Labyrinthula zosterae infections of Eelgrass

Case Study 2

Emerging disease outbreaks associated with changing climate conditions have potential to reshape marine communities. Zostera marina L.8.9 (eelgrass) and other seagrasses are key components in the lower intertidal/ shallow subtidal biome in temperate coastal waters10. Seagrasses produce ecosystem services that fuel coastal fisheries and waterfowl migration, which in turn contribute to the sustainability of human populations by enhancing local livelihoods related to mass market food production, recreational gathering of seafood and ecotourism11. Labyrinthula zosterae has repeatedly been identified as the causative agent in an outbreak of the eelgrass wasting disease in the North Atlantic12, 13,Florida, and Europe, and is a highly complex syndrome strongly influenced by environmental conditions. This epidemic, associated with climate shifts such as rising temperatures, has a cascading effect on biodiversity and productivity in coastal regions dominated by perennial Z. marina stands 8.

In 2003, significant declines in Z. marina populations were reported in the San Juan Archipelago, WA14, 15. One hypothesis is that the protist, L. zosterae may be involved. Preliminary studies by Wyllie-Echeverria and colleagues have isolated L. zosterae from 6 sites in the San Juan Islands and surrounding areas and established methods for inoculation studies. The development of these preliminary methods, including histology, allows development of reliable diagnostics for estimating the prevalence of L. zosterae in Puget Sound eelgrass populations, and testing the response of this host-parasite system to changing environmental conditions.

Field Sampling

Our case study will begin with exploratory field sampling at 3 sites (False Bay, Beach Comber, Picnic Cove) in the San Juan Islands, in collaboration with Sandy Wyllie-Echeverria. Last week we did some exploratory work to test the reliability of gross signs indicative of L. zosterae (henceforth laby) infections and collected preliminary samples. We will examine histology from these samples in lab and then conduct further sampling; so that we feel gross signs in the field are reliable.

We will work at Sandy’s monitoring sites at False Bay on San Juan Island and Beach Haven on the north side of Orcas Island. We might later add a third site (Picnic Cove on Shaw Island). Sandy already has data on the genetic composition of seagrasses at these sites. At each site, we will conduct at least three 10 meter transects parallel to shore. If possible, we will replicate the two shallow and deep transects, to detect a significant difference in prevalence with tidal height


Field sampling design (DIAGRAM)



Density measurements

• Quadrats (1m2) at 0,5, and 10 meters
• Count # of flowering plants
• Total # of shoots
• # of Lacuna and Phyllaplysia egg masses and adults (possibly limpets?) on 5 randomly chosen vegetative shoots
  • measure length of longest shoot

Lesion measurements (excluding flowering shoots)

• 5-10m down center of transect with 0-6" on either side of the transect
• Measure length of longest blade in shoot
• Measure maximum length of each lesion

Laby ID and collections

• Multiple blades from each transect and surrounding area will be collected, photographed and sampled for laby identification. Paired samples will be taken for histology (to confirm laby infections) and PCR (to determine laby identification) and we will attempt to isolate the laby in culture (total n=30).
• A 2 cm piece of each leaf will be preserved in desiccant and archived to confirm the genetic status of the infected leaf (see Wyllie-Echeverria et al. 2010)
• All shoots should be examined for gross observations for health within 0 (Beach Comber) - 6 (False Bay) inches of the transect line. Record the longest shoot measurement and length of all lesions on all blades of the plant.

Environmental parameters

• Temperature sensors (Tidbits) will be installed at each tidal height and we will use HYDROLAB DS 5 to assess Temperature, DO, pH, Chlorophyll, Turbidity and Salinity at each site.
  • The shallow sites were open out of water making it impossible to measure water quality directly on the shallow transects. In these cases, we sampled as close to the shallow transects as possible while keeping the probes submerged in water.
• GPS points were taken at the 0, 5 and 10 m mark in the shallow and deep transects. We also took points in a polygon area around the transects. Ashton will use these points to create a map in ArcMap.

