COORDINATED PHENOMENA IN THE MARINE DEMOSPONGE CHONDROSIA RENIFORMIS: PHYSIOLOGICAL, MORPHOLOGICAL, BIOMECHANICAL AND BIOCHEMICAL ASPECTS.

Although sponges (phylum Porifera) are still often considered to be simple, inactive animals, both larvae and adults of different species show clear coordination phenomena triggered by both extrinsic and intrinsic stimuli. Moreover although sponges lack a nervous system, they show a range of behavioural responses that are affected by exogenously applied neuroactive compounds. Chondrosia reniformis (Nardo, 1847), a common Mediterranean demosponge, lacks both endogenous siliceous spicules and reinforcing spongin fibers and has a very conspicuous collagenous mesohyl mainly composed of collagen. This sponge can react to different stimuli by changing its tensile state: it can rapidly stiffen after mechanical or chemical stimulation or destiffen and, following accidental detachment of the substrate, produce long slender outgrowths. These phenomena involve different responses induced by different external events, but both have been attributed to alteration, under cellular control, of the interactions between the collagen fibrils of the mesohyl. The ability to regulate the viscoelastic properties of the connective tissue matrix is a well- investigated phenomenon in other animal phyla, particularly the Echinodermata in which the mechanical, morphological and molecular aspects of mutable collagenous tissues (MCTs) have been extensively analysed. The present work was intended to investigate the coordinated phenomena of the sponge C. reniformis with an integrated approach that includes: physiology, histology, biomechanics and biochemistry. We focused our attention on different aspects: namely, 1) the reaction to mechanical stimulation; 2) the mesohyl mechanical properties; 3) the possible presence of contraction/expansion events; 4) the role of γ-amino butyric acid and glutamate; 5) the presumptive factor(s) that regulates the sponge tensility. Our results demonstrate that the stiffening reaction that follows mechanical stimulation consists of a passive shrinkage and an active stiffening of the mesohyl given by modification of the ECM mechanical properties and possibly involving the contraction of the canal system. In fact our experiments reveal that the passive compression involves mainly the canal system and the volume recovery occurs when the stiffening effect fade. A significant tensility difference is present between undisturbed and stimulated sponges and evidences on the presence of signal transmission that requires a continuous exopinacoderm (outer epithelium) are reported. This thesis provides information on different mechanical parameters: namely, viscosity, stiffness, breaking strain and breaking stress. As far as recovery process is concerned we suggest that the sponge shape and volume recovery are given by the striking mechanical behaviour of its mesohyl that shows unusual elastic properties. By means of digital time lapses movie we confirm the presence and describe the pattern of contraction/expansion cycles occurring in C. reniformis. Contrary to other species our animal model does not display stereotypical contractions and lacks a clear contraction periodicity. Both glutamate (Glu) and γ-amino butyric acid (GABA) are present in different cell phenotypes. Different experiments suggest that, despite the capability of the two substances to slightly increase sponge viscosity, both GABA and Glu seem to be not involved in the regulation of the stiffening phenomenon and in the recovery process that allows the sponge to return to its original shape and volume. On the other hand the two substances, as demonstrated in other sponges, are able to induce contraction events. There are no differences between the two molecules when comparing the contraction magnitude; however our biomechanical approach highlights significant differences in the forces generated during the contractions, in particular Glu generates higher force than GABA. We hypothesize that the two molecules act on different effectors that produced similar effects though different mechanisms. Finally we demonstrate that at least one protein is responsible of the stiffening of the sponge mesohyl by interacting with collagen fibrils.