Tuesday, July 31, 2012

Keira- Larval bisection, repair, and regrowth (Week 5)

I've recently started a project involving the bisection of Patiria miniata (sea star) larvae. The photograph to the right shows a whole larva before bisection. The red line shows the approximate location of the bisecting cut. 
The cut is done with a pulled needle or the edge of a hypodermic needle. Pulled needles are extremely thin glass, the end of which is flexible enough to move the larvae around and roll them over, while further up, the needle is firm enough to cut through the larva. I broke roughly 25 of the pulled needles in one afternoon learning how to use them, which George (my PI) warned me would probably be the case. It was a pretty steep learning curve, but now I can bisect many larvae (maybe 50 to 100) without breaking a needle. I have found that as the larvae get older, the thickening of their esophagus makes it increasingly difficult to bisect. To remedy this issue, I have been using a hypodermic needle to bisect the older larvae. The tip of a hypodermic needle has an edge that is basically a tiny razor blade, which allows me to make precise and thorough cuts. 

Posterior end of the bisected larva. The stomach is lightly pink from a recent meal of Rhodymenia palmate, the red marine algae that these larvae eat. 

Anterior end of the bisected larva. The open hole has been formed due to cutting through the esophagus as it opens into the mouth. 

I've been fixing the larvae about 5 days after wounding, which is when we begin to see significant remodeling. Initially, the larvae repair the wound by covering it. Then the ciliary bands start to grow to join back together. Next, tissue is built to complete the broken digestive system. Given enough time, each half of the bisected larva is able to regenerate completely and mature to metamorphosis. Given this impressive ability not only to repair, but to fully regrow, the intracellular signaling pathways that govern wound repair are of great interest. The fixed larvae will be stained with a few antibodies to target the signaling pathways of interest.  I am continuing my testing of antibodies on the Dendraster excentricus larvae. I will have more information about these trials as well as confocal pictures next week.

Monday, July 30, 2012

Getting more acquainted with Fidalgo Bay

Betty addresses a captive audience at the Cap Sante and Shell Tank Farm Waterfront park area.
      On Tuesday evening Anna, another student from SPMC, and I met up with the group from the Bikespot and headed out with them on the Trail Tales Interpretive Ride.   Trail Tales is a series of (usually) walks throughout the summer  that discuss different themes every week and this week was ecological.   A lot of the ride was along the Tommy Thompson trail.  It was led by Betty Kuehn and Betty Carteret and they educated the group on the history and cleanup of sites on the Anacortes shoreline.  It was really interesting and informative.
      Anacortes boomed with lumber mills and shipping in a time when environmental impact wasn't even a thought.  Today they are reclaiming their waterfront and cleaning up past generations left behind pollution.  The Department of Ecology leads the project and has taken a bay-wide approach rather than individual sites.  They are focused on cleaning up toxins including, dioxins, furins, PCB's, heavy metals, wood debris, petroleum hydrocarbons and more while also replacing natural habitats.
     The Tommy Thompson Trail is the very same trail that runs over the trestle where our field work has been.  In fact the last stop of the night was at the west end of the trestle and the history of this part of the bay was briefly discussed. Overall it was really nice to learn both more about the history of the town and bay but also about the clean up that has already started.
    On Wednesday Crista and I did a cyanotype of my oyster shells as a part of her project.   It was a lot of fun and the result is striking.  We laid Olympia shells in the center and surrounded them with Pacific shells and let it develop in the sun for ten minutes before sprinting through the building to a water bath.
Crista shows off finished product ready to dry.
        That afternoon Dr. Brian Bingham gave a wonderful talk on how to give a good presentation including graphics and power point as well as personal presentation.  I feel more confident now in my ability to put together a visually appealing and cohesive presentation.  Also there was cake because we don't do anything here at SPMC without food.
          Wednesday night and Thursday morning were more data and I feel like I am making significant headway.  One part still baffles me a bit where I am creating a site map and layers of illustration representing densities of Olympia oysters, clam shell and oyster shell from each sampled quadrat that we took along the entire site.  Crista is helping me with illustrator creating the site maps then Nam will help with my data point plots which helps calm my nerves. 
    In the evening I took a ride into town, down the Tommy Thompson and out across the trestle.  Mount Baker looked just beautiful across Fidalgo Bay in the fading light.