PROTOCOL FOR SETTING UP THE HYDROLAB
We are using the a Hydrolab DS5 to measure a set of in situ water quality parmeters at all of our eel grass monitoring sites. The hydrolab is normally connected to a computer on a boat to measure parameters continuously. However, we would like to launch the hydrolab to record data remotely. Here is the protocol for launching the hydrolab.

1. Turn on Acer laptop and plug USB into computer (DON'T GET USB CABLE WET! Keep it is in the red dry bag while in the field).
2. Open Hydra software on desktop
3. Click "Operate Sonde" & wait for security level to go from 0 to 2 (highlighted with a green box). While you are waiting, switch out the clear storage cup for the weighted sensor guard.
   1. Important: Make sure you have have tap water available to immerse the probes in after you sample. The probes need to be kept moist, so do not keep them out of water for a long period of time.
4. Click "Log Files" tab
5. Click "Create" in bottom left
6. Give the file a name
7. Set logging date, time, launching interval (min= 30sec) and add the parameters you would like to record.
8. Save settings & click "Enable"
9. Detach cable and shut computer down.
10. Put cable back in dry bag.
11. When you are finished collecting data:
    1. Switch the cage for the storage cup (make sure it is filled 3/4 with tap water).
    2. Turn on computer, attach usb cable and open hydra.
    3. Select the log file that you created and download the data. The data are stored in the C drive.

The laby-eel grass pathosystem provides a good opportunity to ask ecological questions in the field, since these are large populations of hosts with a high prevalence of infection. From preliminary assessment, we determined to test the following hypotheses:
H1: Laby infections are more prevalent at higher than lower tidal heights.
H2: There is a water temperature difference between higher and lower transects.
H3: Laby infection prevalence is highly host size dependent.
H4: Laby infection intensity varies among sites in the San Juans.
H5: Laby infections are significantly aggregated at a 1 meter scale.

Future work:
On each transect, we may want to check every eelgrass shoot within approx. 4” of the tape. Each shoot will be sized (into 5 categories) and its severity of infection recorded. Severity of infection will be on a 4 point scale: 0 (healthy), 1 (1 small lesion), 2 (2-3 moderate lesions), 3 (over 50% of blade affected, and/or more than 1 blade in a shoot affected).

At a later time, if we think it is workable at one of the sites, we would like to map the spatial distribution of infected blades and test for aggregation with Ripley’s K statistic. Ripley’s K statistic, has worked well in the past for similarly distributed infections on seafans (Jolles et al., 2002). The Ripley’s K statistic tests whether the distribution of infected individuals is aggregated with respect to hosts. It differs from a simple nearest neighbor statistic, in that it takes account of the fact that hosts may also have an aggregated distribution. The tractable aspect of having a sessile host like a plant or a coral, is that you know its position when it was infected, so the pattern of aggregation can provide information about infection and host resistance processes. Aggregated distributions can be produced by a local infection process and also by aggregations in the distributions of susceptible hosts. To generate the data for this test, we would map the exact spatial distribution of every eel grass shoot and its infection status.


The low tides for Friday Harbor are at:

Monday July 30 9:07 -1.7 FALSE BAY, San Juan Island
Tuesday July 31 9:57 -1.9 BEACH COMBER BAY, Orcas Island
Wednesday Aug 1 10:43 -1.8 PICNIC COVE, Shaw Island
Thursday Aug 2 11:27 -1.5 FALSE BAY, San Juan Island

For field work we will need:

3 transect tapes
2 plexiglass sheets for mapping
temperature, pH meter
data sheets, clipboards, pencils
plastic bags for samples

You will need either very high boots or footware that can get wet walking through eelgrass beds (Keens or Chacos or sneakers – NO flipflops) and shorts.

Laboratory materials and methods

Laboratory prep of samples
Histology—Blades will be labeled with transect and site number, photographed and preserved for histology. A small slice will be made at the lesion edge, loaded into a histology cassette, preserved in 4% formalin for 24 hours, then transferred to 70% ethanol. When the histology comes back in about a week, you will score each slide for the presence of laby. This will allow us to confirm that the signs we used for our field surveys align with actual laby infections. The results of the PCR below will confirm the identity of the labyrinthulid.

