C in a solution containing 1.5% glutaraldehyde, buffered with 0.05 M sodium cacodylate (pH 7.3 plus 1.7% sodium chloride). After brief rinsing, they were postfixed for 1hr at 4 °C with 1% osmium tetroxide in 0.05 M sodium cacodylate at pH 7.3. All specimens were rinsed briefly and dehydrated in a graded series of ethanol solutions, cleared in propylene oxide and embedded in Epon resin. Semithin sections were stained with toluidine blue and thin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (Philips CM 10), at 80 kV. 3. Results Figure 1 shows a thick section where the cuticular layer stained with toluidine blue appears as a thin sheet presenting an irregular outline due to folds and digitations. It is lightly more evident than the main ground substance. As in Figure 2, in TEM observations dense fibrous material loosely intertwined seems to form the cuticular wall. The cuticle thickness ranges from about 0.10 μm to 0.20 μm. At exposed surface filaments look like branched ribbons and aggregate to form a continuous electron-dense layer. No pointed spines or a film of encrusting foreign agents are found on the exterior surface. Figure 2A shows scattered in the subcuticular matrix, strongly electron-dense, roundish, unbounded granules of an average diameter of about 0.1μm. Sometimes the dense material occupies a large area but it is not homogenously packed, and bacteria or algae are seen embedded in it (Fig. 2C). 66 © FORMATEX 2014 Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________ Fig. 1 Microscopical section through the tunic of young S. canopus showing the outer surface or cuticle (c) as a thin sheet with irregular profile. The ground substance (gs) and a group of encrusting agents (e) in the subcuticular region can be seen. Magnification: 100x Fig. 2 TEM view of the tunic. (A) The cuticle (c) from young samples consists of fibrous material that aggregates from outside to form fibers in a net-like structure. In the subcuticular layer (sc) roundish, unbounded, dark granules (g) scattered through the matrix. (B) The tunic of adult samples is more densely packed and thicker. In C a mammillate protrusion where the boundary between the cuticle and the subcuticular region is not well defined; fibrous material seems no tightly compressed and numerous bacteria are entrapped in it. Magnification: (A) 17,000x; (B) 8,000x; (C) 5,000x © FORMATEX 2014 67 Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________ Figures 2 and 3 show the observed ground substance that is quite different from that of adults (Figs. 2B, 3B) because of the low density of the matrix; the densities of the fibers are heterogeneous; bundles are loose and not regularly oriented and some translucent areas can be seen. Fig. 3 High magnification of the ground substance showing loose fibrils and fine bundles that in young Styela (A) are not so interlaced each other as seen in adult (B). Magnification: (A) 26,000x; (B) 22,000x. Few tunic cells are found in the matrix of the ground substance. In figure 4 a cell showing various clear vacuoles with remnants of dark content and others in being about to be discharged. Fig. 4 Micrograph of a free tunic cell presenting clear vacuoles with remnants of dense substances. Magnification: 5,800x 4. Discussion The consistency of the tunic in S. canopus is almost leathery and it is thick as in other ascidian species belonging to the families of Styelidae and Pyuridae [7]. Roughly, the young specimen tunic morphology does not differ very much from that of the adult, but the ultrastructural observations in this study have shown a cuticle considerably thinner than the latter. Although it appears as a peripheral thickening of the ground substance it does not show the compactness as in adult tunic cuticle. As a protective structure the cuticle hardens the tunic surface to prevent mechanical assaults and invading microorganisms, but on the other hand, the process of growth of the animal must maintain the plasticity and flexibility of the tunic for the dynamic changes in length along the axis. The cuticle does not present yet the biological film of epibionts that usually lie onto its surface in adults [7] but some bacteria have gained access to the inner of the tunic. Moreover, the fibers in the ground substance are not so highly strengthened arranged in bundles as in adults. The presence of cells devoid of structures and whose granular inclusions have completely dissolved, support the idea of their involvement in supplying materials for the construction of the entire tunic [4]. The bundle formation here observed is structurally similar to the mechanism of new wall formation occurring in colonial botryllid and other ascidians during process of regeneration and allorecognition reaction, when around the rejection lesion, the fibers aggregate to form a continuous boundary separating the lesion from the contacting colonies [8-11]. On account of the insufficiency of previous observations on young specimens, TEM approaches in the present preliminary study provide several data and hints useful to compare the architecture and the function of the tunic among ascidians species. The latter are considered a sister group of vertebrates being classified in the phylum chordates, subphylum Urochordata [12] so understanding the morphological aspects of this peculiar tissue is interesting from the view-point of comparative morphology, philogenetic relationships and also evolution of the immune response. Acknowledgements This work has been supported by grants from the Italian Ministero della Università e della Ricerca and the University of Palermo research grant to M.A.D. and G.DL. 68 © FORMATEX 2014 Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________ References [1] Goodbody I. The physiology of ascidians. 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