The biological activity of bonellin, an integumentary chlorin in Bonellia viridis

Abstract

The characterization of bonellin and some aspects of its physical and chemical properties that are of direct relevance to this study are discussed. The effect of bonellin on diverse biological systems was investigated. Bonellin and anhydrobonellin methyl ester are photodynamically active, whereas the copper complex of bonellin is not. The erythrocyte haemolysis, effects on ecinoid sperm and development, and inhibition of cleavage of B.viridis embryos by bonellin are the result of photo dynamic activity. Quencher studies suggest that the mechanism is by singlet oxygen formation. The present study while confirming previous work on oxygen uptake in B.viridis entire animals, also revealed that muscle minces containing the integumentary pigment, as well as the viscera, show an increase in oxygen uptake on illumination. This increase is not due to an increase in metabolic rate. It is suggested that the measured increase in oxygen uptake when B.viridis is illuminated in vivo, is mainly due to bonellin sensitized photooxidation reactions. This study shows that bonellin pigmentation develops in the gastrula, i.e. before the feeding stage. The increasing density of pigmentation in ontogenesis, which is most pronounced from the gastrula to the early trochophore stage is the result both of increasing intracellular pigment content and of proliferation of the pigment-laden cells. In larvae undergoing male differentiation, there is progressive depigmentation, which initiates in the anterior region. In larvae undergoing female differentiation, besides the small pigment cells of trochophore origin in the integument, there is the accumulation of pigment in clusters of cells in the coelom. These cells subsequently migrate to the integument. Eggs cultured under continuous illumination, notably the high frequency end of the visible spectrum, do not develop green pigment, although sexual differentiation is otherwise normal. The larvae resulting from the above light culture revealed an orange-brown pigment in vacuolated cells similar to those that normally contain the green pigment. The effects of adult female specimens and various extracts on sex determination of B.viridis larvae were studied. Larvae can settle on the trunk of the. adult female in vivo and differentiate into males, although settlement on the proboscis is preferred. Trunk extracts were as effective as proboscis extracts in "masculinizing" the larvae. The secretion liberated by the irritated adult female in vivo is more effective in masculinizing thee larvae than the crude pigment extract from macerated tissues which in turn is more effective than pure bonellin. The· natural secretion produced, both a high proportion of completely masculinized larvae and also a fast rate of sexual differentiation. The masculinizing ectohormone in viva is not bonellim, although it may be a derivative of it. A tentative hypothesis on the variability in response of larvae of different spawning to a particular environmental factor, as well as t:he significance of the permanently undifferentiated larvae is discussed.