Oct 25, 2022

Public workspaceSchistosoma mansoni cercariae sexing

DOI
dx.doi.org/10.17504/protocols.io.rm7vzby12vx1/v1
  • 1Wellcome Sanger Institute
Sarah K Buddenborg
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzby12vx1/v1
Protocol Citation: Sarah K Buddenborg, Matt Berriman 2022. Schistosoma mansoni cercariae sexing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzby12vx1/v1
Manuscript citation:
in preparation
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: October 24, 2022
Last Modified: October 25, 2022
Protocol Integer ID: 71750
Funders Acknowledgement:
Wellcome Trust
Grant ID: 098051
Wellcome Trust
Grant ID: 206194
Abstract
This DNA extraction method for Schistosoma mansoni cercariae is based on the HOTSHOT method https://health.uconn.edu/mouse-genome-modification/protocols/hotshot-method-of-dna-preparation/. DNA isolation is followed by PCR amplification of the "W1" female W chromosome repetitive region. The 476 bp W1 repeat was identified by Webster, Mansour, and Bieber (1989). PCR primers for the W1 repeat were designed by Gasser, Morahan, and Mitchell (1991). Existing primers for the S. mansoni actin gene are used as a positive control (Delcroix et al, 2006).

W1a - 5' CAA CAC AGT GAA ATT CTT CC 3' (positions 10-29)
W1b- 5' GAA TTC ACC ACT CGA CAT TC 3' (positions 463-482)
CITATION
Philippa Webster, Tag E. Mansour, David Bieber (1989). Isolation of a female-specific, highly repeated Schistosoma mansoni DNA probe and its use in an assay of cercarial sex. Molecular and Biochemical Parasitology.
LINK
10.1016/0166-6851(89)90169-2

CITATION
Robin B. Gasser, Grant Morahan, Graham F. Mitchell (1991). Sexing single larval stages of Schistosoma mansoni by polymerase chain reaction. Molecular and Biochemical Parasitology.
LINK
10.1016/0166-6851(91)90187-b

CITATION
Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorák J, Hsieh I, Bahgat M, Dissous C, McKerrow JH (2006). A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite.. The Journal of biological chemistry.

Materials
Custom DNA oligos ordered from IDT - LabReady (Normalized to 100µM in IDTE pH 8.0):
W1a - 5’ CAA CAC AGT GAA ATT CTT CC 3’
W1b - 5’ GAA TTC ACC ACT CGA CAT TC 3’

SmAct-F - 5’ CAG TGT TCC CTT CCA TCG TT 3’
SmAct-R - 5’ GGA CAG GGT GTT CTT CTG GA 3’

2x Alkaline Lysis Reagent
25ml H2O
125µl 10N NaOH
20µl 0.5M disodium EDTA
Make fresh every 1-2 months
Store at TemperatureRoom temperature

1M Tris-HCl
6.057g Tris-HCl
To 50ml with nuclease-free H2O
Store at TemperatureRoom temperature

2x Neutralizing Reagent
23ml H2O
2ml 1M Tris-HCl
Store at TemperatureRoom temperature


ReagentMilliQ waterContributed by users
ReagentCostar® 6-well Clear TC-treated Multiple Well Plates Individually Wrapped SterileCorningCatalog #3516
ReagentFeatherweight forcepsBioQuipCatalog #4750
ReagentProtein LoBind® 5.0 mL with snap capEppendorfCatalog #0030108302
ReagentProtein LoBind® 15 mL conical tubeEppendorfCatalog #0030122216
ReagentpluriStrainer Mini 100 µmpluriSelectCatalog #43-10100-40
Reagent3ml Sterile Graduated Transfer Pipets individually wrappedFisher ScientificCatalog #13469108
ReagentPlatinum II Taq Hot-Start DNA Polymerase (Invitrogen)Thermo Fisher ScientificCatalog #14966001
ReagentSodium Hydroxide solution 10NMerck Millipore SigmaCatalog #SX0607N
ReagentEthylenediaminetetraacetic acid disodium salt solution BioUltra for molecular biology pH 8.0 ~0.Merck Millipore SigmaCatalog #03690
ReagentTris hydrochlorideSigma AldrichCatalog #10812846001
ReagentNuclease-free WaterSigma AldrichCatalog #W4502-1L

Equipment
Centrifuge 5810 R
NAME
Refrigerated centrifuge
TYPE
Eppendorf
BRAND
5811000065
SKU
https://www.eppendorf.com/gb-en/eShop-Products/Centrifugation/Multipurpose-Centrifuges/Centrifuge-5810-5810R-p-PF-240994
LINK

Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
https://online-shop.eppendorf.us/US-en/Temperature-Control-and-Mixing-44518/Instruments-44519/Eppendorf-ThermoMixerC-PF-19703.html
LINK
Any heat block will suffice
SPECIFICATIONS

Equipment
Mastercycler X50
NAME
thermocycler
TYPE
Eppendorf
BRAND
6311000010
SKU
https://www.eppendorf.com/pt-en/eShop-Products/PCR/Thermocyclers/Mastercycler-X50-p-PF-217186
LINK
any thermocycler will be sufficient
SPECIFICATIONS







Safety warnings

Safety information
Biomphalaria spp. snails with a patent Schistosoma mansoni infection are classed as CL2/BSL2
Cercariae from shedding snails are infectious and proper PPE should be worn at all times