Mt. Baker across Fidalgo Bay.
     Riding across the trestle was nice, hearing the waves gently lapping and feeling the warm evening breeze.  I was surprised when I reached the far end and saw oil in the water.   I shouldn't have been surprised after all the oil we released from the mud when we were doing our survey but I still was. 
Beautiful view along a gorgeous trail.
     Looking up from the water I couldn't look away from the view, what a gorgeous evening in an amazing place.  I foresee more rides out here in my remaining time.
Lunch in the library.
       When we first arrived at SPMC the staff had a potluck lunch for us.  Friday to show our appreciation for all they do for us we had a potluck lunch for them. It was a lot of fun and a lot of good food of course.  
Watermelon, feta, basil salad - amazing!
     After lunch the dive crew headed out to see if they could find any Olympia oysters in a channel that runs near the trestle.  Apparently genetic data shows that younger Oysters at the trestle site have some varied genetics from what was planted pointing at other Olympias nearby and we were on the hunt for them.
The entire trestle takes my 180 degree pan function.
Anne, Annie, Dr. Dinnel and Nate Schwack.
     Anne and Annie dove for quite some time searching in the mucky water with very low viability looking for oysters.   Dr. Dinnel also had them collect some shell and bring it back up for us to look for small juvenile on them that could be hard to spot underwater.  The ladies brought up a good batch of shells but none of them had any spat on them.  
Anne and Annie diving the trestle

The shell collection.
      One fun part of going to the trestle was that we went by Shannon Point on the water and got a new view of our temporary home. It was a great afternoon and a wonderful end to another great week out here at Shannon Point Marine Center.  This week I realized how quickly our time is coming to an end and was saddened.  I wish we could stay out here all summer and maybe even longer.  My mood is buoyed by the thought that I am now a Western student and will hopefully be taking classes out here shortly. 

Saturday, July 28, 2012

Chris - Week 4: SEM Continued

During week 4, I continued my examination of cilia using scanning electron microscopy. In particular, I prepped and scanned 12.5 hour old larvae and 2-3 day old pilidia. The latter group was "cracked" by Svetlana. Essentially, she split them open so that their internal anatomy could be examined in more detail.

The 12.5 hour old larvae were supposed to display an intermediate level of ciliation between the 10 hour and 15 hour age groups. However, they ended up most resembling the 10 hour cohort, possibly due to differences in sea table temperature between the various cultures we were trying to compare.
Fig 1: An angled anterior/posterior view of
a 12.5 hour old M. alaskensis larva

Fig 2: An angled anterior/posterior view of a 12.5 hour old M. alaskensis larva

   The larva shown in Figures 1 and 2 both display cilia, particularly around the equatorial region along the periphery of the specimens. Note the patchy distribution of cilia. 

Fig 3: A lateral view of a "cracked" 2-3 day old
M. alaskenis pilidium

Fig 4: A lateral view of a "cracked" 2-3 day old
M. alaskenis pilidium
Figures 3 and 4 show M. alaskensis plidia  that have been "cracked" in order to reveal some of their internal structures. In Figure 3, note the various ciliated bands, both internal and external.

Tuesday, July 24, 2012

Michaela - Past few weeks

These past few weeks have been full of cleaning the lab, mainly, and getting our equipment ready. Lets just say I'm an expert at cleaning the systems and they look sparkly clean! The past few weeks we have been doing incubations with the coralline algae looking at pH, DO and temperature. We also measured the algae after it had respiration time to see how the algae responded to photosynthesis. My mentor and I had been waiting for CO2 cylinders for about two weeks. We have them now and have been testing the equipment with the CO2 in the water. Some of the equipment wasn't working properly, such as the CO2 meter. Getting the meter looked at tomorrow and hopefully it will be reading properly because its time to start these experiments!

