Shelly’s Notebook: Fri. Oct. 11, Var.low pH x ambient Juvenile Geoduck Pt. Whitney

Experiment Day 36

  • Got to the hatchery at 1pm

Water chem

  • Water level in low pH header tank was low again. Spoke with Matt and it’s still unclear what’s going on with the system. He suggested it could be a clog since the source tank is consistently full and is only feeding the totes and the two conicals. It’s just strange that it has now switched from the low pH header tank being low to the ambient heater tote being low, and now back to the low pH header tote being low. Matt suggested these inconsistencies may have been happening this past fall too only not obvious because if the flow slowed the tank would not drain, where now the header totes have pumps so can drain because of that.

Low pH header: uc?export=view&id=1lDb-6_hAw_ILfrckHl-s64JFKOqOLzi9

  • Took discrete measurements at 1:30pm.
  • for TA, I took directly from the discrete measurment water after filtering it through 20uM. The discrete measurement water samples were directly from the headers and inside the silos (using clean TA cups).
    • Discrete measurments looked as they should
    • I ran TA of 8 samples (4 samples from last week and 4 from today)
      • It took a long time to get the acid flushed through the lines without bubbles. It seems like there was air getting in the outflow line from the burette, so I ended up disassembling and reassembling the tubing and burette. Eventually, after 30 flushes, this seemed to do the trick. I will need to change out the HCl next time. I started the TA run around 3:30pm.
    • Again, I didn’t take measurments for volitional spawned low pH vs. ambient conditioned juveniles (from Sam’s summer experiment) because I was short on time.

animal check

Var.low pH treatment

ambient offspring:

  • 2.1: uc?export=view&id=1Dl26zkZ0omYU1DrivWVDFWkgw8ZIoEwM
  • 2.2: uc?export=view&id=1VzpxOBXA9-htyDlihPmaczB0wUEx4K0Y
  • 2.3: uc?export=view&id=1CuFpKxoAGJAGDNEgXePUUUnoLsvmKU7c
  • 2.4: uc?export=view&id=1VzpxOBXA9-htyDlihPmaczB0wUEx4K0Y
  • 2.5: uc?export=view&id=1eZD5sFVmhBLjpG3s4Q1apxZYuA94ZfWu

var.low pH offspring:

  • 2.8: uc?export=view&id=1tcwtRGhSFBcbymC_lDVgEtijZ4Cx6fJQ
  • 2.10: uc?export=view&id=1_ayq6WmpZe80SYgyCYV90rrMgyEqpGoj
  • 2.11: uc?export=view&id=1myV3z6WLt7IbYwsPEBa9SohzJuCOrzeG
  • 2.12: uc?export=view&id=1u-ooRz_DpwNpu8MwQ9wwjRr8q6Md3EBP

Ambient pH treatment

ambient offspring:

  • 3.1: uc?export=view&id=1K_hT8ndhLvqXU7ZQMOngPLg2q0vPEqmG
  • 3.2: uc?export=view&id=1xqhnXG9zqHEbPNl-mOpy7mBh9YhXWPVG
  • 3.3: uc?export=view&id=1_WN840bj-1vNXO_sa-OJi5WO4SQRhUkb
  • 3.4: uc?export=view&id=13WmBPLwAceH_VavVuJnQ8K8OdQA-_ERo

var.low pH offspring:

  • 3.8: uc?export=view&id=1rznNDIEvbtykKwh2jyEYvrMaW0KxzGyq
  • 3.9: uc?export=view&id=15bQEuG8OHIEsc4oHvs7vhStsKkiM3AcW
  • 3.10: uc?export=view&id=1HZCcZsdW-YuxuFapsiEtFuZbeKur3-CE
  • 3.11: uc?export=view&id=1evzif64Ov50sZLdFN05sd1yEfifhKj5-
  • 3.12: uc?export=view&id=1Tljbz2M3PhCvLP28a3Ko9XSWm0Gcm1AA

Offspring sibs from var.low and ambient conditioned broodstock

  • these are the left overs after we divied up the offspring at the start of the experiment
  • for reference, animals are on 1 mm mesh
  • ambient extras were mostly from smaller initial size class
  • var.low pH extras were mostly from larger initial size class

var.low pH offspring: uc?export=view&id=1Cn03La0L3G_HBABgc8Dw49njeQ8sL49i

ambient offspring: uc?export=view&id=1_43foSk05lvIijuwmMx-meTWNV5ZgoIa

2018 juveniles (Sequim bay outplant vs. hatchery reared)

var.low pH treatment

  • 2.6: uc?export=view&id=1UvuoE3JJYOH5J32s99qpDQUSEcZ5hRjy
  • 2.7: uc?export=view&id=1ViLGb4eh7A2lcpN4mTsJL0iazFw-G-nY

ambient treatment

  • 3.6: uc?export=view&id=1-Lq_YxPH4bo0m9_CIgBQapuIY6_LOHR9
  • 3.7: uc?export=view&id=1I2cqdMfuXsEc7L6aAyJbPWkJOpZXc8XC

