Mar 31, 2023
Snail husbandry for maintaining the Schistosoma mansoni life cycle
- Sarah K Buddenborg1,
- Geetha Sankaranarayanan1,
- Lisa Seymour1,
- Matt Berriman1
- 1Wellcome Sanger Institute
Protocol Citation: Sarah K Buddenborg, Geetha Sankaranarayanan, Lisa Seymour, Matt Berriman 2023. Snail husbandry for maintaining the Schistosoma mansoni life cycle. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7yjd8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 30, 2023
Last Modified: March 31, 2023
Protocol Integer ID: 79779
Keywords: snails, biomphalaria, schistosoma
Funders Acknowledgements:
Wellcome Trust
Grant ID: 098051
Wellcome Trust
Grant ID: 206194
Abstract
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.
Materials
Coarse grit poultry oyster shells
Aquariums (maximum density of snails is ~1 adult snail per 1L water volume)
Air compressor or air source
Marina blue airline tubing (pet store or Amazon)
25mm air stone cylinders (pet store or Amazon)
Artificial lotus leaves (pet store or Amazon)
Temperature-regulated room, aquarium heaters, or incubators to house snail tanks (temperature at 25°C)
MilliQ waterContributed by users
Blunt featherweight forceps wide tipBioQuipCatalog #4750
Sodium AlginateMerck MilliporeSigma (Sigma-Aldrich)Catalog #W201502
Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C1016
MgSO4 x 7H20Merck MilliporeSigma (Sigma-Aldrich)Catalog #M2773
Potassium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P0772
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5761
Iron(III) chloride hexahydrateContributed by usersCatalog #236489
Snail gel food (see recipes)
1x Aquarium Water (diluted from 10X; see recipes)
10X AQUARIUM WATER
5.56g CaCl2
12.28g MgSO4-7H2O
0.43g K2SO4
4.2g NaHCO3
480µl FeCl3-6H2O (0.5g/100ml water)
Fill to 10L and store in 1L bottles
Dilute to 1x: 9L MQ water + 1L 10x
1L diH2O
4g sodium alginate
16g barley grass powder (health food store or Amazon)
4g wheat germ (health food store or Amazon)
4g fish flakes/pellet pre-ground mix (pet store or Amazon)
2g powdered milk (health food store or Amazon)
2 liter 2% CaCl2 solution (40g CaCl2 + 2 liter H2O)
- Add sodium alginate to 1L diH2O and heat on magnetic stirrer with large stir bar, stirring rapidly (temp should be ~ 80°C)
- Add barley grass powder and stir until homogeneous
- Add the pulverized wheat germ/fish flakes/powdered milk mixture
- Stir for several minutes until thoroughly mixed
- Divide mixture between 2 trays so that it is about 15mm deep and cool at 4°C 3-4hrs until gel is set
- Gently flood with 2% CaCl2
- Place at 4°C overnight (can combine multiple slabs of gel into one tank to save space)
- Pour off CaCl2 and rinse 2x with diH2O
- Slice into cubes of approximately 15mm x 15mm and store in re-sealable bag at 4°C. Use within 1 week and do not overfeed.
Safety warnings
For all procedures described, wear appropriate PPE:
- Lab coat
- Long-cuff gloves
- Waterproof vinyl apron (when working with pre-patent and patent snails)
- Face shield (when working with pre-patent and patent snails)
IMPORTANT. ALL pre-patent and patent snails must be treated as if they are infectious (i.e. wear face shield and waterproof vinyl apron). Infected snails will release small amounts of cercariae all the time, even if kept in the dark.
Before start
Thoroughly rinse and autoclave oyster shells (can be re-used indefinitely by soaking in 10% bleach overnight, rinsing thoroughly, and autoclaving)
Preparing a new aquarium tank
Preparing a new aquarium tank
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
infected_new tanks.avery uninfected_new tanks.avery
infected_new tanks.avery uninfected_new tanks.avery Rinse a generous handful of autoclaved oyster shells with water until it runs clear and add to the base of the tank
Fill aquarium 1/2-3/4 full with 1x Aquarium Water (see recipe in "MATERIALS" section)
For infected snails, keep water level at or just below 1/2 full to reduce chance of splashing when handling
Place a clean airline and air stone in aquarium and put in appropriate heated incubator
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
Add one 15ml falcon tube styrofoam rack to breeder tanks (snails also love to lay eggs on artificial lotus leaves)
Add snails to the tank (<100 baby snails/10L water; <10 adult breeder snails/10L; <30 juvenile parasite-exposed snails/10L)
Snail feeding (2x/week)
Snail feeding (2x/week)
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)
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
Snail tank housekeeping
Snail tank housekeeping
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
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
Replace 10% of the dirtiest tanks each week
Snail egg collection and baby snail maintenance
Snail egg collection and baby snail maintenance
Fill a 100ml beaker or small dish with 1x Aquarium Water
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)
Repeat for all styrofoam racks from breeder tanks
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
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
Split any tanks that have >100 baby snails to promote growth
Decontaminating infected and uninfected snail tank and tank water
Decontaminating infected and uninfected snail tank and tank water
Remove airline with air stone and place in tank. If applicable, keep styrofoam rack in tank.
Remove any snails that might be kept – leave dead shells. Add 20ml of 5000ppm Klorsept and leave overnight (minimum 6 hours)
After this period, dispose of the decontaminated water using the sieve or colander to collect oyster shells and dead snails
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
Styrofoam racks, air stones, and airline should be sprayed with 70% EtOH, rinsed liberally with diH2O, and air dried
Put decontaminated and rinsed oyster shells and dead snails in an autoclave tray and autoclave when full
Protocol references
CITATION
LINK
PMID 3455251CITATION
CITATION
Citations
Barbosa ND, Pimentel-Souza F, Sampaio IB. The effect of seasonal temperature and experimental illumination on reproductive rate in the snail Biomphalaria glabrata
PMID 3455251Standen, O.D. Some observations upon the maintenance of Australorbis glabratus in the laboratory.
10.1080/00034983.1951.11685473