María-J. Uriz

Location of toxicity within the Mediterranean sponge Crambe crambe (Demospongiae: Poecilosclerida)

Within-specimen location of toxicity in Crambe crambe (Schmidt) has been addressed by complementary procedures on specimens collected in north-east Spain (Western Mediterranean) in winter of 1993. The toxicity of the distal (ectosome) and basal (choanosome) sponge parts have been analysed and the main cellular types present in these two layers have been studied by light and electron microscopy. The toxicity of the three main cell types, separated by the gradient-density method, has also been analysed. Three main fractions, each of them enriched in a different cellular type, were obtained: Fraction 1 (interface between 2 and 5% Ficoll) contained 90±0.9% (mean±SE) of spherulous cells and 10% of different cell types consisting of choanocytes (5±0.54%), and unidentified cells or cell debris (5±0.84%); Fraction 2 (interface between 5 and 8% Ficoll) was enriched in choanocytes (70±0.95%), and also contained spherulous cells (11.8±0.73%), archeocytes (6.2±0.74%) and unidentified sponge cells (12±0.74%); Fraction 3 (interface between 8 and 11% Ficoll) mainly consisted of archeocytes and archeocyte-like cells (75±0.66%), together with spherulous cells (7±0.74%) and other unidentified sponge cells and cell aggregates mainly formed by choanocytes (18±0.41%). Toxicity [measured in toxicity units, TU, using the Microtox® procedure] was significantly higher in the sponge ectosome (12.45±1.4 TU) than in the choanosome (2.58±0.92 UT). Only the abundance of spherulous cells in the sponge tissues correlated well with the pattern of toxicity observed, and this was corroborated by the toxic behaviour of the three cellular fractions obtained: the one enriched in spherulous cells was highly toxic (9.08 UT), whereas those enriched in choanocytes and in archeocytes were almost inactive (0.48 UT) or totally innocuous, respectively. All these results point to the spherulous cells being responsible for the storage (and possibly production) of the toxic compounds in C. crambe. Toxicity is concentrated in the sponge periphery. Spherulous cells are also concentrated in this area and can also be observed outside the sponge exopinacoderm. These results correlate well with the assumption of a defensive role of toxicity, since encounters with potential epibionts, predators and competitive neighbours take place through this peripheral zone. However, we found two types of spherulous cells (orange and colourless, respectively) coexisting in the same sponge zones as well as in Cell Fraction 1. Thus, we cannot at present determine whether one or both types are responsible for the toxicity encountered, although it is likely that the two correspond to different states of the same cell type.

Trends in space occupation by the encrusting sponge Crambe crambe: variation in shape as a function of size and environment

The relationship between sponge size, habitat and shape was studied in the encrusting sponge Crambe crambe (Schmidt, 1862), which is distributed widely throughout the shallow Mediterranean littoral. Examination of sponge patches in shaded and well-illuminated habitats showed that the degree of peripheral irregularity of the edges of a patch is directly related to patch size. This relationship is valid only for sponges of >100 mm2 in area. Photophilic and sciaphilous sponges display different growth forms. The pattern of growth is interpreted in terms of competition for space. The directional growth of sciaphilous sponges may be due to the presence of dominant neighbours that are good space competitors, and the irregular growth of photophilic sponges to the absence of such neighbours.

Quantitative Assessment of Natural Toxicity in Sponges: Toxicity Bioassay Versus Compound Quantification

Microtox® assay was used to assess the natural toxicity of two sponges, Dysidea avara and Ircinia variabilis. The activity of crude extracts and major metabolites were compared. Methanol extract of D. avara was more toxic than that of acetone and was as toxic as pure avarol, thus suggesting that the toxicity of the sponge was mainly due to this metabolite. We also quantified palinurin, the major metabolite of I. variabilis, in specimens from several habitats. With the same methanol extracts used for palinurin quantification, we ran the Microtox® assay and found a positive significant regression between toxicity and concentration of this metabolite. Pure palinurin was tested at the same concentration present in the extract, and the toxicity recorded was higher than that of the methanol extract. As with avarol from D. avara, palinurin is the main secondary metabolite that confers toxicity to I. variabilis. The results confirm that the standardized Microtox® assay is an accurate and reproducible tool for assessing the toxicity of crude extracts and pure metabolites of marine organisms. These results also suggest that methanol is more suitable than acetone for the detection of species toxicity by Microtox® The method is faster and easier to perform than chemical quantification even when the sponge chemistry is known, and is appropriate for studies on variation in natural toxicity over a range of environmental conditions.

Measuring toxicity in marine environments: critical appraisal of three commonly used methods

Toxicity quantification is important in environmental monitoring, in the field of natural products, and in chemical ecology. The sensitivity and precision of three commonly used methods detecting toxicity in marine environments were compared, using the toxic marine spongeCrambe crambe as a test organism. The paper disk diffusion method (run with marine bacteria) showed the least sensitivity and did not permit toxicity levels to be quantified. The sea urchin and the MICROTOX® tests showed greater sensitivity, and the latter had the higher precision. The relative performance of these methods is discussed. It is concluded that the MICROTOX® bioassay displays the best characteristics for toxicity quantification.

Temporal and spatial co-occurrence in spawning and larval release of Cliona viridis (Porifera: Hadromerida)

This is the first report of egg release by the oviparous excavating sponge Cliona viridis . Adult specimens of excavating (α) and massive (β) sponge forms for the presence of oocytes were monitored from 11 May to 12 July 2000, in a shallow-rocky coast of the north-west Mediterranean. The immediate environment around the sponge was sampled for the presence of eggs. Spawning occurred synchronously in the study area at temperatures above 19°C. Oocytes were released in clusters enclosed in adhesive maternal tissue. They contained symbiotic zooxanthellae. Free, adhesive egg-masses drifted in the water or adhered to erect algae in the vicinity of the sponge. Morula stages and larval release are described.