Grace’s Notebook: Geoduck Egg Development – 3 days later

Today we took a look under the scope of the eggs in the silos from Friday’s egg development trial (Notebook post).

Looking at the eggs

I didn’t really look at the ones that were fertilized.

Benoit and I looked at eggs from the three silos that were not fertilized (although I did see polar bodies, cleavage).

Treatment group (all not fertilized) D-hinge? Notes
H50 yes don’t look great; lots of ciliates; some trochophores
C maybe lots of ciliates; remnants of trochophores; potential D-hinges, but hard to know because a lot of things could have been eaten
50 no some trochophores; lots of ciliates

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Grace’s Notebook: Day 1 of Trial 2 of Geoduck Stripspawn experiment – KCl dose + duration

Today was Day 1 of the second trial of geoduck stripspawn. We created an experimental design that takes into account both KCl dosage, and duration of the dose exposure. Overall things went pretty well (details in post), and on Wednesday, I’ll check for D-hinge development.

Google doc with plan

(written in prep for the day, things changed and will be noted in this post)

8:30am – Pt Whitney

Started at Pt Whitney, biopsy punching gonad tissue and checking for sex and ripeness.

I started out getting two or three males, and used a new biopsy punch with each geoduck. I placed the biopsied males in the right tank and grabbed new geoduck to biopsy from the left tank.

I then found two females, and then I found one that looked like a hermaphrodite, which was pretty cool! I’ve never seen that before:

img

Then I kept looking and finally found another female, though it may not have been as ripe as the other two. I left a little after 9:45am.

10:15am – Taylor Hatchery

I started out with cleaning the work area, cleaning the 20 20um screened mini-silos, gathering materials. Benoit, Michelle, and Molly helped by getting a filtered saltwater line available for me to use in the work area.

Once I had the filtered saltwater, I started setting up the tripours with the different mM KCl dosages, as well as some next to them full of filtered saltwater. While I was doing this, Benoit was setting up buckets in the bucket room with airstones and water in which the fertilized eggs will grow out in at the end of the dosing and fertilizing.

Setting up the dosages:

I filled the tripours up to 800ml (any more than that and the water will spill over once the 20um silo is placed in). When making the dosed saltwater, I made them as though they’d be filled to 1000ml so that the math would be easier.

I made them in a 3L pitcher, so I did them in sets of two. Example: to make the 20mM, I placed 20mL 2M KCl and 1960ml filtered saltwater. I then dispersed it into two tripours, up to 800ml. I did that again, and then moved on to the next dose.

KCl mM 2 M KCl stock to add (ml) ml saltwater to add
20 10 980
50 25 975
60 30 970
80 40 960

Tripour treatments:
img

Check female geoduck ploidy

Before dissecting the females and stripping the eggs, I measured the shells:

female number length width ratio diploid? (yes if ratio < 1.62)
1 127 73 1.739 no
2 121 71 1.704 no
3 124 80 1.55 yes

Benoit ran the ctenidia samples from all three females on the flow (? is that what it’s called) to check for ploidy. All three had the same ploidy, but it isn’t known if they are all three diploid, or all three triploid. Benoit believes they are likely all three diploid.

Stripspawn females

I dry stripped some eggs from all three directly into the screened silo sitting in tripour 19 (50mM with no hydration), and timed for 20 mins.

During those 20 mins, I stripped eggs from the three females into some saltwater. Benoit helped get a lot of eggs out. One female was much more ripe than the other two. Of the remaining two, one female was not very ripe at all unfortunately.

Benoit helped with screening and cleaning the eggs, and counted them using the coulter counter. The first counts showed that there was about 4 million eggs in the 3L pitcher.

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Kaitlyn’s notebook: ANOVA on heath stack juveniles

I preformed an ANOVA on the lengths of the juveniles in the heath stacks at Pt.Whitney. I did a post-hoc test (Tukey HSD) to determine significance between he treatments.

length-treatment

length-treatment

homogenization

With blocking the anova gives a p=0.028 for treatment and p=0.245 for tray.

Here is the Tukey HSD p-values on the anova with blocking:

