Goal stocking density for challenge —> 50 larvae/ml

• 100 larvae per 2ml well
• at least 12,000 larvae for jars

Materials needed:

• larvae
• dry shipper
• genomic sampling materials
• mesh or filter paper
• cooler and ice pack
• paper towel
• RE22 pathogen
• FSSW (enough for rinsing and challenges in plates and jars) at 28-30ppt
• pipettes and tips
• Shellfish diet
• clean beakers or containers to suspend larvae in (from Blount)
• Sedgewick-Rafter slides
• 24 or 6 well plates - tissue culture lab (Fisher Cat No. FBO12927)
• sterile jars (for genomic sampling challenge)
• Microscope
• counters
• small falcon tube sieves (2)
• falcon tubes

Procedure:

At Blount 104:

1. Drain down buckets and concentrate larvae in known volume (by treatment)
2. Count and estimate number of larvae in known volume
3. Take initial genomic samples
4. Filter known number of larvae on to mesh or coffee filter wrapped in a wet towel
5. Place larvae in a cooler with an ice pack for transport
6. Gather any needed supplies and transport to CBLS

At CBLS (in the Gomez-Chiarri Lab and PPP Lab)

1. Prepare the RE22 pathogen in FSSW and in the proper concentration
2. Prepare dilute shellfish diet solution (25ul Shellfish diet into 50mL FSSW) gently inverting to mix
3. Prepare each jar and well by adding the appropriate amount of FSSW (*maintain equivalent stocking densities of the larvae between inoculated and control wells/jars)
4. Rinse larvae onto falcon tube sieve (by treatment)
5. Wash larvae with 4 x 4 mL FSSW using a sterile eye dropper plastic pipette
6. Place larvae in a 50 mL Falcon tube. Fill up with FSSW to 25 mL
7. Fill falcon tube up the rest of the way with 25ml of dilute shellfish diet solution
8. Calculate the number of larvae/ml in each falcon tube and use that number to calculate the volume of larvae needed to add to each well and each jar
9. Add larvae to all wells and jars
10. Add the proper volume of RE22 pathogen to challenge wells/jars and ensure that all wells/jars are at the same stocking density of larvae
11. Set plates and jars to incubate on shaker overnight and note the time
12. Morning next day (as early as possible): Start checking the larvae for mortality by looking at a couple of control wells (make sure they are all mostly alive) and a couple of wells with RE22 only, starting 14-15 h after addition of RE22. If it looks like at least around 50% of the larvae in the RE22 group are dead, proceed to then to count the number of dead and live larvae.
13. To count - take 1 mL of sample from a well after mixing by gently pipetting up and down to mix them well.
14. Place the 1 mL of larvae on top of Sedgwick rafter.
15. Count dead/live using the microscope.
16. Repeat for each well.
17. Once 60-80% mortality has been assessed in plate treatments, begin genomic sampling from the jars using the Puritz Lab procedure for filtering and flash freezing larvae.

NOTE: Make sure that you don’t wait until all larvae in the RE22 treatment are dead: you want to target a mortality of 60 - 80% (no more - no less). If necessary, count all RE22 wells first before counting controls.