TITLE: Schistosoma mansoni cercariae sexing
AUTHORS: Sarah K Buddenborg

[DESCRIPTION]
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)
 

 

 


[BEFORE_START]
- Pre-heat heat block to 95 °C 
- Pre-cool centrifuge to 4 °C 
- Fill an ice bucket with ice

[STEPS]
SECTION: Cercariae Collection
1. While holding with wide-tip featherweight forceps, rinse patent Biomphalaria glabrata snails, individually, with MilliQ water


 

SECTION: Cercariae Collection
2. Place rinsed snails into individual wells of  6- or 12-well plates
SECTION: Cercariae Collection
3. Add 3-4ml of MilliQ water to each well
SECTION: Cercariae Collection
4. 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


SECTION: Cercariae Collection
4.1. 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. 
SECTION: Cercariae Collection
4.2. For snails that are shedding low amounts of cercariae (<100), place into a new tank with food and re-shed in one week
SECTION: Cercariae Collection
5. Put an "X" on the wells of the snails that are shedding and uniquely number each. This numbering must be preserved throughout. 
SECTION: Cercariae Collection
6. Prepare numbered 5ml Eppendorf tubes each fitted with a 100µm pluriStrainer Mini
SECTION: Cercariae Collection
7. Using a new sterile transfer pipet for each snail, pipette water containing the cercariae through the pluriStrainer
SECTION: Cercariae Collection
7.1. Rinse the well and snails with fresh MilliQ water and collect this water as above to fill each 5ml tube to the top
 

SECTION: Cercariae Collection
8. Discard pluriStrainers, close, and submerge 5ml tubes containing cercariae into ice for 30 min
SECTION: Cercariae Collection
9. Centrifuge tubes at 2000 rpm, 30 min, 4 °C 

SECTION: Cercariae Collection
10. 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. 
SECTION: Cercariae and Cell Lysis
11. 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)
 

SECTION: Cercariae and Cell Lysis
12. Incubate at 95 °C  for ~1 hr or until cercariae dissolve, mixing at least every 15 min. Centrifuge using a mini-centrifuge if needed

SECTION: Cercariae and Cell Lysis
13. After tissue is dissolved, place on ice to cool
SECTION: Lysate Neutralization
14. Add 1x volume of 2x Neutralizing Reagent and briefly vortex to mix (i.e. add 50µl 2x Neutralizing Reagent to 50µl of lysate)


 

SECTION: PCR amplification of sex-specific region
16. Run the PCR program as follows for both primer sets:
 
Initial denaturation94°C2 minDenature98°C5 secx25Anneal and extend60°C15 secHold4°C~
 
SECTION: PCR amplification of sex-specific region
15. 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)

 


  

 

SECTION: Analyze PCR Amplicons
17. 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.