Extraction & PCR of laby DNA—An adjacent slice of blade will be preserved in 95% ethanol for subsequent PCR (labeled, screw-cap vial filled with 95% non-denatured ethanol).

For this lab we will use a commercially available kit which relies on the silica membrane extraction, called the Qiagen ® Stool kit.

Materials list:

Tissue samples in 95% ethanol
Qiagen Stool kit
Pipettes
Sterilized microcentrifuge tubes
Marking pens
70ºC incubator
Centrifuge
Vortex
Scalpels, blades, and tweezers
Weigh boats
Trays
Bleach bottles
Ethanol jar

Method: DNA Extraction (Qiagen Stool Kit)

1. Pre-label all the microcentrifuge tubes you will need for this procedure. You will throw away most tubes used, so you need to only minimally label these tubes. You will need a final 1.5 mL microcentrifuge tube, please label this with the following information: your sample accession #, your initials, and the date. Please put the sample # on both the top and the side of the tube. Remember to always run a BLANK extraction as a negative control.
2. Remove blade from ethanol using good sterile technique and cut a small piece of laby-infected blade approximately the length of a pencil eraser (try to minimize the amount of eelgrass – grasses contain pigments which could potentially inhibit subsequent PCR reactions). Weigh the sample and record in your notebook. Mince into small pieces and place them into a 2 mL microcentrifuge tube. Take care not to cross-contaminate samples.
3. Add 1.4 mL Buffer ASL to each sample. Do this by adding 700 µl of Buffer ASL, vortexing for a minute and then adding another 700 µl of Buffer ASL. Once all ASL has been added, vortex continuously for 1 min or until the sample is thoroughly homogenized. Please note: It is important to vortex the samples thoroughly as it will insure maximum DNA concentration.
4. Heat for 5 min at 70º C.
5. Vortex for 15 s and centrifuge sample at full speed for 1 min to pellet blade particles.
6. Pipet 1.2 mL of the supernatant into a new 2 mL microcentrifuge tube and discard the pellet.
7. Add 1 InhibitEX tablet to each sample and vortex immediately and continuously for 1 min or until the tablet is completely dissolved. Incubate for 1 min at room temperature to allow inhibitors to absorb the InhibitEx matrix.
8. Centrifuge sample at full speed for 3 min to pellet inhibitors bound to InhibitEX.
9. Pipet all the supernatant into a new 1.5 mL microcentrifuge tube and discard the pellet. Centrifuge the sample at full speed for 3 min. *in this step, the transfer of small quantities of pelleted material will not affect the procedure*
10. Pipet 15 ul Proteinase K into a new 1.5 mL microcentrifuge tube.
11. Pipet 200 ul supernatant from step 9 into the 1.5 mL microcentrifuge tube containing Proteinase K.
12. Add 200 µl Buffer AL and vortex for 15s. *DO NOT ADD Proteinase K directly to Buffer AL* It is essential that the sample and Buffer AL are thoroughly mixed to form a homogenous solution.
13. Incubate at 70 ºC for 10 min.
14. Remove your samples from the incubator and briefly centrifuge. Add 200 µl of 95% molecular grade ethanol to the sample and vortex for 15 seconds. Briefly centrifuge the tubes.
15. Carefully apply this mixture to a QIAamp spin column. If a white precipitate has formed, make sure to add this to the column. Do not wet the rim of the spin column (this can allow cross-contamination of samples in the centrifuge). Centrifuge at 8000 rpm for 1 minute.
16. Place the QIAamp spin column in a clean 2 mL collection tube.
17. Carefully open the QIAamp spin column and add 500 µl of buffer AW1 without wetting the rim. Centrifuge at 8000 rpm for 1 minute.
18. Discard the 2 mL collection tube containing the buffer and place the spin column into a new collection tube.
19. Open the spin column and add 500 µl of buffer AW2 without wetting the rim. Centrifuge at full speed for 3 minutes.
20. Place the spin column into your final microcentrifuge tube, add 100 µl of buffer AE to the column and allow to incubate at RT for 5 minutes.
21. Centrifuge at 8000 rpm for 1 minute.
22. Remove the spin column and throw it away. You now have a tube of DNA! J

