For this project, you will end up having 2-3 Pisaster ochraceus in your care; you must also keep your eyes on the sea tables in Lab 4 that they all have adequate flow, that the temperature differential remains about 3°C between the central tables and those at the end with heaters (and help keep the seastars fed and happy!). I will show everybody the components and what makes it tick in case anything needs to be adjusted, but please help keep this running smoothly! FYI: my phone # is on a sticky in Lab 4 if I can be of help!
You have all probably heard about Sea Star Wasting Disease (SSWD). The interaction between elevated sea temperature and disease is likely in this case, and right now there are only about 10-15% as many of the beautiful sea star Pisaster ochraceus around San Juan Island as there were only a few years ago.
An interesting and surprising finding, however, is that there is genetic diversity in P. ochraceus that seems to influence the susceptibility of an individual to SSWD. This diversity is easy to monitor: if we amplify the elongation factor 1-alpha locus using PCR, the products can be run on a 2-3% agarose gel and individuals are scored as homozygotes or heterozygotes. Note, however, there is only one type of homozygote, as this locus is apparently overdominant, meaning that one allele is homozygous lethal!
So how is it working? There is no particular reason to associate a "housekeeping" gene with a particular virus or other disease-causing agent. However, EF1A is involved in the general stress response of eukaryotes, including how it aids in rapid generation of proteins like heat shock proteins, and has also been implicated in viral replication as it must be upregulated to help generate new viral proteins.
Our hypothesis is that this genotype is an indicator of how well an individual responds to stress; bear in mind it is often not the pathogen that gets you, but the inflammatory immune response! To check this out, we are going to first see if there are any energetic/activity differences between individuals when they are stressed by heat. After the class, I (JPW) will also follow the change in transcription of the EF1A family during heat stress.
What we will do is collect Pisaster in week 2 of the class. Ideally we have 2-3 individuals for each student in the class; we really need a minimum of 2 per individual, and colleagues at FHL are going to help us reach that goal (which would have been very easy only a few years ago). We will bring them back to Lab 4 to perform the following experiment:
[note that now the experiment is running, we collected n=22 individuals from the same part of the SJI coastline (all within perhaps 1000km of each other), and then did our assays in a common garden (albeit with short acclimation time, no husbandry). This should minimize the influence of environmental variance on trait variance.]
1. place in ambient sea table (11-12.5°C, depending on day), let acclimate to lab for one day. measure distance from madreporite to tip of nearest arm (shortest distance from any arm tip to madreporite). This is the "A" arm and going clockwise are the B, C, D, E arms. Measure B, C, D, E as well - 5 measures for the "combination" to uniquely ID that individual. Also take a photo: Sophie George will be using our genotypes for her crosses as well.
2. This distance and *any* other distinguishing features of YOUR sea star will help us maintain its unique identity. So an individual AND ALL TISSUE SAMPLES must be labeled with this identity (your 3-letter initials and individual number, with your lab notebook maintaining the other data necessary to uniquely ID that individual), and you must make sure you can maintain that identity using visual cues - color, patterns, etc. You should perhaps take a good photo of your 2-3 stars to recognize patterns, etc.
Tissue sample #1 will be about 100µl volume of tube feet sliced away with a clean razor blade into a small tube of ethanol. LABEL. We will use this tissue sample to genotype the seastar.
Tissue sample #2 will be about 200µl volume of tube feet sliced away with a clean razor blade into a tube of RNALater (5-10x volume of tube feet). LABEL. We will refrigerate overnight and then put in -80°C freezer. This will eventually be used for an RNA-seq study (see Janies et al 2016 for echinoderm resources; they used tube feet...)
3. now let the individual relax in the AMBIENT sea table overnight
4. on the next day you will do 'righting response trials' with your individuals, simply flipping on their aboral (back) side and timing how long it takes them to fully get part of all 5 oral surfaces down (lets agree that the trial starts when you flip them, ends when contact is made by the 5th arm, not just its tube feet stretching). Keep them away from the sea table walls! please repeat 3 times if time permits, time the trial with your phone or a stopwatch. More on these trials:
5. when done with these righting response data, put your seastar into the +3° sea tables (set up per Eisenlord et al 2016)[it has actually been varying between +2°-+4° over ambient...daily aeration/cleaning hose-downs to remove detritus). they will acclimate and heal over there for ~2 weeks. Enter data into the Google Docs above.
WATCH YOUR STARS DAILY. ANY SIGNS OF SSWD OR OTHER HEALTH CONCERNS PLEASE REPORT. please feed daily - mussels, clams, barnacles etc. about one mussel/clam sized meal per day...
6. after *7 days was the final, with some only being in for 6-7 days*, we will repeat step 4 (righting responses), take measurements of the original cut you made to see how well the seastar has healed, and take an additional tissue (tube feet) sample from that individual to place into RNALater as before.
So overall, our stars experienced ~one week of elevated temperature (+2-4°C), which should have changed their RNA profile (and physiological performance). Note that our trials happened at ambient temperature water both "before" and "after" so the only difference is their genotype (TBD) and their acclimation profile (how they have altered expression), as well as any other factors like: have they spawned? do they need to spawn? size? do they have SSWD? age? and so on. These are things you can ponder for your report.
Is the response variable within-star the mean time to flip? the minimum? you decide!
In between behavioral trials, we will genotype each individual using PCR as in Wares & Schiebelhut 2016.
(Which doesn't give the details, but punts to Pankey & Wares 2009, which also insufficiently gives the details! Here they are, thanks to my grad student Karen:
94° 0:20, 50° 0:30, 72° 1:00 (repeat 32 times)
You will be asking whether there is variation among the different genotypes (across all individuals in the class) for righting response time and wound healing. To clarify some of the individual variation in other factors that may affect righting response (size? whether they have spawned? color?) you will want to ask how much their righting response has changed between the two trials.
work to be done by Katelyn Chandler fall 2016