Pathogen Filtration Experiment 2014
Introduction:
Disease agents include a variety of taxa and of interest to this class are microbes: viruses, bacteria, fungi and protists. Given the generally small size of these organisms/pathogens, they may also serve as prey for filter feeding marine species. Bivalves often filter larger prey such as those >3-4 µm but are also known to consume and utilize bacteria as food sources (e.g.
Silverman et al. 1995) and some may also filter viruses (Canzonier 1971, Faust et al. 2009).
Other marine invertebrates, such as sponges, may also filter bacteria and viruses (Hadas and
Marie 2006). We aim to examine the capacity of bivalves to filter marine microbes.
As we learned in Week 1 on Bio 533, Labyrinthula zosterae is a pathogen of eel grass, Zostera marina and if found locally in the San Juan Archipelago (Groner et al. 2014). We have several strains of L. zosterae in culture (provided by Dr. Sandy Wyllie-Echeverria) and will collect young eel grass seedlings from False Bay, San Juan Island.
Hypothesis:
We will test the hypothesis that the Pacific oyster Crassostrea gigas and, possibly, the local mussel Mytilus trossulus are able to reduce loads of L. zosterae in seawater.
Materials:
L. zosterae cultures
Hemocytometer
Tissue culture flasks
Cell scrapers
Scalpel blades and handles
Forceps
Bunsen burners or ethanol burners
Starter for burners
Vortex
Media – Modified SSA solid and liquid media (see week 1 lab for methods)
Bivalves: Pacific oysters (1cm and 5 cm) and Mussels (from FHL dock)
Beakers of appropriate size for the size of the bivalve
Supor filters
Filter housing
Sterile Seawater (25 psu) supplemented with penn-strep (2x = 20 ml/1L) (=SW+PS)
Tubing for filter housing
Vacuum pump
TE preservation buffer
Dry ice and coolers
Stir bars
Multi-stir plate
Eppendorf tubes
Tube racks
15 ml Falcon tubes
Lab tape
Sharpies
DNeasy extraction kit
PCR water
Laby primers
PCR master mix and Taq

Experiment Set up
Question: What is the purpose of each of these treatments?
Seawater only
Sea- water and bivalve s
Seawater only
Sea- water and bivalve s
Seawater only
Sea- water and bivalve s
Sea- water, bivalve
& Laby
Sea- water, bivalve
& Laby
Sea- water, bivalve
& Laby
Methods:
Set Up (Blue has already been done)
1.
Grow L. zosterae (Laby) in liquid and or solid media
2.
Select bivalves and place 10 individuals in meshbags
3.
Select beakers based on bivalve size
4.
Set up experimental beakers and label as treatments shown above
5.
A) Scrape and vortex Laby from liquid culture flask,
-Centrifuge at 1000xg for 2mins in 50ml centrifuge tube,
-Decant supernatant (and save for now),
-Add 5 ml of SW+PS to pellet and vortex 1 min.
Or B) Scrape Laby off solid plates and resuspend into 6 separate 5mL of SW+PS
6.
Count cells with hemocytometer and tell Carolyn, Lisa or Maya your concentrations
7.
Dilute Laby into SW+PS in a beaker to get target concentration of 10
8
cells per mL
- FOR EXPERIMENT - Note: wait for your instructors before diluting  .
- Spin 1ml of 10
8 concentration, decant supernatant, freeze pellet at -20C
8.
Make 1:10 serial dilutions in sterile seawater to 10
-10
of Laby - FOR qPCR –
-Count cells from each dilution with hemocytometer
-Spin remaining Laby dilutions at 1000xg for 5mins, decant supernatant, freeze pellet at -20C
9.
Add SW+PS to beakers that will not get Laby.

10.
Filter 25 mL aliquot from each beaker onto a separate supor filter.
-Filter 5 ml preservation buffer through filter.
-Immediately place filter into 1.5 ml microcentrifuge tube.
-place on dry ice. Store samples at -80C.
11.
Add bivalves and stir bars to appropriate beakers.
12.
Add algae to prime oysters for feeding. Let bivalves feed for several hours.
Filtration Time Series
13.
At 0hr, 4hr, 8hr, 12hr, & 24hr remove 1ml SW from each beaker into eppendorf tube
-count Laby cells from each beaker on hemocytometer
-centrifuge remaining cells (1000xg for 5mins), remove supernatant
-freeze at -20C
14.
After 24hrs, remove oysters and excise gills and a piece of digestive gland.
-Place one piece of each on a SSA plate – to culture Laby -
Place another piece in Eppendorf tube and Freeze at -20C.
(Note: DNA from gills will extracted with the Qiagen DNeasy kit and the digestive gland DNA will be extracted with the Qiagen Stool kit)
Eelgrass Exposure
15.
Add 6 eel grass seedlings to each beaker leave for minimum 3 days.
-replace water in beaker with fresh sterile seawater everyday
-Remove Eelgrass and score for lesions
Molecular Anaysis
16.
Extract DNA from your standard curve samples and frozen aliquots as per Qiagen
DNeasy protocol for blood (pages 31-34 EXCEPT we elute with only
100 µl of elution buffer total ).
17.
Make 1:10 serial dilutions of the DNA in PCR water to 10
-10
. Run a conventional PCR using DNA from each dilution.
18.
Run gel and determine your laby concentrations.
19.
Compare presence of Laby on SSA plates to presence of Laby via PCR
References.
Silverman et al. 1995)
Canzonier 1971
Faust et al. 2009
Hadas and Marie 2006.
Groner, M.L 2014.