Tues. Sept. 24, Geoduck Broodstock Histology

This post is in reference to the histology done on the Fall-Winter 2018-2019 Broodstock conditioned in constant low pH. This is data analysis for the manuscript on pH effect on reproductive development

Past histology analysis

• Kaitlin’s staging data

• kaitlyn’s March 6 histology post

• pictures are here: http://owl.fish.washington.edu/hesperornis/P_generosa/

  • Kaitlyn will upload the missing data from 20190221 (038,042,053,056,057,059,and 060) to owl

Scoring females with imageJ

• went through all pictures and separated female images from male images
  • removed all black background in preview using wand tool
    • images with black background removed are here: https://github.com/shellywanamaker/P_generosa/tree/master/amb_v_constantlow_brood_data/histology_scoring/img/black_removed
• downloaded ImageJ2 (a.k.a. fiji)
• protocol:
  1. open image file
  2. get total image area
     • draw a circle around the imaged area then go to analyze -> measure
  3. get image area that is follicles only
  4. get image area that is eggs only
• for steps 3 and 4 I followed this demonstration
  • go to image -> adjust -> color threshold and adjust the hue, saturation, and brightness so that only follicles are highlighted or only eggs are highlighted (i found swithching back and forth between red and black helped). Then click select (areas should be highlighted now in yellow). Then go to analyze -> measure.
  • areas selected are shown here: https://github.com/shellywanamaker/P_generosa/tree/master/amb_v_constantlow_brood_data/histology_scoring/img/selected_area
• imageJ measurements are here: Female_area_measurements.csv
• propagated this excel spreadsheet with the measurements: HistologyScores.xlsx

Analysis of scores

• Did a quick analysis of the scores in R
• markdown here:pHFemaleBroodstockHistoAnalysis.md
  • rmarkdown here: pHFemaleBroodstockHistoAnalysis.Rmd
  • rproj here: histology_scoring.Rproj
• CONCLUSIONS:
  • low pH females showed less area taken up by follicles and eggs indicated low pH may lead to reduced reproductive development, however the difference was not significant