Polymerase Chain Reaction (PCR)

Primer Design:

Using Geneious software, information was gathered from examining the L. zosterae 154 sequence (west coast strain; GenBank: AF265335.1) against the L. zosterae MBL 93-2 sequence (east coast strain; GenBank: AF265334.1). An alignment of the 2 sequences were conducted and new primers were designed in Geneious using Primer 3 in a non-conflicting region (200-1180 bp) for the primers to lay down. Below is the new primer information:




conventional PCR (cPCR):

1. Calculate the total number of reactions, making sure to account for a positive control and no template negative control.
2. Using the recipe below, calculate the amount of each ingredient to add to your master mix.

Ingredient	Amount per Rxn		# of Rxns X 1.1*		To add to Master Mix
GoTaq Mix	12.5 uL	X		=
BSA	1.5 uL	X		=
Forward Primer	0.8 uL	X		=
Reverse Primer	0.8 uL	X		=
PCR H2O	7.4 uL	X		=

*includes 10% more & don’t forget to account for positive and negative controls
3. Using sterile technique, add all ingredients to a microcentrifuge tube that can hold the total volume.
4. Briefly vortex and spin down.
5. Aliquot 23 uL master mix into each tube or well.
6. Add 2 uL template to each tube, making sure to keep track of what sample is where.
7. Cap, briefly vortex, and spin down.
8. Place in thermal cycler and run using the following thermal profile:

	Time	Temp (°C)
1.	10 min	95
2.	30 sec	95
3.	30 sec	50
4.	60 sec	72
5.	Repeat steps 2-4, 40 times
6.	10 min	72
7.	Hold @ 4 deg C

Gel Electrophoresis:

1. Weigh 1.5 g agarose and add to 200 ml flask
2. Add 100 mL 1 X TBE
3. Bring to a gentle boil in the microwave for ~ 3 minutes (1.5min, stop, gently swirl, 1.5min)
4. Add 10 µL of SybrSafe, swirl gently, allow to cool for a few minutes and pour into the gel mold
5. Place combs in and allow gel to set for ~ 15min
6. Add 1X TBE to the gel box (~ ½” over the top of the gel)
7. Pipet 7 µl of 100 bp molecular weight ladder in the far end wells
8. Add 5 µl of loading dye to your PCR products *UNLESS dye is already incorporated into your master mix buffer. Change tips for every sample to avoid contamination.
9. Pipet 7 µl of your PCR product + loading dye into each well.
10. Run at 115V for 45 mins or until dye is ¾ way down the gel.
11. Carefully remove gel and examine under UV light (wearing nitrile gloves and lab coat).
12. Photograph your gel.

Isolation and Culture Techniques:
We will conduct laboratory inoculation experiments with cultured Labyrinthula isolated from your field collections. If the inoculations are successful, we may conduct experiments in the Friday Harbor Laboratories OA facility under variable temperature and OA levels to further characterize disease-OA-temp relationships.
Labyrinthula zosterae isolates can be cultured either in solid or liquid media. Recipes for both are below. Cultures need to be transferred every week or two depending on room temperature for solid media and about 2-5 days for liquid media. All cultures will be grown at room temperature and exposed to light during the day.

Modified SSA Agar
1. Filter sterilize at least 1 L of seawater using to 0.45um or smaller
2. Check salinity using a refractometer and adjust to 25ppt using Nanopure water (or DI water). Adjust slowly, mix well, and check salinity using refractometer frequently.
3. Add 1 L of 25ppt filtered seawater to a 2 L flask. Add an autoclavable stir bar and the following:

• "USB Nobel Agar": 12g
• Germanium dioxide: 1.5 mg
• Yeast extract: 0.1g
• Peptone: 0.1g
• Glucose: 1.0g

4. Loosely cover with foil and autoclave for 20 minutes on the liquid cycle.
5. While media is in the autoclave heat up water bath to 37 oC and thaw 10 mL aliquot of Horse Serum and 25 mL 100X Pen/Strep.
6. Once autoclaved, temper the media for 30 minutes to between 50 and 55 oC after mixing gently - avoid creating bubbles in the agar.
7. Add thawed 10 mL horse serum and 25 mL 100X Pen/Strep.
8. Mix or stir adequately, making sure not to create any bubbles and pour the plates.
9. Let agar cool and then store at 4 oC until use.