Growth over time

I did some preliminary analysis on shell length and wet weight data (that I collected for the respirometry trials I’ve done over 28 days) to try to get an idea if either measurement shows a trend over time. Shell Length over Time unnamed-chunk-23-3.png

Wet weight over time unnamed-chunk-23-4.png

Conclusions Over 28 days:

  • No consistent increase in shell length or wet weight over time.
    • This could be due to too small a sample size at each time point. From 9/6/2019 on, sample size = 5 animals.
  • Apparent difference in shell length and wet weight between treatments:
    • For animals in variable low pH treatment, offspring from variable low pH conditioned parents generally show larger shell length and greater wet weight than offspring from ambient parents
    • For animals in ambient treatment, offspring from variable low pH conditioned parents generally show larger shell length and greater wet weight than offpring from ambient parents.
    • Offspring from ambient parents in variable low pH treatment show the least growth over time
  • Need to confirm these results with larger sample size

Concerns

I am concerned that the var.low pH treatment may be leading to high mortality. The shell clearing I observed in previous weeks seems to continue until there is no shell and the animal dies, as evidenced by the debris in silos 2.1-2.12. These animals are also extremely fragile, so I decided against any cleaning after discussing with Matt. I had originally planned to stop this experiment after 60 days (Oct 31 proposed stop date), however I am concerned to continue much longer if var.low pH treatment is causing high mortality. It’s possible it’s a food thing, and they may need much more food to compensate for the amount of energy they are putting into tolerating the var.low pH condition. I could try pumping more food directly into the headers. Reducing the flow would likely help maintain more food, however turning down the header flow could lead to uneven header tank levels and turning down the flow into the silos reduces the pressure in the manifold leading to uneven silo flow. I will consult with the team about what to do.

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cat slurm-1388268.out dDocent 2.7.6 Contact…


cat slurm-1388268.out
dDocent 2.7.6

Contact jpuritz@uri.edu with any problems

Checking for required software

All required software is installed!

dDocent run started Thu Oct 10 14:41:43 PDT 2019

At this point, all configuration information has been entered and dDocent may take several hours to run.
It is recommended that you move this script to a background operation and disable terminal input and output.
All data and logfiles will still be recorded.
To do this:
Press control and Z simultaneously
Type 'bg' without the quotes and press enter
Type 'disown -h' again without the quotes and press enter

Now sit back, relax, and wait for your analysis to finish

Trimming reads and simultaneously assembling reference sequences
mawk: cannot open uniq.fq1 (No such file or directory)

dDocent assembled 69805 sequences (after cutoffs) into 0 contigs

Using BWA to map reads

Creating alignment intervals

***** ERROR: Requested column 4, but database file - only has fields 1 - 0.

Using FreeBayes to call SNPs
ls: cannot access mapped..bed: No such file or directory
0% 0:0=0s
rm: cannot remove ‘mapped.
.bed’: No such file or directory
could not open "raw..vcf" exiting
mv: cannot stat ‘raw.
.vcf’: No such file or directory

Using VCFtools to parse TotalRawSNPS.vcf for SNPs that are called in at least 90% of individuals
WARNING: RunAsUser for MSP ignored, check group ids (egid=20746, want=51)
can not chdir(/var/spool/clientmqueue/): Permission denied
Program mode requires special privileges, e.g., root or TrustedUser.

Creating plots mirrored along the y-axis

Good code example: https://www.r-bloggers.com/making-back-to-back-histograms/

[code]#!/bin/bash ## Job Name #SBATCH...

#!/bin/bash
## Job Name
#SBATCH --job-name=nl-02
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes (We only get 1, so this is fixed)
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=20-00:00:00
## Memory per node
#SBATCH --mem=100G
#SBATCH --mail-type=ALL
#SBATCH --mail-user=sr320@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/sr320/1009b/


module load ddocent.module


#source /gscratch/srlab/programs/scripts/paths.sh

cd /gscratch/scrubbed/sr320/cragig_wd/

/gscratch/srlab/programs/dDocent-2.7.6/dDocent \
config_cragig.txt

#sbatch, #source

Shelly’s Notebook: Thur. Oct. 3, Pt. Whitney Juvenile Geoduck var.low pH experiment

  • Got to the hatchery at 12:30
  • Had lab meeting from 12:30-1:15

Water chem

  • Water level in header tanks look normal. Last couple weeks it must have been a fluke, or perhaps related to low tide?
    Low pH header: uc?export=view&id=1ZbhTRaP733ENU08XPnchCwa4Wj8anAECambient header: uc?export=view&id=14MmxEBUwSBvIsM43YRST0fQ8xYOFIqdh
  • Took discrete measurements and TA at 3:40pm.
  • for TA, I took directly from the discrete measurment water after filtering it through 20uM. The discrete measurement water samples were directly from the headers and inside the silos (using clean TA cups). I poisoned samples to run at future time.
    • Discrete measurments looked as they should
    • I didn’t take measurments for volitional spawned low pH vs. ambient conditioned juveniles (from Sam’s summer experiment) because I was short on time.

animal check

Var.low pH treatment

ambient offspring:

