TITLE: Snail husbandry for maintaining the Schistosoma mansoni life cycle
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
Maintenance and general husbandry of schistosome-susceptible snails, including but not limited to Biomphalaria glabrata fresh water snails. The purpose of this SOP is to outline the weekly protocols required for general husbandry of a Schistosoma mansoni life cycle through the use of Biomphalaria glabrata snails.

[BEFORE_START]
Thoroughly rinse and autoclave oyster shells (can be re-used indefinitely by soaking in 10% bleach overnight, rinsing thoroughly, and autoclaving)

[STEPS]
SECTION: Preparing a new aquarium tank
1. Label clean aquarium with appropriate information in permanent marker (i.e. date, owner, age, numbers of individuals, exposure date, expected patent date, experiment number, etc). Example labels are attached    
SECTION: Preparing a new aquarium tank
2. Rinse a generous handful of autoclaved oyster shells with water until it runs clear and add to the base of the tank
SECTION: Preparing a new aquarium tank
3. Fill aquarium 1/2-3/4 full with 1x Aquarium Water (see recipe in "MATERIALS" section)
SECTION: Preparing a new aquarium tank
4. Place a clean airline and air stone in aquarium and put in appropriate heated incubator
SECTION: Preparing a new aquarium tank
5. Connect airline and assure that the appropriate amount of air is going to the tank and all other tanks connected to the same airline. Note that changing the number of tanks in a connected airline results in decreased or increased air pressure to other tanks so adjusted accordingly
SECTION: Preparing a new aquarium tank
3.1. For infected snails, keep water level at or just below 1/2 full to reduce chance of splashing when handling
6. Add snails to the tank (<100 baby snails/10L water; <10 adult breeder snails/10L; <30 juvenile parasite-exposed snails/10L)
5.1. Add one 15ml falcon tube styrofoam rack to breeder tanks (snails also love to lay eggs on artificial lotus leaves)
SECTION: Snail feeding (2x/week)
7. Place one cube (15mm x 15mm) of snail gel per 6-8 adult snails or two per 100 baby snails (use size and number of snails to estimate amount of snail gel to give to each tank)
SECTION: Snail feeding (2x/week)
8. To prevent overgrowth of bacteria, do not overfeed snails. If any snail gel is leftover >1 day after a feeding, then the snails are being overfed and reduce as necessary
SECTION: Snail tank housekeeping
9. Visually inspect the snail tanks during feedings for heavy bacterial and/or waste contamination (turbid and/or smelly water, large amount of debris collected in oyster shells) and/or heavy rotifer infestations. If any of these is detected, rinse the snails thoroughly with fresh 1x Aquarium Water and prepare a new tank, airline, and bubbler for the snails
SECTION: Snail tank housekeeping
10. Once a week top up water on tanks to their appropriate levels using MilliQ H2O. To top up the water use the hose connected to the MilliQ water source
SECTION: Snail tank housekeeping
11. Replace 10% of the dirtiest tanks each week
SECTION: Snail egg collection and baby snail maintenance
12. Fill a 100ml beaker or small dish with 1x Aquarium Water
SECTION: Snail egg collection and baby snail maintenance
13. Working with one styrofoam rack at a time, use featherweight forceps to gently remove the egg masses from the rack and place in the beaker (slide forceps under egg mass and lift away gently)
SECTION: Snail egg collection and baby snail maintenance
14. Repeat for all styrofoam racks from breeder tanks
SECTION: Snail egg collection and baby snail maintenance
15. Check the egg masses under the microscope for rotifer infestation. Discard individual egg masses with heavy rotifer infestations. Rinse egg masses if rotifers are present in the water until none are seen under microscope
SECTION: Snail egg collection and baby snail maintenance
16. When finished collecting, empty the beaker into a prepared nursery/baby tank for that particular week (1 new nursery/baby tank per week) rinse container well with 1x Aquarium Water to remove stuck baby snails/egg masses
SECTION: Snail egg collection and baby snail maintenance
17. Split any tanks that have >100 baby snails to promote growth
SECTION: Decontaminating infected and uninfected snail tank and tank water
18. Remove airline with air stone and place in tank. If applicable, keep styrofoam rack in tank.
SECTION: Decontaminating infected and uninfected snail tank and tank water
19. Remove any snails that might be kept – leave dead shells. Add 20ml of 5000ppm Klorsept and leave overnight (minimum 6 hours)
SECTION: Decontaminating infected and uninfected snail tank and tank water
20. After this period, dispose of the decontaminated water using the sieve or colander to collect oyster shells and dead snails
SECTION: Decontaminating infected and uninfected snail tank and tank water
21. Rinse tank with diH2O and then spray tank with 70% EtOH. Scrub tank with green scouring pad, rinse  thoroughly with diH2O and allow to air dry
SECTION: Decontaminating infected and uninfected snail tank and tank water
22. Styrofoam racks, air stones, and airline should be sprayed with 70% EtOH, rinsed liberally with diH2O, and air dried
SECTION: Decontaminating infected and uninfected snail tank and tank water
23. Put decontaminated and rinsed oyster shells and dead snails in an autoclave tray and autoclave when full