Tag Archives: larvae

Bringing Ocean Acidification System online

I was out at Manchester yesterday to help Laura out with getting things going.

tldr- Water is flowing, rising temperature seems to be an issue. This could be attributed to air temp and/or pumps.

When we arrived water temperature was up to 19C after about a week. We decided to drain down system refill, calibrate Durafets, and monitor system over next few days with respect to temperature and pH.

Here is the system draining..
[youtube https://www.youtube.com/watch?v=CtDfMPkK69E?rel=0&showinfo=0]

 

As the system drained we calibrated with NBS buffers (7-4-10). In actuality I think they were only calibrated at 7 and 4. Need to confirm calibration system with Honeywells.

Probes are designated pink, blue, green, and yellow. Two in treatment tanks and two in each of experimental systems. As we placed in 7 buffer they initially read as follows

pink – 6.6

blue – 6.98

green – 6.82

yellow – 6.89

After all were calibrated we went through buffers and just read.

img_3940

 

2016-01-29 09.51.48

 

Here are more #s

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Tour time (if you listen closely you can hear a narration)
[youtube https://www.youtube.com/watch?v=K5xkpAAVSuI?rel=0&showinfo=0]

 

As the system started to refill with ambient water (10c) this is how the pH probes read.

2016-01-28_13_48_31_jpg_1C5BE253.png

This is without any C02 input. We then “sample calibrated” experimental system to read same pH

Camera_Uploads_1C5BE2A3.png

At the end of the day pH was set to 7.5 in treatment tank and we will monitor to see how temperature and pH holds (assuming it can adjust with high flow rates). For more on this day check out Laura’s post.

I will leave you with an inside look at treatment tanks. Note that the first tank in the video (Tank #2) has less water coming in from the head tank as compared to Tank #1.

[youtube https://www.youtube.com/watch?v=ctiNrf2DKEU?rel=0&showinfo=0]

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Re-Reproducing differential methylation analyses, again

Having just given a talk on reproducibility, I am in the midst of responding to reviewer comments about what we did (12 months ago!) and boy can I say every minute of putting this notebook together was worth it. I even found where we ran the entire notebook, so all result files are easily accessible. Beyond praising Claire, I will document my follow up analysis here.

Essentially the want more quantitative information on differential methylation beyond ..

OlsonandRoberts2015_9_docx_1BC2FBAE.png

Makes sense.

Here is what was originally done.

olson-ms-nb_BiGo_dev_ipynb_at_master_·_che625_olson-ms-nb_1BC2FCC5.png

For example the file named linexon contained 16 exon_intersect_DML_lin_u.txt. The 4 files were concatenated to produce lintable ….

eagle_fish_washington_edu_cnidarian_olson-ms-nb-master_12_1714_wd_lintable_1BC2FE30.png

and a little awk

awk 'FNR==NR{sum+=$1;next}; {print $0,sum}' lintable{,} > lin_total
awk '{print $2, $1, $3, (($1/$3)*100)}' lin_total > lineage_DMLs

to create lineage_DMLs

eagle_fish_washington_edu_cnidarian_olson-ms-nb-master_12_1714_wd_lineage_DMLs_1BC2FE65.png


Analogously here are the developmental_DMLs….

eagle_fish_washington_edu_cnidarian_olson-ms-nb-master_12_1714_wd_developmental_DMLs_1BC3F775.png


And we certainly need to now how many all_CGs we have…

eagle_fish_washington_edu_cnidarian_olson-ms-nb-master_12_1714_wd_all_CGs_1BC3F807.png


Table

Feature Family specific DMLs Developmental specific DMLs
Transposable Element 17 16
Promoter Region 2 3
Exon 16 12
Intron 25 46

I know we did this before, but I believe the reviewers want a break-down, or list of which specific transposable elements. This is a long shot if I can find this…
2 minutes later https://github.com/sr320/ipython_nb/blob/master/BiGo_larvae_manuscript4.ipynb.


To be sure files are accurate, I will intersectbed again. Based on recollection there is likely not a difference in proportion based on all TEs. This brings up a an important point of how to record “negative” data that does not go into a paper.

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Oyster food!

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Collecting & cleaning larvae on nylon screen.

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Larval Care – Pacific oyster larvae at PSRF Manchester

It’s Saturday. Yep, Saturday…

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Saturday morning at the hatchery.

 

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Prepared a spreadsheet for Dan to use to calculate necessary quantities of algae (two species) to achieve target concentrations:

Google Sheet: AlgaeFeedingCalcs

Here’re some pics of the day:

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping. Here’re some pics of the day:

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No code required

Today Sam and I took care of the daily maintenance of F2 Olympia oysters grown at the Ken Chew Shellfish Hatchery at Manchester, WA. In short, larvae are being described from Fidalgo (North: NF), Oyster Bay (South: SS), and Hood Canal (HC) broodstock. Though I had done most aspects before, never without Katherine, thus she provided instructions.