  • 2.1: uc?export=view&id=1UkfsMqngRvpS-pI_6NiJSbfIjqt31gGa
  • 2.2: uc?export=view&id=1MSc0ef1YU2-l-qHSu_yL4ZjLgoO3ck9L
  • 2.3: uc?export=view&id=1clcxYbysOFRWIXkEuihZ7oCdR096ns6z
  • 2.4: uc?export=view&id=1IuPASSPJu-E_V5NvA5ckz0IcxbJopABX
  • 2.5: uc?export=view&id=1pCy7T5QrEXDTXeA6eT89J5GLlQoEXOG_

var.low pH offspring:

  • 2.8: uc?export=view&id=1w5T8QNKAMCwAP8Uo0rV7unGO-4eJU9IK
  • 2.10: uc?export=view&id=1XwjldKpHXI_p819lHWV3G2-fCNLfRwnE
  • 2.11: uc?export=view&id=1ID8842QDCpE8KoX5jUixGDvKhCPCqDQy
  • 2.12: uc?export=view&id=1pYkWuzZNvGQsM_C_-VsgViBrQcyYtzit

Ambient pH treatment

ambient offspring:

  • 3.1: uc?export=view&id=1g3QhE1wvOglEh7RG_tdEnEkJcI8bkUbN
  • 3.2: uc?export=view&id=1JAgl0tJ-V2K-8rDHCEYpRIWGCISj9eF7
  • 3.3: uc?export=view&id=18l_z6f-SV4LLpEs17HydL4MG8Uzlhf7F
  • 3.4: uc?export=view&id=1r0IwPuTEG89OLgojbUqTUhDiSd78FyBf

var.low pH offspring:

  • 3.8: uc?export=view&id=1-06hRZHSO6kA0cO4mSsmgWw5_YNUoBZP
  • 3.9: uc?export=view&id=1QLqZNPZXreCWpG1N4Qmcm4V7PJ0AXg8K
  • 3.10: uc?export=view&id=1JYkir0REv1JbAIMRisHY5cDXi4T_KrpD
  • 3.11: uc?export=view&id=1t24OHP-BgSVlZv4_LoHHMNr_KR4vjUT9
  • 3.12: uc?export=view&id=1uq3AVN_bEjtbdHAwM-Jws5dVopMZAOTJ

Sequim bay animals

  • 3.7: 1 mort. uc?export=view&id=1G0z2F_GIWlgrEfBQlvsXkJlj7FAvWHkx

Volitional spawned low pH vs. ambient conditioned juveniles (from Sam’s summer experiment)

  • H1T1 (low pH): uc?export=view&id=1uUSjKDKEw2wUF5lCExPcUvXPw7sTSelu
  • H1T2 (amb): uc?export=view&id=1zwFeJFQNjbW_T7X7eHIzzJWLWoJRHgKQ

System status

This is how things looked right before I left:

Low pH setup

  • uc?export=view&id=17muQJn2be8GQymch_y5NaFRjR9VS2M-Z
  • uc?export=view&id=1f8qW_5pEH1UDOuUI8gJgRV2ewiboR4f4

Amb. pH setup

  • uc?export=view&id=1vMSrx2MpHjaFukX0f7SQhettJBIAdpd9
  • uc?export=view&id=1NtM--LeOIfnrnJYXORgOCkNYhm5XbrKS

Respirometry

SDR plate order

  • uc?export=view&id=10YEeIo1aC_rsQisTbiulpoCC2o6BBdr5

Size photos

  • uc?export=view&id=1V6rvj0KsWOcu3pLh-VlIcZ1CnizSwMMq
    • I accidentally smashed the third column first row animal when getting it out of the vial 😦
  • more size photos are here

Data and analysis

  • I haven’t processed this yet, but will post soon.

Overall observations

  • Survival seems to be OK despite the empty shells on the surface. This is more obvious now that the animals are visibly growing.
  • Animals reared in the low pH condition show clear areas on their shells where perhaps calcium has been removed. Photos from the animal check and from the respirometry size photos show this.
  • Low pH conditioned animals appear larger than ambient conditioned, but many ambient animals are beneath the surface so this needs to be more thoroughly measured (which I’ll do in a couple weeks after 2 total months of conditioning). Besides the shell clearing, the low pH animals seem to look better than the ambient animals, but again that observation is disregarding animals that are buried.
  • It’s interesting that the low pH condition has more live animals hanging out on the surface compared to the ambient condition.

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Sam’s Notebook: FastQC-MultiQC – C.bairdi RNAseq Day9-12-26 Infected-Uninfected

Ran the initial FastQC/MultiQC analysis on the C.bairdi RNAseq data we received earlier today.

Sam’s Notebook: Data Received – C.bairdi RNAseq Day9-12-26 Infected-Uninfected

Finally received the RNAseq data back from NWGC that we submitted on 20190521!

Here’s a table with the library names and the FastQ naming schemes.

NWGC Sample ID Investigator Sample ID
329772 D9_infected
329773 D9_uninfected
329774 D12_infected
329775 D12_uninfected
329776 D26_infected
329777 D26_uninfected