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Pisaster Genotypes and Righting Response

TL;DR


Data!


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!

Now...

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.

schematic of Pisaster

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: Download file "Held_et_al-2009-Invertebrate_Biology.pdf"

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...

as of 6/21 consistent 3-3.5° diff between treatments

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° 2:00

94° 0:20, 50° 0:30, 72° 1:00 (repeat 32 times)

72° 5:00

chill/end)

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.


Eventual RNA-seq work

compare with Janies et al 2016 EchinoDB

work to be done by Katelyn Chandler fall 2016


Thanks for help!

Sophie George, Morgan Eisenlord, the FHL community!

Comments

/groups/eccofhl/search/index.rss?tag=hotlist/groups/eccofhl/search/?tag=hotWhat’s HotHotListHot!?tag=hot4/groups/eccofhl/sidebar/HotListjohnwaresjohn wares2016-06-30 15:22:33+00:002016-06-30 15:22:33updated32johnwaresjohn wares2016-06-29 02:28:51+00:002016-06-29 02:28:51updated31johnwaresjohn wares2016-06-29 01:42:23+00:002016-06-29 01:42:23updated30johnwaresjohn wares2016-06-29 01:42:02+00:002016-06-29 01:42:02updated29johnwaresjohn wares2016-06-28 15:02:12+00:002016-06-28 15:02:12updated28johnwaresjohn wares2016-06-24 04:48:13+00:002016-06-24 04:48:13updated27Added tag - hotjohnwaresjohn wares2016-06-23 17:37:21+00:002016-06-23 17:37:21addTag26johnwaresjohn wares2016-06-22 23:47:31+00:002016-06-22 23:47:31updated25johnwaresjohn wares2016-06-21 18:50:55+00:002016-06-21 18:50:55updated24johnwaresjohn wares2016-06-21 02:38:03+00:002016-06-21 02:38:03updated23johnwaresjohn wares2016-06-21 02:36:50+00:002016-06-21 02:36:50updated22johnwaresjohn wares2016-06-21 02:31:29+00:002016-06-21 02:31:29updated21johnwaresjohn wares2016-06-21 02:18:48+00:002016-06-21 02:18:48updated20johnwaresjohn wares2016-06-19 16:28:21+00:002016-06-19 16:28:21updated19johnwaresjohn wares2016-06-19 16:27:22+00:002016-06-19 16:27:22updated18johnwaresjohn wares2016-06-19 05:03:34+00:002016-06-19 05:03:34updated17johnwaresjohn wares2016-06-19 00:19:10+00:002016-06-19 00:19:10updated16johnwaresjohn wares2016-06-18 19:37:20+00:002016-06-18 19:37:20updated15johnwaresjohn wares2016-06-18 18:56:54+00:002016-06-18 18:56:54updated14johnwaresjohn wares2016-06-18 16:39:15+00:002016-06-18 16:39:15updated13johnwaresjohn wares2016-06-18 15:55:16+00:002016-06-18 15:55:16updated12johnwaresjohn wares2016-06-18 10:37:56+00:002016-06-18 10:37:56updated11johnwaresjohn wares2016-06-17 05:16:48+00:002016-06-17 05:16:48updated10johnwaresjohn wares2016-06-17 05:16:30+00:002016-06-17 05:16:30updated9johnwaresjohn wares2016-06-17 05:15:18+00:002016-06-17 05:15:18updated8johnwaresjohn wares2016-06-17 05:11:21+00:002016-06-17 05:11:21updated7johnwaresjohn wares2016-06-17 05:09:25+00:002016-06-17 05:09:25updated6johnwaresjohn wares2016-06-16 19:53:48+00:002016-06-16 19:53:48updated5johnwaresjohn wares2016-06-16 19:08:22+00:002016-06-16 19:08:22updated4johnwaresjohn wares2016-06-16 19:07:35+00:002016-06-16 19:07:35updated3johnwaresjohn wares2016-06-16 19:05:26+00:002016-06-16 19:05:26updated2First createdjohnwaresjohn wares2016-06-16 18:37:09+00:002016-06-16 18:37:09created1wiki2016-06-30T15:22:33+00:00groups/eccofhl/wiki/ec337FalsePisaster Genotypes and Righting Response/groups/eccofhl/wiki/ec337/Pisaster_Genotypes_and_Righting_Response.htmljohn wares32 updatesPisaster Genotypes and Righting Response TL;DR Data! For this project, you will end up having 2-3 Pisaster ochraceus in your care; you must also keep your eyes on t...Falsejohnwares2016-06-30T15:22:33+00:00johnwaresjohn wares2016-06-29 15:51:46+00:002016-06-29 15:51:46updated15johnwaresjohn wares2016-06-29 04:26:27+00:002016-06-29 04:26:27updated14johnwaresjohn wares2016-06-28 