Tukey multiple comparisons of means
95% family-wise confidence level

AE-AA 0.54267725
EA-AA 0.25261761
EE-AA 0.72476397
EA-AE 0.91813748
EE-AE 0.10032636
EE-EA 0.03504769

H0_T-H0_B 0.9999666
H1_B-H0_B 0.9999930
H1_T-H0_B 0.9303406
H2_B-H0_B 0.9999448
H2_T-H0_B 0.9970459
H3_B-H0_B 0.9994060
H3_T-H0_B 0.9988241
H1_B-H0_T 0.9980676
H1_T-H0_T 0.8107845
H2_B-H0_T 0.9963141
H2_T-H0_T 0.9703042
H3_B-H0_T 0.9875827
H3_T-H0_T 0.9999998
H1_T-H1_B 0.9588824
H2_B-H1_B 1.0000000
H2_T-H1_B 0.9996848
H3_B-H1_B 0.9999842
H3_T-H1_B 0.9708530
H2_B-H1_T 0.9898375
H2_T-H1_T 0.9991863
H3_B-H1_T 0.9962770
H3_T-H1_T 0.4563048
H2_T-H2_B 0.9999849
H3_B-H2_B 0.9999999
H3_T-H2_B 0.9641553
H3_B-H2_T 0.9999998
H3_T-H2_T 0.8273796
H3_T-H3_B 0.9074722

Tank Treatment
H0_T EA
H0_B EE
H1_T AE
H1_B AA
H2_T EA
H2_B AA
H3_T AE
H3_B EE

Laura’s Notebook: June 2019 goals

Accomplished last month:

O. lurida 2017-2018 project (OA/Temp carryover)

  • Revised MS based on co-author feedback and for general improvements
  • Sent for 2nd round of comments.

O. lurida 2018 project (Temp/Food carryover)

  • Finished 1st draft discussion
  • Correlated CV for larval shell length/width with # larvae released that day (is CV higher with more larvae?) Answer: no.

Bypass & admin:

  • Compiled draft application, including draft dissertation
  • Scheduled committee meeting

NSF GRFP

  • Met with Krista N. re: NSF GRIP, interesting Dungeness crab project looking at larval epigenomes in various pCO2
  • Submitted NSF GRFP funding request for next year (final year)

Oly methylation data:

  • Pulled matrices for Katherine

Oly QuantSeq data & future steps:

  • Expanded plan of attack. Would like to include some juvenile Port Gamble whole-body samples to compare those with high pCO2 parental histories vs. no history, in the location where parental history correlated with survival

Goals / To Do List

Admin

  • Prepare committee meeting presentation, meeting agenda, list of items needed / questions
  • Revise bypass application based on committee input
  • Submit bypass
  • Schedule qualifying exam
  • NSA quarterly newsletter

O. lurida 2017-2018 project (OA/Temp carryover)

  • Write cover letter for journal submission
  • Revise MS based on 2nd round of co-author feedback
  • Submit!

O. lurida 2018 project (Temp/Food carryover)

  • Calculate degree-days until larval release onset for all spawning tanks (if possible).
  • Figure out how to do broodstock survival analysis, then do it
  • Write Intro
  • Finalize plots
  • Send to co-authors

Oly QuantSeq:

  • Start testing QuantSeq analysis pipeline with Oly genome, Salmon (removing the multiple read/transcript auto-correct), Trinity’s isogroup designation file.
  • Schedule benchwork, order supplies

Oly epigenetics project

  • Wrap my head around action plan, steps for analysis

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Grace’s Notebook: Geoduck Egg Development Test at Taylor Hatchery

Today I looked into trying to figure out why we saw polar body development as early as we did last week during the first trial of KCl-dosage experiments (Day1, Day2). Details of the experiment are below, but there was egg development in the negative control (no sperm, no KCl), so I’m not sure what this all means.

Preparation

Pt Whitney
I got to Pt Whitney around 9:30am (took the 7:55am ferry) and grabbed 28 geoduck (Molly wanted 25 to spawn). I placed them all in the same cooler.

Taylor
I arrived a little after 10am, and started by discussing the plans with Brent and Benoit. Brent helped sort through the geoduck to help find me three diploid, then I sexed them and stopped once I had three ripe females.

New biospy technique that was SUPER easy:

Benoit noticed that the water in the cooler (in which I brought over all geoduck) looked super milky.

We realized they likely tried to spawn in the drive over, so it was decided that I would clean the entire experimental area, as well as use a bleach/water solution to clean the geoduck prior to dissection. I also rinsed the gonad/visceral mass once dissected with fresh water just to be extra cautious that no sperm made it’s way there.

I also realized I should have made more stock KCl beforehand, so I just made some at Taylor.

I then set up the tripours that I’d be using, rinsed the 20um screen silos that I’d use to treat the eggs, made sure the area had been rinsed, bleached, and rinsed again, then got started by opening up all three geoduck so that I’d be ready to quickly dry strip eggs from all three directly into the 50mM KCl instant dose treatment tripours. Timed for 20mins.

While the instant dose were sitting, I stripped the rest of the eggs and then screened them with a 90, and caught them on a 20.

I put them all in one tripour and had them sit in saltwater to hydrate for 30mins.

Then it was time to take the instant dose eggs out of the 50mM, so I set up two tripours next to them filled with fresh saltwater, then once 20mins was up, I picked up the 20um screen cylinders and placed them into the saltwater.

