Sample collection and sorting
Copepods were collected in Prince William Sound, Alaska in the summer and fall of 2019 during the Northern Gulf of Alaska Long Term Ecological Research (NGA LTER) cruises (https://nga.lternet.edu/). Females “PWS2/June” were collected on June 30th, 2019 at the sampling site PWS2 (Latitude 60° 32.1’N; Longitude 147° 48.2’W) (R/V Sikuliaq, cruise number: SKQ201915S), and the “Pleiades/September” females were collected on September 12th and 13th, 2019 at PWS2 and near the Pleiades Islands (Latitude 60° 16.7’N; Longitude 147° 59.2’W) (M/V Tiglax, cruise number: TGX201909). Copepods were collected with a Midi MultiNet (0.25 m2 mouth area; 150 μm mesh nets) towed vertically from near the bottom to the surface at 0.5 m/sec (PWS2: 798 m; KIP2: 588 m). Upon retrieval, net samples were immediately diluted using filtered seawater collected from depth and kept between 4-6°C to minimize thermal stress. All females selected for the experiments were sorted under a dissecting microscope . Females were placed in groups of three into 750 mL Falcon tissue-culture flasks and incubated under dim light in an incubator for up to 4.5 weeks. Experimental temperatures were at or below deep-water temperatures in Prince William Sound (temperature settings: 4°C for June and 6°C for September). A subset of females was used in the DNA replication experiments; the remaining females were imaged for measurements of prosome length and lipid sac area.
Experimental design and timeline
For the timeline, two to four females were incubated in low concentrations of 5-Ethynyl-2’-deoxyuridine (EdU) for 24 hours at eight time points in June (Figure 2, 0-24, 24-48, 36-60, 72-96 hours and 2, 3, 4, 4.5 weeks), and at six time points in September (0-24, 24-48, 72-96 hours and 1, 2, 3 weeks) to track the numbers of cells with DNA replication in the ovary and oviducts from collection (diapause) to 4.5 weeks post-collection. Furthermore, after checking the females from the June experiment, three time points were added in the first 24 hours with shorter EdU incubation periods (0-3, 0-6, 0-14 hrs) to establish the start of DNA replication post-diapause. Prior to preservation and processing for confocal microscopy, females were examined by light microscopy for any visible 199 morphological changes and imaged for female size and lipid sac area measurements.
See the related dataset https://www.bco-dmo.org/dataset/907880 for the morphological changes and female size and lipid sac area measurements.
EdU protocol
The EdU incubations at the time points listed above were used to obtain a timeline of the formation of oocytes post-diapause. For each experimental time point two to four females were carefully pipetted out of the experimental flasks, imaged, and transferred into well plates with 2 ml of filtered seawater with 0.5 mg of EdU per copepod in June. This concentration was found to be high, and the EdU concentration was adjusted to decrease labeling brightness. Thus, in September, the concentration of EdU was decreased to 0.06 mg of EdU per copepod. The lower concentration improved viewing in the confocal microscope. Females were incubated in this solution for 24 hours except for the first three September time points (0-3, 0-6, 0-14 hrs). After the incubation, females were removed from the EdU, fixed in 4% paraformaldehyde in Sorensen’s Phosphate Buffer pH 7.2 (PB) and labeled using a ThermoFisher Click-iT EdU Alexa Fluor 594 Imaging Kit (catalog number: C10639) following the manufacturer’s instructions. Samples were washed for 15 minutes thrice in PB then in 0.5% Triton X-100 in PB for three 15-minute long permeabilization washes. EdU labeled cells were fluorescently tagged with Alexa Fluor 594 dye using a copper-catalyzed click reaction. Three additional 15-minute washes in PB were done before samples were stored in VECTASHIELD Antifade Mounting Medium containing DAPI, a nuclear DNA counter-label to EdU. Samples were stored at 4°C until mounting and imaging. Because DAPI in VECTASHIELD frequently did not permeate into the ovary, dilutions of VECTASHIELD with DAPI or Hoechst 3342 in phosphate-buffered saline were used to fully label the ovary prior to imaging on the confocal microscope.