We originally had CO2 going into our sump and the CO2 wasn't going in the water long enough. Leigh built a cylinder that has the air stone in the bottom and incoming water coming in from the top (left). This makes the bubbles move up through the water against the current and this air bubbles stay in the water longer. Since this change everything has started to come together and after the CO2 cylinders arrived we have started to circulate the water with CO2 from the sump into the whole system. 

I'm excited to see how this experiment will go and looking forward to help get it going. The experiment should start this week if we get the CO2 meter fixed and I will post a new blog about it soon.

Keira- How does the confocal microscope take such pretty pictures? (Week 4)

This week I got to test some fluorescent antibodies with the confocal microscope. While many did not work or yielded inconclusive results, some worked very well with lovely results. These are photographs of Patiria miniata (sea star) taken on the confocal microscope.
Figure 1: P. miniata (front view)
Figure 2: P. miniata (side view)
The blue and orange colors are added to show the contrast; the images are actually in greyscale when scanning specimens. The colors represent the two different dyes used on these larvae. Most of the color you see (blue in Fig 1, orange in Fig 2) is fluorescence emitted from Hoescht 33342. This is a dye that intercalates with DNA, so it stains the nucleus of every cell. This helps discern the structure of the organism. In these Patiria, the two ciliated bands are shown by the areas of greatest color density. Patiria have cells that are monociliate, meaning that each cell can form only 1 cilium. Thus, in order to form a ciliated band, the larva must have a great density of cells in that area. The cells are comparatively sparse in the rest of the larval body.

The infrequent dots of contrasting color (orange in Fig 1, white in Fig 2) shows light emitted by from the fluorescent antibody. Last week my blog post discussed primary and secondary antibodies. The primary antibody in this case was phospho-histone.

Picture credit:
Histones are the "spools" around which DNA winds. Most of the time, DNA is loosely arranged in the nucleus, but when the cell is getting ready to divide, it starts organizing the DNA in order to form chromosomes. The simplified cartoon to the right demonstrates this idea. The "phospho-" prefix means that the histones have been activated by the addition of a phosphate group, an energy rich molecule comprised of four oxygen atoms in a tetrahedral arrangement around a phosphorus atom. The activated histones begin organizing the DNA in preparation for mitotic division.

The phospho-histone antibody binds specifically to these activated histones, thereby tagging only the cells which are actively dividing or that have just finished dividing. The secondary antibody binds to the phospho-histone and then can be excited to fluoresce, which shows us where the dividing cells are.

The confocal microscope works by scanning one layer at a time and saving the information about light emitted by the sample specifically in that layer. By scanning thin slices through the sample and then compiling the data, we are able to see 3-D images such as the ones shown in Figures 1 & 2. Two lasers alternate light emission on the sample.  One laser is set to excite the Hoescht dye and the other to excite the phospho-histone antibody. The confocal microscope "reads" the fluorescence emitted from the sample in each of the channels.  The fluorescence information from each channel is compiled separately. The images can be overlapped to show both the Hoescht dye and the phospho-histone, thus illuminating all the nuclei as well as individual dividing nuclei on the same larva.

Kailey - Tidepooling and getting muddy.