04:28:38+00:002016-06-28 04:28:38updated13Added tag - hotjohnwaresjohn wares2016-06-24 15:41:01+00:002016-06-24 15:41:01addTag12johnwaresjohn wares2016-06-19 16:30:30+00:002016-06-19 16:30:30updated11johnwaresjohn wares2016-06-14 13:46:34+00:002016-06-14 13:46:34updated10johnwaresjohn wares2016-06-14 13:43:30+00:002016-06-14 13:43:30updated9Added tag - clinejohnwaresjohn wares2016-06-14 13:35:17+00:002016-06-14 13:35:17addTag8Added tag - phylodiversityjohnwaresjohn wares2016-06-14 13:35:16+00:002016-06-14 13:35:16addTag7Added tag - pcrjohnwaresjohn wares2016-06-14 13:35:08+00:002016-06-14 13:35:08addTag6Added tag - barnaclesjohnwaresjohn wares2016-06-14 13:35:07+00:002016-06-14 13:35:07addTag5johnwaresjohn wares2016-06-14 13:34:06+00:002016-06-14 13:34:06updated4johnwaresjohn wares2016-06-14 13:32:58+00:002016-06-14 13:32:58updated3johnwaresjohn wares2016-06-14 13:31:23+00:002016-06-14 13:31:23updated2First createdjohnwaresjohn wares2016-06-14 13:18:57+00:002016-06-14 13:18:57created1wiki2016-06-29T15:51:46+00:00groups/eccofhl/wiki/6f73aFalseBalanus diversity cline/groups/eccofhl/wiki/6f73a/Balanus_diversity_cline.htmljohn wares15 updatesBalanus diversity cline Background A few studies published DNA sequence data from barnacles (B. glandula) collected 15-20 years ago; the best example is here: ...Falsejohnwares2016-06-29T15:51:46+00:00Added tag - limpetsjohnwaresjohn wares2016-06-24 15:41:25+00:002016-06-24 15:41:25addTag10Added tag - diversityjohnwaresjohn wares2016-06-24 15:41:23+00:002016-06-24 15:41:23addTag9Added tag - distributionjohnwaresjohn wares2016-06-24 15:41:21+00:002016-06-24 15:41:21addTag8Added tag - ecologyjohnwaresjohn wares2016-06-24 15:41:20+00:002016-06-24 15:41:20addTag7Added tag - nichejohnwaresjohn wares2016-06-24 15:41:18+00:002016-06-24 15:41:18addTag6Added tag - hotjohnwaresjohn wares2016-06-24 15:41:17+00:002016-06-24 15:41:17addTag5johnwaresjohn wares2016-06-21 16:40:11+00:002016-06-21 16:40:11updated4morgankellyMorgan Kelly2016-06-20 14:33:01+00:002016-06-20 14:33:01updated3morgankellyMorgan Kelly2016-06-20 14:28:46+00:002016-06-20 14:28:46updated2First createdmorgankellyMorgan Kelly2016-06-20 14:11:36+00:002016-06-20 14:11:36created1wiki2016-06-21T16:40:11+00:00groups/eccofhl/wiki/d254bFalseTransect study/groups/eccofhl/wiki/d254b/Transect_study.htmljohn wares10 updatesTransect study In this exercise, you will use transect surveys and environmental data to create climate envelope models for a group of species of your choosing (it ...Falsejohnwares2016-06-21T16:40:11+00:00Added tag - hotmorgankellyMorgan Kelly2016-06-24 15:41:56+00:002016-06-24 15:41:56addTag5Added tag - adaptationmorgankellyMorgan Kelly2016-06-24 15:39:33+00:002016-06-24 15:39:33addTag4morgankellyMorgan Kelly2016-06-14 13:41:13+00:002016-06-14 13:41:13updated3morgankellyMorgan Kelly2016-06-14 01:37:59+00:002016-06-14 01:37:59updated2First createdmorgankellyMorgan Kelly2016-06-14 01:37:42+00:002016-06-14 01:37:42created1wiki2016-06-14T13:41:13+00:00groups/eccofhl/wiki/60339False1. Tigriopus heat tolerance/groups/eccofhl/wiki/60339/1_Tigriopus_heat_tolerance.htmlMorgan Kelly5 updates1. Tigriopus heat tolerance Background: Both genes and the environment contribute to most phenotypes. This complicates studies of local adaptation: local adaptation occurs when ...Falsemorgankelly2016-06-14T13:41:13+00:00hot/groups/eccofhl/search/index.rss?sort=modifiedDate&kind=all&sortDirection=reverse&excludePages=wiki/welcomelist/groups/eccofhl/search/?sort=modifiedDate&kind=all&sortDirection=reverse&excludePages=wiki/welcomeRecent ChangesRecentChangesListUpdates?sort=modifiedDate&kind=all&sortDirection=reverse&excludePages=wiki/welcome0/groups/eccofhl/sidebar/RecentChangesListmodifiedDateallRecent ChangesRecentChangesListUpdateswiki/welcomeNo recent changes.reverse5searchlist/groups/eccofhl/calendar/Upcoming EventsUpcomingEventsListEvents1Getting events…