In this project, we exposed broodstock to PolyIC immersion right before spawning (2 h). This project tracks the offspring of these parents to test for immune priming parental effects. This is a brief overview of the project.
Oysters were spawned at Point Whitney using their production workflow. Spawning was conducting using pooled egg and sperm from Control and Treated broodstock using approximately equal number of parents contributing to each pool.
Larvae were reared in 10,000 L tanks (n=1 per treatment) at Point Whitney using their production workflow for the first three days. Larvae were fed (nano-pav and CC mix) and maintained using their production practices. At 3 days post fertilization, larvae were screened at 60 um and sampled for RNA/DNA. Larvae were then distributed into 1,000 L tanks (n=2-3 per treatment).
At 8 days post fertilization, larvae were dropped from each tank and screened onto 105 um screens and sampled for RNA/DNA. Growth rates and survivorship (>90%) for all larval groups were high. Food was increased as larvae grew. Larvae were returned to tanks (n=2-3 per treatment).
At 14 days post fertilization, larvae were dropped from each tank and screened onto 240 um screens and sampled for RNA/DNA. Growth rates and survivorship (>95%) for all larval groups were high! Larvae were prepared for shipment to Kona facility for setting in Hawaii and shipped.
Setting was conducted in Kona and treatment groups were kept separate. Larave were kept in Kona until 22 November 2024 when they were shipped back to Point Whitney. Setting was very high (>20-25%). Spat were then allocated to n=3 upwellers in the indoor system at Point Whitney. Control spat were stocked with 0.9L of spat per upweller; Treated spat were stocked with 0.5-0.75 L of spat per upweller.
On 4 December 2024, we evenly spread the density of spat in n=2 upwellers per treatment (0.9 L in all tanks). Two additional tanks contained the excess of control and treated mixed together. Spat were imaged for size.
Seed were held at Point Whitney Shellfish Hatchery until they were transported to University of Washington for experiments on 26 Feb 2025 and 22 Mar 2025. Seed were held in tanks with Instant Ocean seawater and fed with algal paste.
We performed the following measurements:
- Oyster growth using size images during rearing
- Metabolism measured via a resazurin assay (see Huffmyer et al. 2026 for the method)
- Survival under a range of temperatures in laboratory assays
- qPCR gene expression under and acute stress experiment
See the manuscript for details on responses measured and statistical methods.
polyIC-larvae/
├── data/ # Raw and processed input data
│ ├── acute-resazurin/ # Plate reader files from acute resazurin assays
│ ├── environmental/ # Temperature logger files and hatchery measurements
│ ├── growth/ # Shell length measurements (CSV + JPEG images)
│ ├── outplant/ # Field outplant data organized by site
│ │ ├── baywater/
│ │ ├── point_whitney/
│ │ └── sequim/
│ ├── qPCR/ # qPCR Cq values, CFX instrument files, and reports
│ ├── resazurin/ # Resazurin metabolic assay plate files and metadata
│ ├── rna-dna/ # RNA/DNA extraction records and sample metadata
│ └── survival/ # Acute thermal survival assay data
├── figures/ # Output figures organized by analysis type
│ ├── environmental/
│ ├── growth/
│ ├── outplant/
│ ├── qPCR/
│ ├── resazurin/
│ └── survival/
├── output/ # Derived data files and rendered analysis outputs
│ ├── qPCR/
│ └── resazurin/
├── protocols/ # Lab protocols
│ └── RNA-Extraction-Methods.docx
├── scripts/ # Analysis scripts (R Markdown, Quarto, Python)
│ ├── hatchery_environment.Rmd
│ ├── loggers.Rmd
│ ├── growth/
│ │ ├── growth.Rmd
│ │ └── growth.qmd
│ ├── outplant/
│ │ ├── growth_SequimBay.Rmd
│ │ ├── oyster_measurement_analysis.py
│ │ └── requirements.txt
│ ├── qPCR/
│ │ └── 01.00-polyIC-qPCR-analysis.Rmd
│ ├── resazurin/
│ │ ├── resazurin-analysis-batch2.Rmd
│ │ └── resazurin-testing.Rmd
│ └── survival/
│ └── survival-analysis.Rmd
└── polyIC-larvae.Rproj # RStudio project file
All R scripts should be run from the project root directory (i.e., with the working directory set to polyIC-larvae/). The easiest way to ensure this is to open polyIC-larvae.Rproj in RStudio before running any scripts. When knitting an R Markdown file, click the arrow next to the Knit button and select "Knit Directory: Project Directory".
scripts/hatchery_environment.Rmd
Reads daily hatchery water quality measurements (temperature, pH, salinity) from data/environmental/daily-measurements.xlsx and summarizes conditions by rearing period. Outputs a summary table.
scripts/loggers.Rmd
Reads raw Hobo Tidbit temperature logger files from data/environmental/loggers/ and joins them with logger metadata from data/environmental/loggers.xlsx. Plots temperature time series for three deployment contexts: Sequim Bay field outplant, Point Whitney hatchery tanks, and UW FTR incubation tanks. Saves figures to figures/environmental/.
