Inducing gemmulation in the freshwater sponge Ephydatia muelleri in culture using theophylline

Freshwater sponges produce overwintering cysts called gemmules that are full of stem cells, allowing them to survive harsh winter months. The gemmules of Ephydatia muelleri can be kept in 3°C for months, and in -80°C for years, while still remaining viable to hatch and develop into a functional sponge, as outlined in the protocol: "Hatching and freezing gemmules from the freshwater sponge Ephydatia muelleri" ( https://dx.doi.org/10.17504/protocols.io.863hzgn). With a recently published chromosome-level assembly of the genome (Kenny et al ., 2020), along with multiple transcriptomes, this makes Ephydatia muelleri an ideal sponge species to establish as a model sponge species that can be used in labs world-wide.

While the gemmules used in the lab are usually collected from rivers and lakes every season for practical reasons, there have been studies showing the gemmulation process can be induced. A classic study looking at this process was conducted by Rasmont in 1974, who used theophylline to induce gemmulation in the freshwater sponge Ephydatia fluviatilis . Using up to 400uM of theophylline, this study showed that gemmulation can be induced in 100% of the sponges used in the experiments, and can occur as fast as 4 days after treatment with theophylline. While this is an interesting and important process to understand the biology of freshwater sponges, the ease and practicality of collecting masses of gemmules from wild populations most likely led to this process being rarely revisited in the lab.

Here, we outline a protocol for inducing gemmulation in the freshwater sponge Ephydatia muelleri using theophylline, establishedby revisiting the study done by Rasmont (1974). In E. muelleri , the gemmulation process takes longer than in E. fluviatilis , with the fastest documented gemmulation detected at 8 days after treatment with theophylline, and taking up to 12+ days in some cases. The gemmulation process usually completes within 4-5 days once the gemmulation process can be detected, and results in a smaller gemmule size than the ones obtained from wild populations. The newly formed gemmules take roughly 6-7 weeks to mature in the 3°C incubator before they can be plated to hatch.

While this is a lengthy process, being able to induce gemmulation in the lab increases the potential for using E. muelleri in transfection studies.

References

Kenny, N.J., Francis, W.R., Rivera-Vicéns, R.E. et al. Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri . Nature Communications. 2020. 11, 3676. https://doi.org/10.1038/s41467-020-17397-w.

Rasmont R. Stimulation of cell aggregation by theophylline in the asexual reproduction of fresh-water sponges ( Ephydatia fluviatilis ). Experientia. 1974. Jul 15;30(7):792-4. doi: 10.1007/BF01924190. PMID: 4367998.

This protocol is based on an 8-gemmule sponge grown in a 5cm Petri dish (Max. volume 20ml) and treated with 400uM theophylline. Both the number of gemmules and total volume of medium can be scaled up or down.

For 10mM stock solution of theophylline:

MW:180.16

For 10 ml of 10 mM stock solution = 18.0 mg (0.018g) in 10ml of dH2O

For 50 ml of 10 mM stock solution = 90.1 mg (0.090g) in 50ml of dH2O

-> Store in 4°C (fresh is always better, but this should last for at least 1 week, up to a month at 4°C)