Apr 04, 2023

Public workspaceSchistosoma mansoni cercariae shedding

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly7bbplx9/v1
  • Sarah Buddenborg1,
  • Geetha Sankaranarayanan1,
  • Magda E Lotkowska1,
  • Catherine McCarthy1,
  • Arporn Wangwiwatsin1,
  • Carmen L Dias Soria1,
  • Lisa Seymour1,
  • Gabriel Rinaldi1,
  • Matt Berriman1
  • 1Wellcome Sanger Institute
  • Schistosoma mansoni
Sarah K Buddenborg
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly7bbplx9/v1
Protocol Citation: Sarah Buddenborg, Geetha Sankaranarayanan, Magda E Lotkowska, Catherine McCarthy, Arporn Wangwiwatsin, Carmen L Dias Soria, Lisa Seymour, Gabriel Rinaldi, Matt Berriman 2023. Schistosoma mansoni cercariae shedding. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7bbplx9/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: April 03, 2023
Last Modified: April 04, 2023
Protocol Integer ID: 79923
Keywords: schistosomula for further experimentation, shedding schistosoma, infected biomphalaria glabrata snail, schistosoma mansoni cercariae, biomphalaria glabrata snail, schistosomula, infected snail, schistosoma, snail, snails in the dark, biomphalaria glabrata, biomphalaria, mansoni cercariae, cercariae
Funders Acknowledgements:
Wellcome Trust
Grant ID: 098051
Wellcome Trust
Grant ID: 206194
Abstract
Schistosoma mansoni cercariae can be obtained from infected Biomphalaria glabrata snails. Although infected snails will release (shed) cercariae constantly at low numbers, it is possible to trigger the release of larger quantities by keeping the snails in the dark for a few days before shedding.

Material obtained from these procedures can be mechanically transformed into schistosomula for further experimentation.

Materials
ReagentMilliQ water
Reagent12-well Clear TC-treated Multiple Well Plates Individually Wrapped SterileCostarCatalog #3513
Reagent24-well Clear TC-treated Multiple Well Plates Individually Wrapped SterileCostarCatalog #3524
ReagentSterile Graduated Transfer PipetsFisher ScientificCatalog #13479108
ReagentBlunt featherweight forceps wide tipBioQuipCatalog #4750
ReagentLugol solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #32922-250ML
ReagentNalgene™ PPCO Wash Bottles, 500mLThermo FisherCatalog #2405-0500PK

Equipment
Zoom stereomicroscope with diascopic illumination stand
NAME
stereomicroscope
TYPE
Nikon
BRAND
SMZ800N
SKU
Lamp or other light source
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

Troubleshooting
Safety warnings
Biomphalaria glabrata snails are fresh water snails. In the laboratory, infected snails can be kept in the dark; however, infected snails will release small amounts of cercariae all the time, even in the dark.

Cercariae are infectious to humans. Please use proper PPE at all times, including a lab coat, waterproof over-gown, long-cuff gloves AQL <=0.65, and face shield.

  • All disposable materials should be placed in biohazardous waste bins
  • Glassware should be immersed in a klorsept solution of at least 50ppm for at least 2 hours, rinsed with diH2O and then autoclaved
  • Any liquids should be treated with klorsept solution of at least 50ppm for at least 2 hours
  • Liquids treated with klorsept should be diluted further and disposed in the drain
Preparing snails for shedding
Place aquarium tank containing snails >=4 weeks post-exposure to S. mansoni on a stable bench top
Note
Snails infected with one miracidium ("monomiracidium-infected") can take up 7 weeks to begin shedding. Repeat sheddings maximum of 1x/week until the 7th week post-exposure.
Using featherweight forceps, remove snails from the tank and rinse gently with 1x aquarium water in a wash bottle

  • This is critical in order eliminate false positives (snails with cercariae on them from a different shedding snail)
Critical
Place snails into labelled 12- or 24-well plates with one snail per well

Shedding snails
Place cover on plate and place snails under LED or fluorescent lighting for up to 2 hours to trigger release of cercariae from the infected snails.
Note
IMPORTANT - While shedding, cercariae are in high density and the water is highly hazardous!

Add enough 1x aquarium water to each well so that they are covered
Check snails for shedding by viewing each well under a zoom stereomicroscope with diascopic illumination stand
Collecting cercariae
Collect water containing cercariae every 30 minutes (alternatively, you can just collect cercariae at the end of 2 hrs shedding; this will give you the most concentrated cercariae suspension) using a transfer pipet and top up wells with new 1x aquarium water

  • If needed (i.e. for monomiracidium-infected snails where separate sexes are needed), keep cercariae from each snail separate and use new transfer pipet for each snail
Dispose of snails that did not shed by putting them in a container klorsept (See "Guidelines and Warnings") or place in new tank if you'd like to re-shed after an additional week.
Return shedding snails to a new tank with food and appropriate labels – make sure their air stones are under the water level and bubbling properly (See protocol "Snail husbandry for maintaining the Schistosoma mansoni life cycle")
Note
Snails should not be re-shed for one week minimum

Protocol references
Paul F. Basch.Cultivation of Schistosoma mansoni In vitro. I. Establishment of Cultures from Cercariae and Development until Pairing
10.2307/3280632