Key R packages: tidyverse, readxl, lubridate, ggplot2, seacarb, cowplot
scripts/growth/growth.Rmd
Analyzes shell length data from data/growth/growth.csv. Plots oyster length over time by parental treatment (control vs. poly(I:C)-treated) with linear trend lines and mean ± SE summaries. Fits linear models per treatment and saves figures to figures/growth/.
Key R packages: tidyverse, ggplot2, readxl, lme4, lmerTest, emmeans, cowplot
scripts/survival/survival-analysis.Rmd
Analyzes acute thermal survival assay data from data/survival/survival_assays.csv. Oysters from control and treated families were exposed to elevated temperatures; mortality was scored at 0, 5, and 24 hours. Uses binomial mixed-effects logistic regression (glmer) with tank and batch as random effects. Includes overdispersion and zero-inflation diagnostics (DHARMa), post-hoc comparisons (emmeans), and model-predicted mortality plots. Saves figures to figures/survival/.
Key R packages: tidyverse, lme4, lmerTest, car, emmeans, DHARMa, ggeffects, ggplot2
scripts/resazurin/resazurin-analysis-batch2.Rmd (primary)
Analyzes resazurin fluorescence data from plate reader .txt files in data/resazurin/plate_files/batch2-seed/. Calculates metabolic rates (area under the curve) as a proxy for metabolic activity, joins with size and survival metadata, and tests for effects of parental treatment and temperature. Saves figures to figures/resazurin/ and derived data to output/resazurin/.
scripts/resazurin/resazurin-testing.Rmd
Exploratory and method-development script for the resazurin assay pipeline. Used to evaluate and validate the plate reader workflow before batch analyses.
Key R packages: tidyverse, readxl, ggplot2, lme4, lmerTest, emmeans, cowplot
scripts/qPCR/01.00-polyIC-qPCR-analysis.Rmd
Analyzes qPCR data from data/qPCR/ to examine gene expression differences between control and treated families. Reads Cq values, calculates delta-delta-Ct fold changes, and tests for differential expression. Saves figures to figures/qPCR/ and processed outputs to output/qPCR/.
Key R packages: tidyverse, readxl, ggplot2, lme4, emmeans
Outplant data are in progress and are not detailed in the manuscript.
scripts/outplant/growth_SequimBay.Rmd
Analyzes growth data from the Sequim Bay field outplant site in data/outplant/sequim/. Compares shell size between control and treated families over time at the field site. Saves figures to figures/outplant/.
Key R packages: tidyverse, readxl, ggplot2, lme4, emmeans
scripts/outplant/oyster_measurement_analysis.py
Python script that uses computer vision (OpenCV) to automatically detect and measure oysters in JPEG images from data/outplant/sequim/size/. Extracts length and width (in mm) for each detected oyster, annotates images with bounding boxes, and saves measurements to a CSV file (oyster_measurements.csv) in the same directory. This is a test script only for automated analyses.
-
Open the project — double-click
polyIC-larvae.Rprojin RStudio. This sets the working directory to the project root automatically. -
Install required packages — on first use, install any missing packages from the R console.
-
Set knit directory — before knitting any
.Rmdfile, click the arrow next to the Knit button and choose "Knit Directory: Project Directory". This ensures that relative paths likedata/survival/survival_assays.csvresolve correctly from the project root. -
Knit or run interactively — either knit the whole document to HTML, or run chunks interactively in the RStudio console. All outputs (figures, derived CSVs) are written relative to the project root.
- All data files use relative paths from the project root. Do not move individual scripts out of the repository without updating paths.
- Raw environmental logger files (
.xlsx) are indata/environmental/loggers/. Logger serial numbers are mapped to experimental groups viadata/environmental/loggers.xlsx. - Growth images (
.jpeg) indata/growth/are organized by date and tank. Measurements derived from these images are compiled indata/growth/growth.csv. - Resazurin plate files are
.txtexports from the FLx800 plate reader instrument. - RNA/DNA extraction records and sample metadata are in
data/rna-dna/and are used as inputs to the qPCR analysis.
protocols/RNA-Extraction-Methods.docx — detailed RNA extraction protocol used for larval and spat samples collected at 3, 8, and 14 days post-fertilization and at subsequent time points.
Note that samples collected using this protocol are not measured in this manuscript - the qPCR sampels were extracted using protocols described in the manuscript.