Modified SSA Broth
1. Filter sterilize at least 1 L of seawater using to 0.45um or smaller.
2. Check salinity using a refractometer and adjust to 25ppt using Nanopure or DI water. Adjust slowly, mix well, and check salinity using refractometer frequently.
3. Add 1 L of 25ppt filtered seawater to a 2 L flask. Add an autoclavable stir bar and the following:

• Germanium dioxide: 1.5 mg
• Yeast extract: 0.1g
• Peptone: 0.1g
• Glucose: 1.0g

4. Split into four 250 mL corning jars (the ones with the orange lids) with lids loose and covered in foil.
5. While media is in the autoclave heat up water bath to 37 oC and thaw 10 mL aliquot of Horse Serum and 25 mL Pen/Strep.
6. Once autoclaved, swirl media to mix well and cool the media for 30 minutes to between 50 and 55 oC.
Add thawed 2.5 mL horse serum and 6.25 mL 100X Pen/Strep to each jar.
Store at 4°C in clean room fridge until use.

References
1. Jiang, Z.J., X.P. Huang and J.P. Zhang. 2010. Journal of Integrative Plant Biology 52(10): 904-913.
2. Palacious, S.L. and R.C. Zimmerman 2007. Marine Ecology Progress Series 344: 1-13.
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5. Wyllie-Echeverria, S. and J.D. Ackerman 2003. Pages 199-206. IN: E.P. Green and F.T. Short (eds) World Atlas of Seagrasses. Prepared by the UNEP World Conservation Monitoring Centre. University of California Press, Berkeley, USA. 298 pp.
6. http://www.ipcc.ch/
7. Nakatsuji, N and E. Bell. 1980. Control by calcium of the contractility of Labrinthula slimeways and of the translocation of Labyrinthula cells. Cell Motility and the Cytoskelton 1 (1): 17-29.
8. Kenworthy, W,J. K, S. Wyllie-Echeverria, R.G. Coles, G. Pergent, C. Perget-Martini. 2006. Pages 595-623. IN: A.W.D. Larkum, R.J. Orth and C.M. Duarte (eds). Seagrasses: Biology, Ecology and Conservation. Springer; The Netherlands. 691 pp.
9. NOAA 2011: http://sharpfin.nmfs.noaa.gov/website/EFH_Mapper/map.aspx
10. Rasmussen, E. 1977. In C.P. McRoy and C. Helfferich (eds) Seagrass Ecosystems: a Scientific Perspective. Pages 1-51.
11. Wyllie-Echeverria, S., Z. Hughes and T. H. Dewitt. (in EPA review).
12. Muehlstein, L.K., D. Porter and F.T. Short. 1988.. Marine Biology 99: 465-472.
13. Muehlstein, L.K., D. Porter and F.T. Short. 1991. Mycologia 83:180-191.
14. Ferrier, L. and Berry, H. (2010). DNR Nearshore Habitat Program.
15. Wyllie-Echeverria, S., S. L. Talbot, J.R. Rearick. (2010). Estuaries and Coasts 33:811-827.
16. Davies, J., Guerry, A., Ruckelshaus, M. (2009) Proc. Coast. Zone 09. Np.
17. Jolles, A., Sullivan, Alker and Harvell. 2002 Disease transmission of aspergillosis in seafans: inferring process from spatial pattern. Ecology 83: 2373-2378.










Light micrographs of healthy (#1, #2) and infected (#6,#8 L. zosterae) Zostera marina blades.