ROD challenge protocol (version 1)

ROD = Roseovarius Oyster Disease

Protocol for an Roseovarius Oyster Disease (Juvenile Oyster Disease) Challenge using Juvenile Oysters Exposed to Diel-cycling Hypoxia and Acidification This document incorporates portions of Katrina (Kat) Kulesh’s protocol from the Summer of 2022. The sections copied from Kat’s protocol are denoted with a “KK”.

Pathogen Preparation - KK

  • Streak a known glycerol stock of Aliiroseovarius crassostreae CV919-312 (formerly called Roseovarius crassostreae CV919-312) on a marine agar plate and incubate
  • Add a colony to a 10 mL mYP30 or SWT broth in a 50 mL Falcon tube and incubate with a loosely screwed on cap and agitation
  • Perform a DNA extraction, then a gel electrophoresis to confirm presence of DNA
  • Perform a PCR on the extracted DNA with both forward and reverse primers,, then a gel electrophoresis using a 1kB ladder (100 mb ladder if can’t find 1kB)
  • Used JOD primers 1339F and 115R
  • Use a negative control with no DNA and only nuclease-free water Find more details on primers, and bands given from gels in cited paper *Maloy, A. P., & Miller, K. B. (2008). Localisation of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected Eastern oyster (Crassostrea virginica). Journal of Invertebrate Pathology, 97, 150-158.

Set up: Two Days in Advance - KK

  • Prepare 15 mL of broth per container one or two days before adding ROD to oysters
  • Prepare set-up in incubator fridge with non-ROD and ROD containers separated by hanging piece of plastic
  • Have each container aerated with disinfected air stones and airlines
  • *Be sure aerators work!
  • Divide juveniles amongst replicate baskets by dry weight
  • Dry with kimwipes or something and measure using scale
  • Add oysters to mesh baskets! - They will acclimate for ~48 hours before the beginning of the challenge
  • Add to the Puritz lab’s mesh baskets and place in bowls filled 0.650 L
  • Careful to not let aerators drydock the oysters, don’t place aerators immediately underneath baskets
  • Feeding: Either mix Reed Mariculture Shellfish Diet in with water to fill bowls or separately dilute and add to bowls
  • Feed daily at similar times if do-able
  • Water changes: Daily 50% (subject to change depending on juveniles’ feeding habits)
  • Use a toothbrush or dedicated bristle column brush per treatment to clean sides of container and mesh basket of algae BEFORE changing water
  • It’s good to periodically shift oysters around in their baskets
  • ***You do NOT need to turn on the incubator- the heat produced from running the aerators keeps it at a consistent 22C when the door is propped open!!!!!!!!!!!!!!!!!!!!
  • Keep a thermostat inside the incubator just in case to be sure it remains at room temperature over the acclimation and experimental period.

Day of Innoculation - KK

Pathogen Prep

I didn’t do this last time, but this could be at Marta’s discretion

  • Centrifuge CV broths at room temperature for 10 mins at 3000 rpm
  • Remove supernatant broth
  • Resuspend in the same amount of FSSW as removed supernatant (or less maybe)

  • THEN, go forth with checking OD and spot plating using this FSSW-resuspended CV broth

  • Check OD of CV in triplicate and use RE22 OD600 calculations page to quantify approximate CFUs/mL
  • Calculate to have concentration of 1*105 CFU/mL CV in challenge containers
  • This spreadsheet Marta made for OD600-to-CFU calculations is wonderful
  • Make sure to use your supernatatn as a blank in triplicate too (either FSSW or mYP30)
  • Spot Plate CV for More Accurate Counts
  • Split an mYP30 plate in thirds, labeled 4, 5, 6 for CV dilutions
  • Dilute CV 1:10 where 0 is undiluted and 1 is 100ul CV with 900 ul FSSW
  • 2 is 100ul from dilution 1 and 900ul FSSW and so forth
  • Mix and add 10ul spots in triplicate to the mYP30 plate
  • 3 x 10ul spots for the 4th dilution
  • 3 x 10ul spots for the 5th dilution
  • 3 x 10ul spots for the 6th dilution
  • Place in incubator overnight and count spots at every quantifiable dilution the day or two following making the spot plate

Juvenile prep

  • Scrub, water change, and feed oysters
  • *Feed as close to the beginning of the challenge as possible
  • Initial (pre-challenge) genomic sampling

Innoculation

  • Add CV at quantified volume to reach a concentration in the ROD-treatment containers of 1*10^5 CFU/mL
  • Record time CV was added

Innoculation Notes from 20230825:

  • Stock OD: 0.548
  • CFU/mL of Stock: 6.58*10^8
  • Amount of Stock Added to each container: 10mL
  • Time Stock Was Added: 3:00PM

PCR on 20230906 confirmed that we did inoculate with ROD.

