Tag Archives: EIMD

Eelgrass, sea stars, and little oysters

Today started with a beautiful morning drive to false bay. The tide was lower today and the wind less intrusive so we were able to compleat all the work planned. Fieldwork was led by Maya Groner and Drew Harvell.

1. Prevelence of disease in mid level plot (n=10 2nd longest blades removed each 10 meter from 0-50). The blades were measured and level of disease assessed back in the lab.

2. Collected Zostera marina shoots for Maya’s experiment.

3. Assessed mid level plot density (# plants on frame at 0, 25, and 50 meters).

3. Located the culling experiment plots and removed blades from center of each. There was noticeably more smithora present at this plot compared to the one we visited yesterday on Shaw. At this site there were 2 sets of plots.

Plot 1: Control1-cull1-cull2-control2, Plot 2: control1-cull1-cull2-control2

The False Bay Zostera marina bed.

The False Bay Zostera marina bed. Tide is not out yet so moat of the eelgrass is underwater still. 

Working hard to get all the culling experiment plots done before the tide comes in!

Working hard to get all the culling experiment plots done before the tide comes in!

Example of clear laby lesion (right) and a lesion that is not clearly laby (left). It is best to be conservative and only count lesions where there is black visible next to the light tissue.

Example of clear laby lesion (right) and a lesion that is not clearly laby (left). It is best to be conservative and only count lesions where there is black visible next to the light tissue.

Trying to locate our culling plots underwater.

Trying to locate our culling plots underwater.

Since the level of disease increases with blade age it is important to always choose the second longer blade so results ads consistent.

Since the level of disease increases with blade age it is important to always choose the second longer blade so results ads consistent.

The laby infection displays as a light patch edged by black, necrotic tissues where the plants defense has responded to the infection.

The laby infection displays as a light patch edged by black, necrotic tissues where the plants defense has responded to the infection.

Measuring and scoring disease stage of Zostera marina blades in lab.

Measuring and scoring disease stage of Zostera marina blades in lab.

Today I also started work on a pilot study to determine if bivales filter the sea star wasting disease pathogen out of the water.

1. Juvanile oysters (1-3 cm) were out planted at Eastsound Dock (high SSWD) and Cresent Bay (low SSWD). N=10 were left for duration of the low tide and 10 left for 24 hours. Oysters were placed on mesh bags just below the surface of the water, next to where pisaster are found with disease. Many thanks the Amy Henry and Natalie Rivlin for thier help placing the bags at Orcas Island. They returned at 6 pm with the first set of oysters, which I then removed from shell, measured, weighed wet tissue, and froze at -80.

Oysters in mesh bags prepared to be placed at the field site.

Oysters in mesh bags prepared to be placed at the field site.

Cute baby oysters courtesy of Colleen Burge!

Cute baby oysters courtesy of Colleen Burge!

SSWD/oyster filtering data collection plan.

SSWD/oyster filtering data collection plan.

I set the experiment up in the ocean acidification lab. It will only run a few days but if the data turns out interesting may be the basis of a larger study.

Oyster exposure to SSWD conducted tanks within the OA lab so the incoming water was filtered.

Oyster exposure to SSWD conducted tanks within the OA lab so the incoming water was filtered.

I will include more on the experimental design tomorrow. Basic set up: 3 control (no stars) and 3 experiment tanks (symptomatic stars),  40 oysters in each bag, 10 oysters removed at each of 4 time points after which experiment is ended. The field and lab data will then be looked at with disease prevelence at the field sites and data from clams.

Tomorrow I will be heading over to Orcas Island to retrieve the remaining oysters (24 hour exposire) and out planting a bag at the FHL dock.

 

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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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Invert dissections

Today we dove right into practical marine invertebrate anatomy and tissue morphology, some of us for the first time after a brief lecture introduction. Monica and I dissected an oyster (species not ID’d) and Pisaster ochraceus.

It was interesting choosing the appropriate sectioning strategy to truly pick apart important anatomical structures; for the oyster we removed the mantle and then cross-sectioned the body, after first prying open the shell with a shucking knife and a large rock as a prying hammer. We were able to easily ID gills, adductor muscle, mantle, and palps before cross-sectioning the body to discover the digestive tract and large gonads, which when placed on a slide under the microscope revealed hundreds of eggs.

The sea star proved a challenge, even with some early symptoms of wasting disease (white lesions, contorted rays) it was difficult to get very far with the scissors and dissecting scalpel; at times it took the two of us to peel the spiny cover away. We did dissect one arm to find the digestive glands and bulbous ampulla (the gonads must have washed out sometime in the dissection). All in all, a challenging and very interesting exploration into the inside life of a marine invertebrate!