After the rest of the eggs were done hydrating, it ended up being 35 mins because I realized I had a few more things to set up.

I used a plunger to mix the eggs, then scooped about 70-80ml of the egg+saltwater and placed them into each of the four remaining screened cylinders sitting in the tripours (2 control, 2 50mM).

I let them sit for 20 mins, then removed the 50mM screens and placed them into fresh saltwater tripours (2:15pm).

At this point, it was time to get some sperm, so Molly helped me find some that I could use from their geoduck.

We started out by placing 2ml of the sperm/saltwater into 1 of each of the treatment tripours (was careful to have the non-fertilized tripours on the other side of the bench, and I wore gloves after fertilization). Fertilization time: 2:23pm.

At 2:50, Benoit took a sample from the control fertilized group and saw that there were about 5/6 eggs with polar bodies at a quick glance, and also noticed there wasn’t enough sperm. So at 2:52pm, I added 3ml more to the three tripours.

Starting at 3:07pm, we started the counts.

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Yaamini’s Notebook: June 2019 Goals

observe

Except it’s me trying to prove I can finish a paper before I leave for Friday Harbor while also prepping to TA and finishing assignments for my Evidence-Based Teaching Methods class……………………….

May Goals Recap:

Virginica Gonad Methylation:

Gigas Gonad and Larvae Methylation:

  • Since FROGER couldn’t come to a consensus about DNA methylation methods, I pressed pause on this project.

Virginica Sperm Methylation:

  • See above.

Other:

  • Identify three GSR candidates to run by Steven

June Goals

Since I leave for Friday Harbor June 16 to TA Ecology of Infection Marine Diseases, I only have four goals.

Virginica Gonad Methylation:

  • Finish the forking draft paper

Other:

  • Meet with Steven to discuss timelines for upcoming chapters and qualfying exams
  • Get a GSR
  • Schedule a committee meeting for when I get back

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Shelly’s Notebook: Thur. May 30, 2019 WOAC Symposium

The West Coast Ocean Acidification Center held its biennial meeting at the Center for Urban Horticulture today.

Here’s the agenda: Symposium 2019 Draft 4.docx

Here’s the abstracts: WOAC 2019 abstracts.docx

Here’s the poster I presented after Paul’s: CrabMetab_Poster_Final.jpg

Here’s the talk I presented: WOAC2019_STrigg_metab.pptx

Some interesting points were:

Simone Alin’s work showing the 2015 blob year actually had “good” conditions while 2017 had “poor” conditions – this manuscript is in prep

Micah Horwith’s DNR work on oyster (gigas and oly) and clam (manila and geoduck) sea grass outplants showed varying results: – variation between sites, but overall consistent: – positive growth for oysters – no effect for geoduck – negative effect for manila clams – perhaps due to clams burying while oysters stay on the surface? – sea grass does not generate more CO2 at night because of biomass accumulation, fixation, and sluffing

Julie Keister and Evelyn Lessard are finding different microbial community composition associated with different seasons, regions, and seawater chemistry from their cruise surveys (diatoms dominate, high in the spring, decline in the fall; dinoflag. high in the summer; noctiluca and ciliates in the spring and summer) – also check out Vera Trainer’s plankton studies about microbe toxicity/physiology changing with CO2 (ORHAB)

Check out:

  • LiveOcean app to see how winds, upwelling/downwelling, and fresh water input impact seawater current
  • Nanoos climatology app to see chlorophyll and when algae populations are more or less present

Nina Bednarsek’s paper on pteropod mortality and OA – she’s also seeing dissolution of carapace in Dungeness crab megalopae by SEM (not published yet) – would be interesting to look at metabolomics data in light of this

Thoughts from meeting

  • Everyone is very interested in community reponse in the field but not sure the best way to study it
  • Everyone is also very interested in trans-generational effects and if adaptation seems possible

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Shelly’s Notebook: Fri. May 31, 2019 Methyl CpGoe analysis

This post is related to github issue #694

The main problem was not being able to combine the ID_CpG_labelled outputs together into one file as Sam was able to do in his original CpGoe analysis using this script.