Experimental maintenence

  • Aim for oyster exposure to CV for ~12 hours before first water change/feeding
  • 1/2 water changes + feeding every day & full water changes on Fridays to avoid weekend water change
  • Mortality estimates on MWF

Maintenance schedule

Day Date Tasks Person
T 8/29/23 1/2 water change Shannon
W 8/30/23 mort & 1/2 water change Shannon
Th 8/31/23 1/2 water change Shannon
F 9/1/23 mort & full water change Shannon
Sa 9/2/23 no maintenance* -
Su 9/3/23 quick mort & full water change Caitlin
M 9/4/23 Holiday - no maintenance* -
T 9/5/23 mort & 1/2 water change Caitlin
W 9/6/23 mort & 1/2 water change Shannon
Th 9/7/23 1/2 water change Shannon
F 9/8/23 mort & full water change Shannon
Sa 9/9/23 no maintenance* -
Su 9/10/23 no maintenance* -
M 9/11/23 mort & 1/2 water change Shannon

*if we are not anywhere near 40-50%, no maintenance or mort count needed

Schedule is subject to change based on how mortality is progressing.

Water change protocol

Notes:

  • Start with control group to avoid contamination
  • change gloves in between treatment groups (CON/ROD)
  • Ethanol gloves and surfaces to avoid contamination, but ensure it is dry before handling juveniles
  • Rinse containers and tools with ASW before using them

Materials needed:

  • Artificial Seawater (ASW, 28-30ppt)
  • Shellfish Diet (SD)
  • 70% ETOH in spray bottle
  • beakers
  • green trays - for transporting cups
  • ASW squirt bottles
  • new clean baskets (if needed)

Procedure:

  1. Ethanol bench and trays and wipe dry with paper towels.
  2. Make ASW+SD. Dilute 1.5ml of SD into 3.2L of ASW (for a full water change make up 3 3.2L jars like this)
  3. Turn off airstones before moving any baskets, to avoid contamination via aerosols
  4. Remove cups for water change - start with control cups
  5. Remove basket and place it in it’s treatment-specific red bin.
  6. Into the “OLD WATER” beaker, pour out either 1/2 the volume or the full volume (depending upon if you’re doing a 1/2 or full water change).
  7. Attempt to rinse out the built up algae and poop from the basket- if possible. Tilting the basket will help to separate oysters from debris.
    • If baskets need to be changed, rinse juveniles into new labeled basket with ASW squirt bottle. The red bins can be used to catch seawater.
  8. Top off the cup with ASW + SD (measured in the “NEW WATER” beaker) for a total volume of ~650ml.
  9. Return basket to it’s cup and place back in the incubator with airstone.
  10. Turn on airstones after all water changes are complete.
  11. Dispose of ROD water – ask Marta!
  12. Dry, bleach, and ethanol the benchtop.
  13. Bleach all glassware, bins, caps, squirt bottles, baskets, etc. following the Puritz Lab bleaching protocol (see below).
  14. Set glassware and other materials to dry on the drying pad.
  15. Double check the air bubbles and make sure the incubator is propped open before leaving for the day.
  16. Check ASW stock, bleach, and sodium thio and text Megan if any needed to be replenished.
  17. Record water change and any notes in lab notebook (located on Puritz Lab bench).

Bleaching protocol

Materials needed:

  • bleach
  • sodium thiosulfate
  • water
  • DI

bleach

Procedure:

  1. Bleach with dilute bleach solution (a quick pour of bleach + water should be good). Be sure to bleach the outside of jars/beakers too with 10% bleach squirt bottle (on Puritz bench).
  2. Let sit for at least 15mins
  3. Pour out bleach
  4. Neutralize remaining bleach with sodium thiosulfate crystals dissolved in water (pour some crystals into each container and add hot water & mix)
  5. Rinse everything very, very well 5-6x in hot water
  6. Rinse in DI water

Mortality estimates

Materials needed:

  • Magnifying glass or dissecting scope
  • Petri dishes
  • tweezers or paint brush
  • counters

Note: This dissecting scope works really great!

scope

Procedure:

  1. Transfer ~50 oysters to their treatment-specific dish
  2. Under magnification, assess whether the first 50 you see (at random) are alive or dead keeping count with counters
    • living oysters will be closed and have some color to them
    • dead oysters can look gapped open, often have no color, and sometimes they float a little
  3. Record alive/dead counts in notebook
  4. Perform for 1-2 baskets per treatment group
  5. Update Megan with % mortality estimates for the day (we are aiming to get 60-70%)
  6. Dishes can be wiped out with a kim wipe – do the control dishes first to avoid contamination!

Final mortality counts and sampling

check back later!

Written on August 24, 2023