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Big picture, small picture

Today in our lab we looked at the big picture (dissections of molluscs and echinoderms) and the small picture (histology slides of diseased and healthy oysters and abalone).  The big picture allowed us to familiarize ourselves with the various body parts of these animals and also to discover just how hard it is to cut through a pisaster ochracheus.  The small picture introduced us to the difficulties and sometimes joys of differentiating tissue types and identifying parasites.

Here is our dissection of a diseased pisaster ochraceus:

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You can see the Anus in the middle surrounded by the stomach, the radial canals/nerves extending down the arms, the yellowish gonads and greenish cecae or digestive glands, and the ampulae (white sacks on each arm) which control the tube feet.

Next, we have a picture of a sectioned and stained abalone infected with Coccidia:

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Coccidiae are obligate intracellular parasites, which infect the kidneys of abalone.

 

 

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The beauty in Anatomy & Histopathology of Mollusks

Today was full of beautiful marine inverts and microscopy! Morgan and I examined the California mussel, Mytilus californianus, and the Pacific oyster, Crassostrea gigas (below). C. gigas M. californianus Using our advanced tools we gained access to what laid between the shells (see below). ADVANCED TOOL

inner anatomy of C. gigas inner anatomy of M. californianus Inspection of the anatomy from the macro level was exciting, but observing the tissue under the compound scope opened up a whole new world!

M. californianus gill (to be replaced with video of gill action, once I figure out how to load videos…)

Later we also examined prepped slides of abalone and observed this sweet design of nature (the radula epithelial tissue), which constantly deposits new layers to the radula, the molluscan feeding structure.

Radula Epithelial

I leave you with one last beautiful specimen we examined today, from another Phylum, the Pacific blood star, Henricia leviuscula. Stunning!

Henricia leviuscula

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Reyn’s Day 2: Dissections and Slides

After a nice orientation to the island and introduction to the course yesterday, we jumped into histology/histopathology head-first.  Luckily, we weren’t thrown into the deep end without at least some floaties.

Today’s lectures included an overview of molluscan (abalone and bivalve) anatomy and diseases as seen in histo.  It seemed much more straightforward on the powerpoint slides than in practice, but we’ll get to that in a bit.

Before we looked at the histo slides, we familiarized ourselves with the fresh specimens through vivisections (I did not know this word was so charged).  While it may seem cruel to cut the inverts while they’re still alive, it is very helpful for us to see the function and location of body parts to translate them to our understanding of histo slides.  The inverts were anesthetized before we did anything with them.

We first opened up some mussels:  Mytilus edulis and Mytilus californianus, assuming my IDs are accurate.  We first cut the hinge ligament and then sliced the posterior and anterior adductor muscles holding the valves together.  After this, we scraped off the mantle on one valve so that the whole body lay on one side.

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Look at that gonad!

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Gonads and ctenidia were removed. We also must have sliced into the stomach by accident because the crystalline style just popped out.

The first image is M. californianus with the left (?) mantle removed.  The deep orange-y stuff on the top is gonad, which we at first thought was ovary, but a quick google search and a look under the microscope (below) revealed that our believed-to-be girl mussel was actually a boy.  We were able to figure out much of the mussel’s anatomy, but its heart escaped us.

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Very, very motile sperm.

Next we dissected a Pisaster ochraceus, which was lovely and purple, albeit showing signs of wasting disease (twisting and lesions).  It was also incredibly ossified, which made chopping into it very difficult.  Fortunately we had the great Morgan to help us get to the goodies inside.

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Morgan helping us out. (I’ll flip it when I figure it out)

The inside of the sea star was pretty neat.  We were able to see gonads (ours presumably male), ampulla for each of the tube feet, and digestive caeca.  It was cool to see how the ampullae controlled individual tube feet by squeezing them.

In the afternoon, we began our ventures into the histo slides.  I had hoped that I would be an expert after the lectures, but the slides proved to be worthy adversaries.  I was often left stumped, trying to figure out which tiny pink spots were which.  It was very satisfying to have been able to find rickettsia (dark spots/colonies in the posterior esophagus of an abalone), Bonamia (little dots in enlarged hemocytes with offset nuclei), and ferritin granules.  It’s clear that I need more experience looking at slides, but it’s very exciting to learn more about a tool that I’ve seen used so often.

Until tomorrow!

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