     Lately I find myself spending a fair amount of time with my data which isn't exciting at all to read about so until it is I will skip recanting my excel adventures and stick to the more exciting adventures.
      Tuesday Crista and I went out again with the abalone crew, the weather was beautiful and spending the morning on the water was fabulous. 
The Fauna
    It was an exciting day as Anne and Annie had finished their required dives with Nate as an instructor and were diving just the two of them.
Divers below.
     After Annie and Anne descended to the abalone site Nate dropped Crista and I off on Allen Island to tidepool and explore the intertidal areas.   It was exciting to jump from the bow to the rocks and then back again when our time was over, contrary to the voices in my head neither Crista or I ended up in the water. 
Practicing over/unders in a tiny tidepool.
Run little guy!
A singular gooseneck barnacle we found,
   It was really nice to explore a beach so untouched by humans.  We only discovered two signs of humanity, a piece of pvc wedged way back in the rocks and a small inflatable boat that had washed up, which we rescued.  We had a great time exploring and watching the hordes of isopods flee from our approach.
The trestle is a popular local bike trail, the Tommy Thompson Trail.
      Wednesday was our last day of fieldwork at the end of the trestle that we had been working on.  Dr. Dinnel and I were joined by Anne, Annie, Nam, and Dickson a tireless volunteer.
Data sample areas as seen in muddy footprints.
We rounded out our data, collecting from shallower areas as well as a rock patch, mud flat and broken barge structure that all sit on the north side of the trestle.

Nam, Anne, Dickson and myself survey the barge site.
Olympia oysters settled on a rock in the rock pile.
Egg capsules, most likely of the invasive Japanese oyster drill.

     We made short work of our day with so many experienced volunteers.  Though we have gotten good at counting substrate and identifying Olympia oysters we still all seem to get pretty muddy by the end of the day.
     Thursday found Dr. Dinnel, Dickson, a few other volunteers and myself at the opposite end of the trestle.
The west end of the trestle and Tommy Thompson Trail.
     The western end of the trestle site where we sampled runs along rocks after the end of the pilings.  Though we sampled both substrate and Olympia oysters as we have been we sampled in the zone between the total mud and the rocky shore.  One group sampled either side of the trail from the end of the pilings every twenty feet for approximately 2000 feet.

Our side of the trail.
The quadrat, pole and bucket of science.
Intrepid volunteers.

Dickson insisted I needed a picture for my mom.
     In the evening it was time for discover scuba at the local pool with Nate Schwarck.  We were pretty evenly split between certified divers and brand new folks.  Those of us certified buddied up and did skill review and had a blast.  I partnered with Umi and it was nice to go back over all those skills that you don't necessarily practice after you get your certification.  It was also a joy to see all the smiling faces of the new divers.   After three hours split between swimming, playing on the awesome water slide and SCUBA diving we all slept well and soon after our return to Shannon Point.
   Friday I spent the day at WWU at transitions attempting to get registered and getting my student ID.  Thank goodness two of my friends from WCC were there as well and we were able to share our frustrations at being last to register and not being able to get classes.   I have a plan and hope.

Kailey - Data, outreach, abalone and food.

     Last week was all about data entry, number crunching, outreach and amazing food.  Early in my week I entered data and read papers about oysters, progress but not outwardly exciting.
    Tuesday I met up with Denise Crowe who does outreach for SPMC and Gemma, a graduate student.  We loaded up the outreach trailer with sea life and headed to WWU main campus.   We participated in a program for younger students to interest them in the sciences called Campus to Compass. There were two groups of students who all seemed to have a great time.
Denise Crowe educating scientists in the making.
Sea cucumber mouth.

     After we returned to Shannon Point and got everything situated I met up with Anne and Annie and we went to Dr. Dinnel's house for dinner.  Anne and Annie are working with Dr. Dinnel, Nate Schwarck and others on native Pinto abalone restoration.  The rumors were true and we were treated to an amazing meal with great company.

Baked oysters with ginger and wasabi were just some of the delicious offerings.
      I crunched some numbers the next day then cleaned and prepared the temperature and salinity sensors we recovered for download later this week.  I made crab rangoon for everyone with fresh crab that Eric and I got diving on Sunday.

