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Cells from the freshwater sponge Ephydatia muelleri were isolated by dissociating hatching gemmules. During the first 24 h the cells reaggregated, but the aggregates progressively disintegrated again to single cells, among which the spicule-forming sclerocytes were recognized. Such cultures were used to study spicule (megascleres) formation in vitro. The isolated sclerocytes formed the organic central axial filament onto which they deposited inorganic silicon. The size of the spicules (200 to 350 microns in length) as well as the rate of spicule formation (1 to 10 microns/h) under in vitro conditions were similar to the values measured in vivo. Immediately after completion of spicule formation, or even before, the sclerocyte could start formation of a new spicule; 5% of the cells were in the process of forming two spicules simultaneously. Cultivation of sclerocytes in the absence of silicon resulted in the formation of the axial filament only. We succeeded in maintaining the sclerocytes in a proliferating and spicule-forming state for up to 3 mo. These results demonstrate that the establishment of short-term cell cultures from E. muelleri is possible; however, future studies must be undertaken to identify the growth factors required for a permanent culture of sponge cells.

In the present paper it is shown that the marine sponges Geodia cydonium and Verongia aerophoba contain the gene coding for P-glycoprotein P170, also known as a multidrug-resistance gene. Western blot studies revealed that polyclonal antibodies raised against hamster P170 cross-react with the sponge polypeptide of Mr 125,000. After endoglycosidase F treatment, the sponge P125 is converted to a polypeptide of Mr 105,000. Northern blot studies, using the human P170 cDNA probe, revealed a size of 4.2 kb for the sponge P125 transcript. The level of this transcript does not change in response to incubation with the aggregation factor. Confocal laser scanning microscopy showed that P125 is a cell membrane bound protein. In addition, sponge membrane vesicles possess a potential to bind in vitro 2-acetylamino-fluorene, vincristine and daunomycin. This process is Verapamil-sensitive, a characteristic known also for the mammalian vesicle associated P170. The data reported demonstrate that the classical multidrug resistance mechanism, described in drug-resistant tumor cell lines, functions also in sponges and may explain the relative resistance of these animals to pollution.

Abstract-Relatively high amounts of inorganic polyphosphates (approximately 55 g of polyphosphate/g of wet weight) were found in the freshwater sponge Ephydatia muelleri, particularly in the gemmules (260 g/g). Here we report that the polyphosphate content of this sponge changes during development and in response to adverse environmental conditions. Germination and hatching of gemmules of E. muelleri is accompanied by a strong decrease (by 94% at day 2) in polyphosphate level and a rise in exopolyphosphatase activity. On the other hand, induction of gemmulogenesis by theophylline results in an increase (by 61%) in polyphosphate content of sponge tissue. An increase in polyphosphate content and a decrease in exopolyphosphatase activity also occur during tissue regression when hatched sponges are exposed to polluted water from river. Nonionic organic compounds extracted from this water were identified as contaminants causing a rise in polyphosphate content of E. muelleri. The results show that measurement of polyphosphate level may be a promising method to detect responses of the freshwater sponge to polluted waters.

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