Growing freshwater sponges from gemmules in the laboratory
Scott Nichols
Abstract
This is a basic protocol for growing freshwater sponges from gemmules in the laboratory. We specifically developed this protocol for working with Ephydatia muelleri , but have used it for other species as well. This protocol is good for cleaning gemmules, and removing contaminating protists, fungi, and bacteria.
Before start
Before Start
Gemmules can be collected seasonally throughout the world. There are published records of freshwater sponge distributions, but it is also possible to use citizen science apps such as iNaturalist to identify locations where they have been reported. A map of freshwater sponge reports in the United States can be found at the following link: https://www.inaturalist.org/projects/freshwater-sponges-of-the-united-states.
Attachments
Steps
Gemmule sterilization
Place a 40-70 µm cell strainer into a clean 10 cm Petri dish filled with cold, lake/spring water.
Cut off the end of a p1000 pipette tip with scissors to increase the size of the opening. Using the trimmed pipette tip, transfer the isolated gemmules into the cell strainer.
Gemmule sterilization: sterilize with hydrogen peroxide, then rinse
Prepare a 60mL solution of 1% hydrogen peroxide in lake water and place in a 50 ml conical tube (fill to the very top of the tube).
Transfer the cell strainer containing gemmules into the hydrogen peroxide solution and incubate for 0h 5m 0s.
Remove the cell strainer from the hydrogen peroxide solution and rinse very thoroughly by placing under a flowing tap of RO water for at least 0h 1m 0s.
Gemmule sterilization: select gemmules for use
Place the cell strainer into a clean 10 cm Petri dish containing lake/spring water.
Using a new p1000 tip, transfer the gemmules from the cell strainer to the surrounding dish.
Gemmule plating: sterilize in Anti-Anti for 24-48 hours
Place the desired amount of spring/lake water containing Anti-Anti in the culture dish where you wish to grow the sponges, and then add the number of gemmules you wish to grow, and place at Room temperature.
Incubate in Anti-Anti for 24 - 48 hours, then replace with pure lake/spring water, or with lake/spring water containing another antibiotic such as 50µg/mL kanamycin or 100µg/mL ampicillin (depending upon your experimental goals).
Gemmule plating: position gemmules in the center of the culture dish
Sponges typically start to hatch between 72 and 96 hours after plating, at which point they attach to the culture well/dish and cannot be moved.
It is important to make sure the gemmules are positioned according to your experimental needs (usually towards the center of the well to enable imaging). If placed near the edge of the well/dish, the sponges may grow vertically and be difficult/impossible to image. A trick for moving gemmules to the center of the well is to swirl the dish for several seconds to create a vortex within the well.
Gemmule plating: Mature sponges are best used between days 7-10
If the sponges are grown in at least 500µL of lake/spring water per gemmule it is usually not critical to change the solution over the course of a 7-10 day experiment.
If you wish to limit autofluorescence for subsequent imaging studies, it is essential to grow the sponges in a dark incubator or drawer to limit the growth of intracellular Chlorella -like algae.
Without feeding, sponges cannot be maintained long beyond 10 days usually. As they age, their tissues often retract and/or the entire sponge will migrate away from the gemmule capsule, leaving behind sponging fibers and spicules. We avoid working with sponges at this stage.