Yaamini created new CpGoe files specific for CAP, CDS, GENE, Exons, and windows features of the genome mentioned in this post and following this markdown file. After having problems with the script adding ID_CpG file headers and joining the files together, I attempted to run this script

on Mox:

  1. First I copied the data and script over to Mox: – mkdir /gscratch/srlab/strigg/data/Cvirg/FROGER_CAP_CpGoe – cd /gscratch/srlab/strigg/data/Cvirg/FROGER_CAP_CpGoe
    • rsync –archive –progress –verbose yaamini@172.25.149.226:/Volumes/web/spartina/2019-05-21-FROGER/CAP_CpGoe .
  2. Next I turned this script (/gscratch/srlab/strigg/data/Cvirg/FROGER_CAP_CpGoe/CAP_CpGoe/2019-05-22-CAP-File-Naming-Script.sh) into a Mox job (/gscratch/srlab/strigg/jobs/20190523_CAP_File_Naming.sh) and ran it on May 23. It errored out in the join step after 3-4 hours of running and joining 26 of the 90 files.
    • error message: “Broken pipe join –nocheck-order ID_CpG_labelled_all ${file}ID_CpG_labelled “
    • slurm file is here: /gscratch/srlab/strigg/data/Cvirg/FROGER_CAP_CpGoe/CAP_CpGoe/slurm-863764.out
  3. I copied everything to my folder on Gannet https://gannet.fish.washington.edu/metacarcinus/Cvirginica/FROGER/
  4. I realized some of the sample names have more than one underscore and having a non-unique column name could have been problematic.
  5. We also learned that certain bash commands (sed and maybe even the join command) may not run the same way on a mac (how Yaamini and I have tried running the script) as they do on a PC (how Sam ran it)
  6. I made changes to the script to

On Ostrich:

  1. I used this jupyter notebook to
    • run the updated script on the CAP data
    • run the updated script on the GENE data to duplicate what Sam already did so I could compare the output of the new script to Sam’s.
  2. QC: In R, I compared output of new script with output of Sam’s orginial script that I reformatted in R.

CONCLUSIONS:

  • updated script gives same output as original script and can now be used on remaining CpGoe analyses (CDS, Exons, windows).

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Grace’s Notebook: June Goals

I leave for FHL on June 16th for a 5-week intensive course on the Ecology of Infectious Marine Disease!! Lots to do before I go. ## Taylor Hatchery Work – Continue strip spawn trials – Read more about mechanisms behind geoduck spawning, why KCl works, etc. ## Oysterseed DIA – Revisit and try to get paper finished – Clean up repo

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Grace’s Notebook: Day 2 Geoduck Stripspawn Project at Taylor – not great results

Today we checked to see what development occurred since Wednesday’s strip spawn and KCl-treatment trials. There were so few eggs in the tripours to begin with, that there ended up being way too small of numbers of D-hinge larvae that I ended up dumping the trial and we’ll have to rethink this and re-do it. Details in post.

Counts using coulter counter

We started out using the coulter counter to see if it would work. We screened the tripours (used 31-33 because those are the extras) on 60micron screens. We realized quickly that there was a lot of larger junk that stayed in the sample, including pollen because the table wasn’t covered over the two days. We then swithed to screening with a 90micron screen into a 60 in order to catch the big stuff. I suspended them in 10ml seawater.

The first counts of the coulter counter were pretty high, but then the next two tripours were very low. Counts listed below:

Counts are #cells/ml in 10ml samples.

Tripour No. Trtmnt Count 1 Count 2 Count 3 Avg
1 0mM KCl 86 75 85 82
16 50mM KCl with hydration step 13 12 11 12
25 80mM KCl 29 43 34 36

The coulter counter was likely counting things that weren’t D-hinge.

Counts by hand

We then decided to do counts by hand. I screened them the same way and suspended them in 10ml seawter just like with the coulter counter.

I sampled out 1ml after mixing well by flipping contatiner upside-down a few times. I put the sample on a slide and put two drops of lugols in order to preserve the organisms so that they wouldn’t be moving around when I was trying to count them.

The counts were also very low. I didn’t count all tripours – I just started out by looking at a bunch of different treatments to see if it was worth me continuing and counting them all. Counts are below:

Counts are #D-hinge/1ml in 10ml samples

Tripour No. Trtmnt Count/ml Total No.
2 0mM KCl 0 0
4 10mM KCl 4 40
8 20mM KCl 6 60
17 50mM KCl + hydration step 7 70
18 50mM KCl + hydration step 10 100
19 60mM KCl 3 30
26 80mM KCl 0 0
27 80mM KCl 3 30
29 50mM KCl without hydration 3 30

The numbers are way too small to have too much meaning. Each tripour originally had ~10,000 eggs, so in terms of percentages, the number of eggs that made it to D-hinge range from 0%-0.01%.

Notes from this trial/things to change for next time

  • Didn’t have a true “negative control”. A true negative control would have been having a tripour of just eggs (no sperm added to fertilize)
  • 10,000 eggs/tripour is sticking with the 10eggs/ml rule that the hatchery has, but maybe it’s too small a scale for capturing any differences in this experiment. Maybe try putting more eggs in the tripours…. or using larger buckets?
  • Keep male and female geoduck separate after biopsy punching gonads to determine sex – may be cause of early polar body sightings (see this post).
  • Use less treatment groups, easier to manage and may be better to start on a much smaller scale, then get more complex as we learn what works

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