Echinometra mathaei and its ectocommensal shrimps: the role of sea urchin spinochrome pigments in the symbiotic association.

Tuleariocaris holthuisi and Arete indicus are two ectocommensal shrimps closely associated with the tropical sea urchin Echinometra mathaei. This study provides a comparison of these two E. mathaei symbiotic crustaceans and particularly focuses on the relationship between T. holthuisi and its host's pigments (i.e. spinochromes), and its dependency on its host. While all the analyses underline a close association between A. indicus and E. mathaei, they reveal a particularly close interaction between T. holthuisi and its host. Chemical analyses reveal that these shrimps present the same spinochrome composition as E. mathaei, and have similar colouration, allowing camouflage. Isotopic composition and pigment loss after host separation suggest that these pigments are certainly assimilated upon feeding on the urchin. Moreover, symbiont isolation experiments demonstrate the high dependency of T. holthuisi on its host and the importance of the host's pigments on their survival capacity. Finally, some host recognition mechanisms are investigated for T. holthuisi and show the probable implication of spinochromes in host selection, through chemical recognition. Hence, all the results suggest the essential roles of spinochromes for T. holthuisi, which, in turn, suggests the potential implication of these pigments in the shrimps' metabolism.

Identification and quantification of spinochromes in body compartments of Echinometra mathaei's coloured types.

Sea urchin pigmentation is mainly due to polyhydroxy-1,4-naphthoquinones called spinochromes. If their molecular structures are well known in test and spines of many species, their abundance and distribution in other body compartments remain unstudied. The aim of this study is to analyse the pigment composition in four body compartments (test/spines, digestive system, gonads and coelomic fluid) of four coloured types of the sea urchin Echinometra mathaei. Qualitative and quantitative measurements by mass spectrometry highlight the existence of 13 different pigments; among which are five isomers of known spinochromes as well as three potentially new ones. The composition comparison shows the largest spinochrome diversity in 'test/spines' body compartments. The spinochrome concentrations vary from 48 to 1279 mg kg-1 of dried body compartment. It is the highest in the digestive system, although it is also important in the organic fraction of the 'test/spines' body compartment. This observation may be explained by higher exposures of some body compartments to external environments and by the protective role fulfilled by spinochromes against microorganisms, ultraviolet radiation and reactive oxygen species. The 'black' type-the most common coloured type in coral reefs-has the highest concentration of spinochromes indicating their importance in Echinoids' fitness by acting as a protective agent.

The Roles of Spinochromes in Four Shallow Water Tropical Sea Urchins and Their Potential as Bioactive Pharmacological Agents.

Spinochromes are principally known to be involved in sea urchin pigmentation as well as for their potentially interesting pharmacological properties. To assess their biological role in sea urchin physiology, experiments are undertaken on crude extracts from four species and on four isolated spinochromes in order to test their antibacterial, antioxidant, inflammatory and cytotoxic activities. First, the antibacterial assays show that the use of crude extracts as representatives of antibacterial effects of spinochromes are inaccurate. The assays on purified spinochromes showed a decrease in the growth of four strains with an intensity depending on the spinochromes/bacteria system, revealing the participation of spinochromes in the defense system against microorganisms. Secondly, in the 2,2-diphenyl-1-picrylhydrazyl antioxidant assays, spinochromes show an enhanced activity compared to the positive control. This latter observation suggests their involvement in ultraviolet radiation protection. Third, spinochromes present a pro-inflammatory effect on lipopolysaccharide-stimulated macrophages, highlighting their possible implication in the sea urchin immune system. Finally, cytotoxicity assays based on Trypan blue exclusion, performed in view of their possible future applications as drugs, show a weak cytotoxicity of these compounds against human cells. In conclusion, all results confirm the implication of spinochromes in sea urchin defense mechanisms against their external environment and reveal their potential for pharmacological and agronomical industries.

Effects of Holothuroid Ichtyotoxic Saponins on the Gills of Free-Living Fishes and Symbiotic Pearlfishes.

Several carapid fishes, known as pearlfishes, are endosymbiotic in holothuroids and asteroids. These echinoderms contain a strong concentration of saponins that are efficient membranolytic repellents to predators. We compared the effects of exposure to saponins from the sea cucumber body wall and from the Cuvierian tubules on the behavior and gill ultrastructure of pearlfishes and free-living fishes. Saponins were extracted from the body wall of two holothuroids, the Mediterranean Holothuria forskali and the tropical Bohadschia atra, and from the water surrounding the Cuvierian tubules of B. atra. Five species of carapids that live in symbiosis with holothuroids and seven species of free-living fishes were exposed to these extracts. The free-living fishes exhibited a stress response and died about 45 times faster than pearlfishes when exposed to the same quantity of saponins. Cuvierian tubules and saponins extracted from the body wall were lethal to the free-living fishes, whereas the carapids were much less sensitive. The carapids did not exhibit a stress response. The high toxicity shown by Cuvierian tubules was not explained by the nature of the saponins that were identified by mass spectrometry, but it is likely due to the higher concentration of saponins in the tubules. Histology and scanning and transmission electron microscopy of the gills of the free-living fishes and pearlfishes showed that saponins act at the level of the secondary lamellae where they induce the detachment of the epithelia, create edema at the level of the epithelia, and induce pores in the epithelial cells that lead to their destruction and the invasion of inner cells (pillar cells and red blood cells). This sequence of events happens 5 min after saponin exposure in free-living fishes and after 1 h in carapids.