010707.500 031505.750 031505.812 051403.500 052420.250 091313.500 091313.562 160801.666 160801.749 161905.250 180519.375 191608.562 081512*900 Defensive mechanisms of holothuroids (Echinodermata): Formation, role, and fate of intracoelomic brown bodies in the sea cucumber Holothuria tubulosa Didier Jans 1 , Philippe Dubois 1 , Michel Jangoux 1,2 1 Laboratoire de Biologie marine (CP 160/15), Université Libre de Bruxelles, 50 avenue F.D. Roosevelt, B-1050 Bruxelles, Belgium 2 Laboratoire de Biologie marine, Université de Mons-Hainaut, 19 avenue Maistriau, B-7000 Mons, Belgium &misc:Received: 20 January 1995 / Accepted: 17 July 1995 &p.1:Abstract. Brown bodies are pigmented aggregates of amoebocytes found in the coelomic cavities of most ho- lothuroids (sea cucumbers). Brown body formation was induced by injection of carmine particles into the peri- visceral coelom of Holothuria tubulosa. Formation be- gins with release of a fibrillar material by the spheru- locytes. This fibrillar material acts as an extracellular matrix upon which amoebocytes and carmine particles collect. Amoebocytes develop an extensive pseudopodial network and progressively condense into aggregates with varying degrees of compactness. While condensing, amoebocytes either phagocytose or encapsulate carmine particles. A destructive process begins once particle ag- gregation is complete, resulting in numerous intracellu- lar residual bodies and extracellular residual body-like structures, depending upon whether the carmine parti- cles were phagocytosed or encapsulated. Induced bodies have the same ultrastructural features as naturally occur- ring ones. Brown bodies are progressively eliminated to the outside through coelo-rectal canaliculi, and the body cavity is essentially cleared of all induced bodies within seven days following injection. &kwd:Key words: Coelomocytes – Brown bodies – Immune mechanisms – Spherulocytes – Holothuria tubulosa (Echinodermata) Introduction The coeloms of some echinoderms contain brown-col- oured bodies of various sizes that either float passively in the coelomic fluid or adhere to the body wall (Hyman 1955; Hetzel 1965). These brown bodies are common in holothuroids and spatangoid echinoids (Hetzel 1963; De Ridder and Jangoux 1984; Canicatti et al. 1989) and have been reported occasionally in other echinoderm groups (Jangoux 1982). Brown bodies are pigmented cell aggregates that retain unwanted material (Arvy 1957; Hetzel 1963). They sometimes include parasitic elements such as turbellarian egg-capsules (Shinn 1985) or gregarine cysts (Coulon and Jangoux 1987), which tend to be ensheathed by several layers of coelomocytes (Shinn 1985). At least two types of coelomocytes, amoe- bocytes and spherulocytes, are directly involved in brown body formation (Canicatti and Quaglia 1991). Formation of brown bodies can be induced experimen- tally by injecting various abiotic and biotic materials, such as particulate dyes (Bertheusen and Seljelid 1978; Dybas and Fankboner 1986), bacteria (Dybas and Fank- boner 1986), and vertebrate erythrocytes (Canicatti and D’Ancona 1989; Canicatti et al. 1989), into the coelom. The spherulocytes respond by releasing the contents of their spherules (Dybas and Fankboner 1986; Canicatti et al. 1989), and the amoebocytes react by either phago- cytosing or encapsulating the foreign matter, depending on its size and abundance. The nature and role of the se- creted substances and the mechanism of coelomocyte aggregation to form brown bodies are largely unknown. This paper describes the development of experimen- tally induced brown bodies, with an emphasis on ultra- structure, and compares the structure of induced and nat- urally occurring bodies. It considers the fate and func- tion of the coelomocytes involved and of the brown body as a whole. Materials and methods Individuals of the aspidochirote holothuroid Holothuria tubulosa Gmelin, 1788 were collected by SCUBA diving in the deepest part of the Lacco Ameno seagrass bed at Ischia Island, Bay of Na- ples, Italy. They were maintained in an open-circuit aquarium at the Ischia laboratory of the Stazione Zoologica di Napoli. Brown body formation was induced by injecting 0.5 ml car- mine suspension (5 mg/ml filtered [0.22-μm] sea water) into the perivisceral coelom of 16 specimens (20–23 cm in contracted Funding provided by grant 9.4517.91 from the FRFC (Belgium). D.J. and P.D. are, respectively, Research Assistant and Research Associate of the National Fund for Scientific Research (Belgium) Correspondence to: M. Jangoux&/fn-block: Cell Tissue Res (1996) 283:99–106 © Springer-Verlag 1996