# Morphometrics – Crassostrea gigas OA Selection Bags Using ImageJ

Due to some sort of data mis-handling, morphometric data that was previously taken for thousands (seriously, THOUSANDS) of Pacific oysters in 2014 was found to be incorrect. Unfortunately, there’s not enough of a “paper trail” to back track to see what/where things might have gone wrong to try to fix the issues. Essentially, they all had to be re-measured!

The one good thing is that all of the oysters were photographed at the time of sampling (along with a ruler), which allows us to go back and measure them.

I re-measured them all using the free imaging software ImageJ.

Oyster measurements taken were length and width. For length, the oyster was measured from hinge to the leading edge of the shell, attempting to measure as close to the theoretical center line of the oyster as possible, while also capturing the two points furthest from each other. The width was measured at the apparent widest part of the oyster and attempted to be perpendicular to the length measurement line.

Each image with the measurement lines was saved as a .tif file and the filename appended with “measured”. Additionally, each image produced a corresponding Excel file named CgOA_measurements_bag_info.xls, where “bag_info” contains information regarding the oysters in that set.

Images were measured by setting the pixel scale using 100mm (10cm) measurement on each image via the ruler in the image. Images were greatly enlarged when setting the scale to improve scale accuracy. Some images did not contain a ruler. Instead, the scale was set using the length of a weigh boat: 89mm (8.9cm). Weigh boat size was gathered from manufacturer specs: VWR Cat#89106-768 (8.9cm x 8.9cm x 2.5cm). Files corresponding to these sets of measurements are appended with “no_ruler” in the filename. The sample sets that were measured in this fashion were oyster bags:

• 492
• 530

The measured images and the individual Excel files were uploaded to the following Dropbox location: Dropbox/Friedman Lab/Carolyn Lab/Manuscripts/2016/Cg OA selection/Data/Sam DATA.

Data from the individual files was aggregated in the following spreadsheet in Dropbox: Dropbox/Friedman Lab/Carolyn Lab/Manuscripts/2016/Cg OA selection/Data/Sam DATA/files to merge/Cg OA selection 9mo sampling All 3 sites_survival data_ FOR SAM to add L and W data.xlsx

Data is still missing (i.e. no labelled image file was present) for the following oysters:

• 458 21-37
• 486 1-20
• 556 20-45
• 588 1-19

Here’s a quick summary of the amount of data I gathered. I’ll provide details of how I used ImageJ to not only measure the samples, but also create a more reproducible means of following the data acquisition process so that we can improve our ability to follow the “paper trail” from who acquired the data, how they acquired it, and allow people to easily review that data. This way, once all this data is transposed to some master spreadsheet, it will still be granularly accessible for any future troubleshooting that might be needed. I’ll do this in a separate post .

So, what did this work produce and how did I determine this information?

Using Bash (i.e. command line in Terminal):

Count the number of image files analyzed (i.e. saved) by ImageJ:

ls -1 *.tif | wc -l

163

Count the number of spreadsheet files produced by ImageJ:

ls -1 CgOA*.xls | wc -l

164

Well, there’s an odd discrepancy. These should be the same number. However, if anything were off, I’d expect the number of images to be greater in number than the Excel files. That would indicate I went through the measuring process, but neglected to save the data. However, this suggests that there’s an “extra” Excel file. It’s possible that I accidentally saved the image to a different location by accident. Will look into this…

Count the number of measurements taken. This will be a two step process.

First, aggregate all the data from the individual data files into a single file:

for i in CgOA*.xls; do awk 'NR>1' "$i" >> all_measures.csv; done The code above uses a for loop to look at each Excel file (files beginning with “CgOA” and ending with “.xls”). Each file ($i) is passed to the program awk, which concatenates/appends the contents of the file (excluding the header line; NR>1) to a new file (all_measures.csv).

Next, count the number of lines (i.e. measurements) in the all_measures.csv file:

wc -l < all_measures.csv

5251

Whoa! That’s pretty remarkable. Over 5000 individual measurements were recorded (length and width for each oyster). That means there were over 2600 oysters!!!

Hopefully we won’t have to re-measure these guys a third time!

# 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!

# 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:

# 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.

# 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.

# 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:

Here’re some pics of the day:

# 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:

# Spawning & Crossing – Crassostrea gigas at PSRF Manchester

Helped Dan initiate his experiment 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:

# Histology – Fixed Oyster Larvae Embedded in Agar

Oyster larvae were received 5/19/2014 in Davidson’s fixative. There were three different groups (A, B, and C), with each group consisting of 5 different tubes. Confirmed with Hamdi that the tubes in each group were just replicate sample collections from the same exact population of oyster larvae.

Made a 4% agar solution in distilled H2O and cooled to 56C.

Transferred 1mL of larvae in Davidson’s fixative from a single tube in each group to their own separate weigh boats. Weigh boats were angled to help pool the larvae solution. Mixed the larvae with an equal volume of 4% agar solution. Allowed mix to solidify. Transferred to the appropriate cassettes (labelled A, B, and C) with a sponge and stored in freshly made Davidson’s fixative solution.

Here’s an image of the interior box lid that explains what the A, B, and C group labelling means:

# qPCR – Colleen’s Oyster cDNA 2GE with OsHV NGS Primers

A sample that was not in the strip of cDNA tubes was not qPCRd in the last run (20110518). Ran that sample (labelled 2GE R 1/5), as well as positive control DNA (07-CB-719-3), 1GE (cDNA) and 3GE (cDNA). Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF)

Overall, samples look good (no signal in NTCs, clean melt curves, reps are good). Data has been sent to Colleen for analysis.