When I arrived the algae tank was empty and pump was off.
IMG_2915_JPG__9_documents__9_total_pages__1B651C27.png
The system was flushed with freshwater (with Sam soaked with said water) along with bleach. The algae tank was filled. The first set of tanks that were processed were the larger larvae (+160um).
Banners_and_Alerts_1B651CE0.png
Using 100um screen larvae were collected, brought up in 800ml and 0.5ml (x3) taken for counts.
IMG_2922_JPG__9_documents__9_total_pages__1B651D6C.png
The remaining larvae were placed back into cleaned systems.

Next, the smaller larvae tanks were processed, using 160 over 100 um screens, with larger ones moving over to +160um tanks (above). For this counts were done for both size classes and DNA samples taken for 160 size class. The smaller larvae placed back in the same tanks.
Banners_and_Alerts_and_IMG_2919_JPG__9_documents__9_total_pages__1B651E4B.png
For DNA samples, 4mls of larvae from 800ml beaker were taken, rinsed with ethanol and placed in 1.5ml centrifuge tube with 1ml of RNAlater (This was also done with fresh larvae- below).
Banners_and_Alerts_1B651F19.png

The last systems tackled were the “new”, fresh larvae from collectors..
oly_1B651FB8.png
For this, larvae were captured using 200um / 100um screen, with larvae moved to larger tanks, counts done, and samples collected for DNA. As expected very little larvae, they were brought up in 400ml, 1ml used for counting, and 8ml used for DNA samples.
Banners_and_Alerts_1B652044.png

Sam counted the larvae, probably still very wet (Sam and the larvae).
sam_1B652232.png

And here are the counts!
IMG_2923_JPG__9_documents__9_total_pages__1B6520EF.png

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There is something about TEs

For purposes of proposaling and reports, I have gone back to look at a small project done in collaboration with scientist at IFREMER looking at pesticide exposure on oyster larvae methylation.


The control library had limited yield so the number of loci with data from the treated and the control library was restricted. However using a liberal 3x coverage for both, we found a total of 823 DMRs (544 hypermethylated and 279 hypomethylated).

igv


Intriguingly, when one accounts for all CGs in the genome, these DMRs are predominantly in transposable elements.

Screenshot_7_9_15__11_15_AM_1B4EF5A5.png

Reminiscent of …

biorxiv_org_content_biorxiv_early_2015_03_13_012831_full_pdf_1B4EF6FD.png


The analysis is not pretty, but here is what I have to offer.

https://github.com/sr320/nb-2015/blob/master/Cg/YE-pesticide/YE-pesticide-summary.ipynb

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Morphometrics – Olympia Oyster OA Larvae Completed

Finished measuring Ostrea lurida larvae using NIS-Elements BR (10x objective and 10x nosepiece setting in software). That’s 2,620 larvae measured!

NOTE: Robyn previously measured 33 tubes worth of larvae, but I’m not certain where that data was saved.

All measurements by me can be found here (Google Sheet): FHL_Oly_larvae_measurements

Measured 30 larvae from each tube of the following samples:

  Box Label Color Tube Label Notes
70 FHL Oly OA x DO Box 2 etOH Friedman Purple 4.3.15 U FB HL.L-1
71 FHL Oly OA x DO Box 2 etOH Friedman Purple 4.3.15 V FB HL.L.2
72 FHL Oly OA x DO Box 2 etOH Friedman Purple 4.3.15 W FB HH.L.1
73 FHL Oly OA x DO Box 2 etOH Friedman Purple 4.3.15 X FB HH.L.2
74 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 Y SS LLH3 Many transluscent shells
75 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 Z SS LL.H.4 Many transluscent shells
76 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 AA SS LHH3 Mostly transluscent shells
77 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 BB SS LHH4
78 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 CC SS LL.L.3
79 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 DD SS LL.L.4
80 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 EE SS LH.L.3
81 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 FF SS LH.L.4
82 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 SS LLL-1 4.3.E
83 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 SS LLL2 4.3.F
84 FHL Oly OA x DO Box 2 etOH Friedman White 4.3.15 SS LHL-1 4.3.G
85 FHL Oly OA x DO Box 2 etOH Friedman Pink 4.4.15 FB release Low CO2 103A
86 FHL Oly OA x DO Box 2 etOH Friedman White SS Low CO2 4.4.15 release 103A All shells partially transluscent.
87 FHL Oly OA x DO Box 2 etOH Friedman Blue SS High CO2 4/5-6/15 release 103B All shells mostly transluscent.
88 FHL Oly OA x DO Box 2 etOH Friedman Pink 44/5-6/15 release FB LCO2 FB 103A

 

Example images from notes above:

 

Sample #74

 

 

Sample #75

 

 

Sample #77

 

 

Sample #85

 

 

Sample #86

 

 

Sample #87

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