Before start
- Pre-heat heat block to Temperature95 °C
- Pre-cool centrifuge to Temperature4 °C
- Fill an ice bucket with ice
Cercariae Collection
Cercariae Collection
1h
1h
While holding with wide-tip featherweight forceps, rinse patent Biomphalaria glabrata snails, individually, with MilliQ water


Note
- Rinsing snails individually prevents cross-contamination of cercariae from another snail
- Snails should be at least 5 weeks post infection with a single miracidium

Place rinsed snails into individual wells of 6- or 12-well plates
Add 3-4ml of MilliQ water to each well
Induce shedding of cercariae by place snails under a direct light for up to 2 hours or until cercariae are visible in well with a naked eye


2h
After 2 hrs, remove snails that are not shedding cercariae and place into a new tank with food. These snails can be re-shed in one week. Some monomiracidium-infected snails will not begin shedding until up to 8 weeks after exposure. At this time, if they are still not shedding cercariae, it is safe to assume they were not infected and can be disposed of properly.
For snails that are shedding low amounts of cercariae (<100), place into a new tank with food and re-shed in one week
Put an "X" on the wells of the snails that are shedding and uniquely number each. This numbering must be preserved throughout.
Prepare numbered 5ml Eppendorf tubes each fitted with a 100µm pluriStrainer Mini
Using a new sterile transfer pipet for each snail, pipette water containing the cercariae through the pluriStrainer
Rinse the well and snails with fresh MilliQ water and collect this water as above to fill each 5ml tube to the top
Note
If you do not have time to complete the DNA isolation, PCR, and gel electrophoresis in a single day, snails can be kept in 6-well plates with a fresh water change daily. Be sure to maintain the numbering of the snails.

Discard pluriStrainers, close, and submerge 5ml tubes containing cercariae into ice for Duration00:30:00
30m
Centrifuge tubes at Centrifigation2000 rpm, 4°C, 00:30:00

30m
After centrifugation, place tubes immediately back on ice and aspirate/pipette off liquid, taking care not to disrupt the cercariae pellet. Try to leave no more than 50µl of water on the cercariae pellet as the more dilute the lysate is, the more difficult amplification will be.
Cercariae and Cell Lysis
Cercariae and Cell Lysis
Add 1x volume of 2x Alkaline Lysis Reagent to each cercariae pellet and mix well (i.e. add 25µl 2x Alkaline Lysis Reagent to 25µl of cercariae in MilliQ)
Note
2x Alkaline Lysis Reagent
25ml H2O
125µl 10N NaOH
20µl 0.5M disodium EDTA
Make fresh every 1-2 months
Store at TemperatureRoom temperature

Incubate at Temperature95 °C for ~1 hr or until cercariae dissolve, mixing at least every 15 min. Centrifuge using a mini-centrifuge if needed

1h
After tissue is dissolved, place on ice to cool
15m
Lysate Neutralization
Lysate Neutralization
Add 1x volume of 2x Neutralizing Reagent and briefly vortex to mix (i.e. add 50µl 2x Neutralizing Reagent to 50µl of lysate)


Note
1M Tris-HCl
6.057g Tris-HCl
To 50ml with nuclease-free H2O
Store at TemperatureRoom temperature

2x Neutralizing Reagent
23ml H2O
2ml 1M Tris-HCl
Store at TemperatureRoom temperature

PCR amplification of sex-specific region
PCR amplification of sex-specific region
Prepare two PCR master mixes on ice (for actin control and W1 primer sets) each with enough for your samples, 1 female positive control, 1 male negative control, and 1 water blank control (i.e. if you have 8 samples, you will need 11 reactions for actin primers and 11 reactions for W primers)

Note
W1a - 5’ CAA CAC AGT GAA ATT CTT CC 3’
W1b - 5’ GAA TTC ACC ACT CGA CAT TC 3’

SmAct-F - 5’ CAG TGT TCC CTT CCA TCG TT 3’
SmAct-R - 5’ GGA CAG GGT GTT CTT CTG GA 3’



Mock-up of layout for 8 samples and their controls in four 8-well PCR strips

ABC
ReagentFinal concentrationVolume per reaction
Nuclease-free water-6.32µl
Platinum II Taq0.04U/µl0.08µl
5x Platinum II Buffer1x2µl
10mM dNTP0.2mM each0.2µl
10uM F0.2uM0.2µl
10uM R0.2uM0.2µl
DNA*-1µl
10µl reaction
*If you think your DNA concentration is really low/high, you can measure first on Nanodrop (blank with Neutralization Buffer) and change input volume as necessary. Keep in mind that using more than 10% of the overall volume may inhibit the PCR reaction.

Run the PCR program as follows for both primer sets:
ABCD
Initial denaturation94°C2 min
Denature98°C5 sec
Anneal and extend60°C15 secTo step 2 x25
Hold4°C~
30m
Analyze PCR Amplicons
Analyze PCR Amplicons
1h
1h
Run a 1% agarose gel to analyze 5-10µl of your PCR product. Males will have PCR product band for actin only; Females will have PCR product band for actin and W1.
1h
Citations
Philippa Webster, Tag E. Mansour, David Bieber. Isolation of a female-specific, highly repeated Schistosoma mansoni DNA probe and its use in an assay of cercarial sex
10.1016/0166-6851(89)90169-2
Robin B. Gasser, Grant Morahan, Graham F. Mitchell. Sexing single larval stages of Schistosoma mansoni by polymerase chain reaction
10.1016/0166-6851(91)90187-b
Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorák J, Hsieh I, Bahgat M, Dissous C, McKerrow JH. A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite.