I spent the week at the Hatfield Center in Newport, OR. Mary Beth Rew Hicks and I dissected tissue from frozen age-0 cod that were collected between 2008 - 2024 near Kodiak, AK (Anton Larson Bay). The goal is to assess genetics through time in this area where beach seine surveying is conducted annually, and where they have been seeing shifts in size distributions since the marine heatwave era (from unimodal to bimodal).

Overview of methods

• Fish were pulled from the freezer by year. Bags thawed gradually in the fridge or on the benchtop / ice packs until they could be separated without breaking fish. When fish were frozen together in blocks they were submerged (still in plastic bags) in room-temp water. Within years, where possible, fish were selected somewhat deliberately to target big fish and little fish, and when frozen in blocks we attempted to select fish in the middle of the blocks to target fish with less chance of thaw-related degradation.
• Fish were individually measured (total length), weight whole (wet weight), then held by the fin and rinsed with ethanol and blotted dry with a kimwipe.
  • Exception: many fish collected from 2022 and 2023 were individually frozen in bags with IDs, in which case we did not re-measure them and used the total length / wet weight data collected in the field when they were caught. That data was manually transferred from the 2022/2023 spreadsheets to our data spreadsheet.
• Fish were then bisected at the tail. The head/gut portion was put back into individual cryo bags and labeled with their new MHW genetics ID (001-405, see data spreadsheet). The skin was peeled off the tail portion and about ~20 mg of muscle tissue was removed and placed into the lysis buffer from the DNeasy blood/tissue kit. Removing the skin decreased the likelihood of contamination from other fish preserved inside the same bag, and targeting tissue inside the fish decreased the likelihood of tissue degradation. We also did not want to touch the liver or other organs, in case folks want to go back and process those in the future.
• Small sections of fin clips preserved in ethanol were used for some additional samples to look at genetics associated with “ecotype” (deep vs. shallow caught juveniles) and for sex-assay development in Pacific cod and Walleye pollock. See separate tabs in the data spreadsheet.
• Between samples instruments were soaked in bleach solution, then rinsed with 2-3 rounds of DI water, and spritzed with ethanol.
• Tissues sat in lysis buffer for up to a few hours while fish were dissected, then proteinase K was added in groups (typically by year) and they incubated for 16-24 hours at 56C. After initial incubations lysates were vortexed and inspected. If tissue was still visually present additional proteinase K was added to those samples and they were returned to the incubator for 2-3 hours.
• Lysates were held in the fridge until extractions.
• We extracted four 96-well plates of DNA, two at a time (all on Friday) following the DNeasy kit.
• DNA concentrations were quantified for all samples using 10 uL.
• Duties: Mary Beth pulled bags from the freezer. Laura selected, measured, and rinsed fish. Mary Beth dissected fish. Laura added proteinase K and managed incubations. Laura and Mary Beth worked together to transfer lysates to 96 well-plates (Laura: plates 2, 4; Mary Beth: plates 1, 3). Mary Beth performed DNA rinses, centrifuge spins, and elutions. Laura prepped Qubit tubes, then both Laura and Marybeth transferred 10 uL of each sample to their respective qubit tubes.
• Mary Beth has the lab notebook with our sample and measurement data. I’ve digitized it here

Some daily notes:

Tuesday, January 14th

• We opted to do 2008, 2012, and 2013. 2008 fish didn’t look great, but 2006 fish looked much worse.
• We dissected 92 fish and stuck them into buffer+proteinase K. This included 45 each from 2013 and 2013 and 6 from 2008. They were stuck in 56C overnight.

Wednesday, January 15th

• In the morning we *added more proteinaseK+buffer to all 2008 samples (#90-#96), and samples 29, 30, 31, and 82 (those had gelatinous mass / sludge at bottom) and put them back in the incubator at 9am. Removed them at 12:00pm after 3 additional incubation hours.
• Prepped more tubes We continued sampling fish and added proteinase-K after each year was complete then into incubator,
• Samples 97-135 (all year 2008 fish) into incubator at 12pm.
  • NOTE: One fish from the next batch (year 2014, #136) went into incubator for lysis along with the 2008 fish.
• Samples 137-175 (year 2014) into incubator at 2:20pm. All 2014 fish were dissected.

Thursday January 16th

• Removed samples from incubator at 8:30am
• Added another 200uL buffer ATL + proteinaseK to two samples: 198 & 234, back in incubator at 9am
• Started on post heatwave samples. Decided to use muscle tissue from freezer fish for consistency and to preserve the fin clips for other needs.
• Many of the 2022 and 2023 fish were already measured in the field and placed into individual bags (labeled with an id, “BL####”). So we did not remeasure them but simply recorded their ID to get measurements from data sheets.
• We accidentally dissected some of the 2022 fish from Cook Bay. We did not extract that DNA. There will be some individually-bagged fish put back in the freezer. If needed by future studies we recorded their total lengths and wet weights.
• Ended day with fin clips from ecotype age-1 cod (deep and shallow), and males/females from pacific cod and walleye pollock

Friday January 17, 2025

• In the morning added 20ul more proteinase-K to all ecotype and male/female samples, returned to the incubator at 8:45am. Removed from the incubator at 11:30am.
• Extracted DNA with DNeasy kits. After adding Buffer AW2 and centrifuging (with tape on top) we let the plate sit at room temperature unsealed (with a kimwipe on top) to make sure ethanol fully evaporates
• Eluted with two 200 uL rinses of elution buffer to maximize yield.
• Prepared Qubit working solution:
  • 384 samples + 2 standards = 386 tubes
  • Working solution is already prepared in this qubit kit!
  • 190uL into each standard and sample tube
  • 10uL of each standard and each sample into designated tube

Number of samples and DNA concentration. All but 1 samples should have sufficient DNA quantities (>1,000 ng)

Year	Heatwave Status	Number Samples
2008	Before	45
2012	Before	44
2013	Before	46
2014	Heatwave	40
2016	Heatwave	46
2019	Heatwave	20
2021	Since	53 - already sequenced
2022	Since	44
2023	Since	46

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Length frequency distributions

I want to determine whether our sampled fish reflect the same size distributions found in all fish in each year. Here are charts for comparison. Left = all fish, right = sampled fish.

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Additional samples

Ecotype samples, age-1
Year	Ecotype	Number of samples
?	Deep	? 16 total
?	Shallow	? 16 total
Sex assay samples
Species	Sex	Number of samples
Pacific cod	Male	9
Pacific cod	Female	9
walleye pollock	Male	9
walleye pollock	Female	9

Ecotype

16 extracted for the “ecotype” question. Need to check in with Mary Beth on how many are from deep and shallow, I don’t believe we have 8 and 8 (not very many available for deep)

Sex assay

36 extracted for the sex assay, 18 each from Pacific cod and walleye pollock (9 males, 9 females from each)

For both the ecotype and sex assay samples, concentrations are lower than the muscle tissue samples taken for the MHW genetics. BUT DNA quantities are sufficient for sequencing (min. Is 500 ng, all samples have >1,000 ng). My guess is that tissue lysis could have been longer or with more enzyme.

Gel notes, from Mary Beth:

Made with SYBR Safe: this gel shows origin at the top but that’s not the best way so future images will have gel origina at the bottom. But all looks good in many of these samples. Possibly a dud from 2013 in sample 025.

This used “Safe stain” which I don’t like as much as the look of SYBR safe. The first ones here are from tube extractions so may have higher concentrations. Shows that 2006 concentration is much lower… Final three are from plate 4, including fin clips from a WP (known male, collected fresh and placed immediately in ethanol) and the last two from age-1 Pcod from August 2024 – again fresh fin clips immediately stored in EtOH. These three of course have nice large-MW bands.

2008 not looking great. 267 did not get loaded well onto the gel so take with grain of salt. Past 2010 things look promising.

This used “Safe stain” which I don’t like as much as the look of SYBR safe. The first ones here are from tube extractions so may have higher concentrations. Shows that 2006 concentration is much lower… Final three are from plate 4, including fin clips from a WP (known male, collected fresh and placed immediately in ethanol) and the last two from age-1 Pcod from August 2024 – again fresh fin clips immediately stored in EtOH. These three of course have nice large-MW bands.

SYBR safe