Methods for culturing the freshwater sponge Ephydatia muelleri: Growing sponges on agarose and priming gemmules v1
Authors: Sogabe Shunsuke , Cornish Taitum , Hill April , Riesgo Ana , P Leys Sally 
Published: April 2022

The freshwater sponge Ephydatia muelleri is an emerging sponge model system. This species has clonal asexual reproduction stages called gemmules that are long-lasting and easily maintained in the lab, and a chromosome-level assembly of the genome (Kenny et al., 2020) with molecular resources (ephybase.ca), making this an easily used species for study by researchers around the world. Sponges grow attached to substrates, and one technical difficulty when working with species are attached to a substrate, is how to transfer samples to and from different solutions and vessels for experimental manipulations. In the case of E. muelleri, transfer is usually done with gemmules before they hatch and attach, or with sponges that are attached to small coverslips. Here, we outline a simple and effective method to grow E. muelleri on 2% agarose, a substrate that allows them to be peeled off with little damage, and transfer to other containers at any stage. Once transferred and left alone, they re-attach readily. E. muelleri development from gemmules are referred to as stages: stage 1 is just prehatching; stage 2 is hatched with no aquiferous system; stage 3 has 'pockets' of water spaces that are growing and merging with each other; stage 4 has an incipient but not fully organized canal system; stage 5 has a fully developed canal system with osculum vent. Although all stages can be easily peeled off the agarose substrate, sponges at earlier stages (2-3) develop at the same rate as undisturbed sponges after transfer, allowing direct comparison with unmanipulated control samples. This method allows different stages of E. muelleri to be transferred into cuvettes for electroporation, or into a solution for pharmacological treatment, or onto a new substrate for imaging. We also describe the best mechanism of timing hatching of gemmules. Gemmules are 'dormant' when kept at 3°C. Once placed at room temperature they undergo development within the collagen capsule, that involves differentiation of different cell types prior to their emergence from the micropyle (opening), as described by Höhr in 1977. This development can be thought of as "priming", or readying them for hatching. 'Priming' gemmules to the same level of hatching readiness, allows more control over timing of experiments. Primed gemmules also appear to be more resilient to both physical and chemical treatments. The gemmules slowly get primed once cleaned, or thawed from -80°C. A general guideline to prime gemmules is: 1 week in a 3°C incubator = 1 less day it takes for the gemmules to hatch after plating 1 day at room temperature = 1 less day it takes for the gemmules to hatch after plating Once they are over-primed (usually after 4-5 weeks in the 3°C incubator), they will start hatching even when kept at 3°C. We suggest that for reproducibility of experiments that concern the stage of development, experiments using E. muelleri gemmules would benefit from using primed gemmules or indicating how primed the gemmules were. Reference 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. Höhr, D. 1977. Differenzierungsvorgänge in der keimenden Gemmula von Ephydatia fluviatilis. Rouxs Arch. Dev. Biol. 182: 329-346.