Most tunicates are characterized by the presence of the tunic, an outer protective specialized tissue, covering the mantle epithelium or epidermis. The tunic consists of a leathery or gelatinous matrix containing microfibrils of polysaccharides linked to proteins, and free living cells randomly distributed within it [29], [30] and [31]. These cells are involved in various biological functions such as tunic synthesis, wound healing, immunological and excretory activities ([32], and references therein; [33]).

The origin of tunic cells is not entirely clear; in general, they are thought to originate from the hemocytes or connective tissue. In C. intestinalis it has been shown that during inflammatory-like reactions [34] hemocytes migrate by diapedesis from the hemolymphatic lacunae trough the mantle epithelium into the tunic leading to a subsequent increase of the tunic cell population [35]. Apart from its role as a support and an adhesive SRT1720 datasheet to the substratum, the tunic is considered as a protective barrier

of the soft body against mechanical damage and infection, CP868596 and a site of self/non-self recognition [36] and [37]. Here, we search for the presence of the natural molecules Ci-MAM-A and Ci-PAP-A in the tunic from naïve C. intestinalis by using immunocytochemistry and employing specific antibodies against these antimicrobial peptides. Moreover, to investigate whether these peptides are actually involved in immune defense, we also analyzed tissue samples of specimens where local inflammatory-like reactions in the tunic have been experimentally induced. The present study aims at extending the understanding of the functions of AMPs in tunicates by investigating their significance in local immune responses aside from their role as potent effector molecules of circulating hemocytes in the

hemolymph. C. intestinalis specimens about 10–12 cm in size were collected from Termini Imerese harbor (Sicily, Italy). Animals free of encrusting marine matter were maintained at 15–18 °C in aerated sea water. To provoke an inflammatory reaction, sheep erythrocytes (1×107 suspended in 0.2 ml phosphate buffered saline (PBS), pH 7.4) were injected into the tunic tissue. Four days later, the specimens showing an immune reaction in the tunic (macroscopically Sclareol seen as a circular or elliptical whitish area visible through the transparent tunic) were chosen for further analyses. Ciona specimens injected with 0.2 ml PBS served as a control. For routine microscopy, cubes of tunic fragments, 1–3 mm3 in size, cut off from different regions of the animal body and from the oral siphon, as well as excised from the injection site were processed by standard techniques which can be summarized as follows: fixed with 1.5% glutaraldehyde (Sigma Chemical Co, St. Louis, Missouri, USA) buffered in 0.05 M sodium cacodylate, pH 7.3, post-fixed in 1% OsO4, and dehydrated in a graded series of ethanol solutions, and subsequently embedded in epoxy resin.