Tag Archives: laby

Learning to work with eelgrass disease

Today we visited the eelgrass bed in False Bay. The work was led by Sandy, Maya Groner, and Drew Harvell. It was a cloudy morning but began to clear when we got to the site. We were coming out to take samples from the culling experiment that Drew, Mo, and I set up. Two transects were set up, each with 4 plots. The two end plots were controls and the two middle plots experimental. In the experimental plots all the laby infected blades were removed to see if this would reduce disease. The water was too high today (mostly due to the wind pushing water into the bay) to reach the plots so we conducted a prevelence survey and will return later this week to sample the experimental plots. The site was extreamly healthy, showing only a few lesioned blades. This is great to see, but I fear Shaw Island where we go tomorroe will not be as healthy!

METHODS FOR EELGRASS DISEASE PREVELENCE: 1. 50 m transect set up with transect tape. Set transects are marked with PVC and GPS. 2. Cut 10 blades every 10 m, choose the 2nd longest blade of each plant sampled, cut blade right above sheath. 3. Collect blades in plastic bags (1 per spot). 4. Assess density at 0 m, 25 m, and 50 m. 5. Repeat 2 times, 5 m apart.

We modified this protocal due to the tide hight. Only one transect was used. 20 blades taken from each spot, though in last one there was not enough to sample.

In the afternoon, we returned to the lab to process the eelgrass blades. Each blade was scored as healthy or lesioned. We measured blade length and width, and the length and width of each lesion. 2 healthy and 2 infected blades were plated. Laby is distinguished from damage by the black ring around clear lesion. Maya says this indicates the plants defense.

LABY PLATING PROTOCAL: 1. Clean surface od blade with razor, 2. Cut section with laby, 3. Dip blade into filtered sea water and swirl to clean, 4. Blot, 5. Quickly submerge in 95% ethanol amd remove (to kill surface laby) 6. Allow to dry and place in plate.

Cell counts were started from the laby Ann Jerrell plated to measure colony growth of different strains.

After lab we had an excellent diagnistics lecture from Carolyn. And Colleen arrived with baby and adult oysters! Oysters were placed in flow through sea water tables with bubblers. Later, we had an excelent set of talks on the research we all do outside EIMD. Really looking forward to more of these. I did not have all the July intertidal data to update the graphs with so only presented the subtidal. Will have the other figures ready for Drew to present in her seastar lecture with the temperature and experiment data.

On Tuesday night I got a set of clams and mussels from Pen Cove to sample as possible sea star disease vectors. I have attached a link of Reyn dissecting gill tissue at midnight for qPCR testing later!

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qPCR – Lexie’s QPX Temp & Tissue Experiment (see Lexies Notebook 4/26/2011)

Ran qPCR with Lexie’s cDNA samples from this experiment with the following primer sets in order to better evaluate her biological reps:

QPX_SPB_F/R (SR ID: 387, 388)

LABY_A/Y (SR ID: 116, 121)

LABY was run as a potential normalizing gene. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Samples were run in duplicate and were labeled according to what was written on the tops of Lexie’s cDNA tubes.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

LABY primers worked, but the melt curves don’t look that good. I’ll let Lexie worry about the rest of the analysis.

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PCR – “Unknown” Dungans/Lyons

This is a repeat of yesterday’s set up with LABY primers, but with an annealing temp of 53C in hopes of improving the number of amplicons generated from additional samples. See yesterday’s PCR run for info on samples.

Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.

The lower annealing temperature clearly resulted in more products. The ~500bp band was cut from each lane and stored @ -20C. All bands will be purified using Millipore spin columns and then sent for sequencing.

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PCR – Dungan isolate (MIE-14v) gDNA from 20090708

PCR of MIE-14v just to make sure that we can’t get a product from this sample, despite NanoDrop readings suggesting that there’s no DNA. Used both LABY and Euk primer sets. PCR set up is here. Anneal temp 50C.

Lane 1 – 100bp Ladder

Lane 2 – Euk

Lane 3 – Euk H2O

Lane 4 – Euk H2O

Lane 5 – Euk H2O

Lane 6 – LABY

Lane 7 – LABY H2O

Lane 8 – LABY H2O

Lane 9 – LABY H2O

Lane 10 – 100bp Ladder

Results: Nothing, as expected. Need to devise a new method of isolating gDNA from these “problem” isolates.

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PCR – Two new Dungan isolates from earlier today

Set up PCRs on:

MIE-14y

VNTc-1.2-C1/G10

Used Euk A/B and LABY A/Y primers. Anneal temp 50C. PCR set up is here.

NOTE: Due to the extremely low concentrations of gDNA from these two samples, I used a large amount of gDNA in the rxns. Check the PCR set up link for actual numbers.

Lane 1 – Hyperladder (5uL)

Lane 2 – VNTc-1.2-C1/G10 (Euk primers)

Lane 3 – MIE-14y (Euk primers)

Lane 4 – H2O (Euk primers)

Lane 5 – H2O (Euk primers)

Lane 6 – VNTc-1.2-C1/G10 (Laby primers)

Lane 7 – MIE-14y (Laby primers)

Lane 8 – H2O (Laby primers)

Lane 9 – H2O (Laby primers)

lane 10 – 100bp Ladder

Results: Nada. Probably because of low [gDNA], but could also be due to PCR inhibitors in the gDNA. Will retry using Amplitaq and less gDNA.

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