A gorgeous day for a dive.
     Thursday morning Crista and I were lucky enough to join Nate, Jay, Anne and Annie on the Zoea and accompany them on a Pinto abalone out-plant site survey.  The weather wasn't cheerful but our intrepid divers made two dives finding, counting and measuring previously planted juvenile abalone.  They discovered one abalone so big there was no way it was an out-plant which is a wonderful sign for a locally very threatened species.

Pre-dive planning and safety check.
Nate, Anne and Ann preparing to descend.
Crista the photographer getting her photo snapped for once.
  You couldn't pry the smiles off Annie and Anne's faces when they returned, they had a good dive and reassuring abalone finds.  On the way back to the marine the fog finally burned off and the day turned gorgeous.

Anne and Annie chatting about the dive.
That is one happy diver.

      On top of surveying abalone out-plant sites Anne is also researching diet options for juvenile abalone.  Her babies had come to the lab the day before and I was surprised how tiny they are, only a few mm.
Anne's baby abalone. (It is red, the green on the bottom is algae.)
     That evening nearly everyone got together and we celebrated Thanksgiving in July. Everyone cooked something and it was really fun to see the differences and similarities in what everyone considered traditional.  It was a really wonderful evening with people I am really grateful to have had a chance to meet and get to know.

Umi captured the group nicely.
Anne cutting the bird.

Monday, July 23, 2012

Thomas - Purse Seineing on the Mighty Columbia

This week’s work took me to the Columbia River for a two day trip to gather a population sample of sub-yearling salmon. These Salmon will be used to continue research on growth, development, genetics, health and a myriad of other things. The collection method on this day was a fishing technique known as purse seining. Purse seining is a good method to take samples in an area because the net is the same size every time you deploy it and it is stationary so unlike a towed trawling net the purse seine net captures organisms in the same size area each time. This is advantageous because it can tell someone what is in that area at that given time.

The operation requires two boats; the main boat and a skiff. The main boat is designed for this purpose and is outfitted with a winch, a boom and a water pump. The deck is large and open on one side to allow for the net to be hauled on board.

The skiff is smaller an looks much like a fishing boat that you might see on a lake with the exception of an anchor point that the net is attached to while it is being deployed. The smaller boat shadows the larger boat for the majority of the day and I was constantly amazed at the degree of skill that the operators of both vessels showed throughout the day.

Day one started out with smooth waters and cloudy skies. After a safety and equipment check we headed out into the mighty Columbia. The drive across the flat water took about 25 minutes. Once we reached the correct location we quickly got to work and began to deploy the net. After some preparations were made a rope which is fastened to one end of the net is thrown to the skiff driver who hooks it to his boat and pulls away.

As the skiff driver pulls the rope the net begins to slither off of the boat and into the water. The bottom of the net is weighted and sinks. The top is lined with floats which float behind the skiff. The 500 foot long net is stretched in a large circle and the skiff driver brings his end back to the boat where it is reunited with its opposite side.

When the circle is complete a large weight is dropped into the water to close the bottom of the net and the net is pulled in by hand, the whole crew helps with this task and it is carefully piled back on the deck for the next deployment. Fish are collected, identified, weighed, and the samples which we set out to collect are quickly frozen and stored for later analysis. Other fish are simply recorded and let go without harm.

The net is deployed many times throughout the tide as a way to identify when the fish are where in the water, in relation to tidal changes. The location also changes to see where the majority of the population is at a given time. Many types of fish were collected from herring to anchovies, salmon and smelt. During the final haul of the first day I was shocked to see the water in the net boiling as it was pulled onto the boat. I found it difficult to focus my eyes on what it was that was causing this phenomenon but as it got closer to the surface I realized that we had caught an estimated 16,000 anchovies! Luckily we only had to count a small portion of them and all were released back into the ocean.

A quick thank you to Brian the skipper of the Pelican who’s expertise got us out and back both days without the slightest difficulty. I do enjoy watching someone who is a master of their craft, perform a skillful task as if it is the most natural thing in the world. What a great opportunity!