TITLE: Growing freshwater sponges from gemmules in the laboratory
AUTHORS: Scott Nichols

[DESCRIPTION]
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.

[GUIDELINES]
Guidelines

Gemmules are temperature sensitive. Keep gemmule stocks at 4 °C to maintain their longevity. There are published reports of defined media for growing sponges, but we typically use store bought spring water, or sterile-filtered lakewater; both work well.

A note about gemmule storage:

Field-collected gemmules should be stored at 4 °C in sterile-filtered or autoclaved lake water. Keep gemmules collected from different adults in separate containers to ensure that you don't accidentally mix different species, and to ensure that gemmules used in an experiment have the same genetic background. If stored in a deli-style fridge, place gemmules in an opaque container to limit the growth of algae. 

Anecdotally, we have observed that gemmules remain viable longer if stored in a minimal volume of water, just covering their surface. 

[STEPS]
SECTION: Gemmule sterilization
1. Place a 40-70 µm cell strainer into a clean 10 cm Petri dish filled with cold, lake/spring water. 
SECTION: Gemmule sterilization
2. 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. 
SECTION: Gemmule sterilization: sterilize with hydrogen peroxide, then rinse
3. Prepare a 60 mL solution of 1% hydrogen peroxide in lake water and place in a 50 ml conical tube (fill to the very top of the tube). 

SECTION: Gemmule sterilization: sterilize with hydrogen peroxide, then rinse
4. Transfer the cell strainer containing gemmules into the hydrogen peroxide solution and incubate for 5 min. 
 


SECTION: Gemmule sterilization: sterilize with hydrogen peroxide, then rinse
5. 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 1 min. 
 


SECTION: Gemmule sterilization: select gemmules for use
6. Place the cell strainer into a clean 10 cm Petri dish containing lake/spring water.
SECTION: Gemmule sterilization: select gemmules for use
7. Using a new p1000 tip, transfer the gemmules from the cell strainer to the surrounding dish.
 

SECTION: Gemmule plating: sterilize in Anti-Anti for 24-48 hours
8. 
 
Even though you have sterilized the gemmules with hydrogen peroxide, it is still possible for cultures to contain contaminating fungi. To prevent fungal growth, dilute the antibiotic-antimycotic (Anti-Anti) to 1x final concentration in lake/spring water.
SECTION: Gemmule plating: sterilize in Anti-Anti for 24-48 hours
9. 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. 
 


SECTION: Gemmule plating: sterilize in Anti-Anti for 24-48 hours
10. 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 50 µg/mL kanamycin or 100 µg/mL ampicillin (depending upon your experimental goals). 
 



SECTION: Gemmule plating: position gemmules in the center of the culture dish
11. 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. 
SECTION: Gemmule plating: position gemmules in the center of the culture dish
12. 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. 
SECTION: Gemmule plating: Mature sponges are best used between days 7-10
13. If the sponges are grown in at least 500 µL of lake/spring water per gemmule it is usually not critical to change the solution over the course of a 7-10 day experiment. 
SECTION: Gemmule plating: Mature sponges are best used between days 7-10
14. 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.
SECTION: Gemmule plating: Mature sponges are best used between days 7-10
15. 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.