Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

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.

Confocal imaging and quantification of cell division Females were mounted in VECTASHIELD with DAPI with their left lateral side facing up except for three individuals that were mounted dorsally. Samples were imaged using a Leica SP8 X Confocal Laser Scanning microscope with a × 20 glycerol immersion lens and a white light laser. DAPI has an excitation peak of 359 nm and emission peak of 457 nm, while Alexa fluor 594 has an excitation peak of 590 nm and an emission peak of 617 nm. Imaging was optimized through gating out of some wavelengths to decrease background and autofluorescence. Samples were imaged by tile scanning through each copepod to locate the entire ovary. Z-stack sections were 1.04 μm apart to ensure that no cells were missed due to large imaging gaps. Whole-mount females were imaged from the start of the ovary and oviducts until the depth at which resolution was lost due to insufficient laser penetration. Using Leica’s merge software, multiple regions with individual z-stacks were merged to form a single z-stack of an ovary larger than the lens’ field of view.

Add Latitude and Longitude columns with values from the “Methods and Sampling” section of the dataset page. Convert lat and lon to decimal degrees with 4 digit precision.

PWS2 lat 60.535 and lon -147.8033
Pleiades Lat 60.2783 and lon -147.9867

Remove blank column at the end of the file

Rename the column ‘Sample ID’ to EdU_sample_ID_number to match the column naming in a related dataset: Post-diapause Neocalanus flemingeri females morphometric measurements and calculations of lipid fullness and lipid volume taken from the R/V Sikuliaq and the R/V Tiglax in the Gulf of Alaska, sub-arctic Pacific from 2019-06-30 to 2019-09-13

Rename the column ‘Incubation length in hours’ to Length_of_EdU_incubation_in_hours to match the column naming in a related dataset: Post-diapause Neocalanus flemingeri females morphometric measurements and calculations of lipid fullness and lipid volume taken from the R/V Sikuliaq and the R/V Tiglax in the Gulf of Alaska, sub-arctic Pacific from 2019-06-30 to 2019-09-13

Rename the column ‘Amount of EdU given in mg’ to ‘Amount_of_EdU’ to remove the units from the header name since the units are noted in the parameters section of the dataset page.

Replace spaces in column names with underscores.

Capitalize the first letter of each full month name in the Collection_month column.

Reformat dates from %m/%d/%Y to %Y-%m-%d

Reorder the fields so that the sample collection information is at the front (Station, Latitude, Longitude, Date_collected, Collection_month), followed by columns about EdU incubation (EdU_sample_ID_number, Amount_of_EdU, Incubation_date_at_start, Length_of_EdU_incubation_in_hours,). And then the results columns (Time_point_in_days and Number_of_cells_replicating).

NSF Award Abstract:
The sub-arctic Pacific sustains major fisheries with nearly all commercially important species depending either directly or indirectly on lipid-rich copepods (Neocalanus flemingeri, Neocalanus plumchrus, Neocalanus cristatus and Calanus marshallae). In turn, these species depend on a short-lived spring algal bloom for growth and the accumulation of lipid stores in order to complete an annual life cycle that includes a period of dormancy. The intellectual thrust of this project measures how the timing and magnitude of algal blooms affect preparation for dormancy using a combination of field and experimental observations. The Northern Gulf of Alaska - with four calanid species that experience dormancy, steep environmental gradients, well-described phytoplankton bloom dynamics, and a concurrent NSF-LTER program - provides an unusual opportunity to identify the factors that affect dormancy preparation. Education and outreach plans are integrated with the research. Educational efforts focus on interdisciplinary opportunities for undergraduate, graduate and post-doctoral trainees. The project will generate content for existing graduate and undergraduate courses. U. of Alaska Fairbanks and U. Hawaii at Manoa are Alaska Native and Native Hawaiian Serving Institutions, and students from these groups will be recruited to participate in the project. Because fishing is a major industry in the Gulf of Alaska, outreach will communicate the role copepods play in marine ecosystems using the concept of a dynamic food web tied to production cycles.

Diapause (dormancy) and the accompanying accumulation of lipids in copepods have been identified as key drivers in high latitude ecosystems that support economically important fisheries, including those of the Gulf of Alaska. While the disappearance of lipid-rich copepods has been linked to severe declines in fish stocks, little is known about the environmental conditions that are required for the successful completion of the copepod's life cycle. A physiological profiling approach that measures relative gene expression will be used to test two alternative hypotheses: the lipid accumulation window hypothesis, which holds that individuals enter diapause only after they have accumulated sufficient lipid stores, and the developmental program hypothesis, which holds that once the diapause program is activated, progression occurs independent of lipid accumulation. The specific objectives are: 1) determine the effect of food levels during N. flemingeri copepodite stages on progression towards diapause using multiple physiological and developmental markers; 2) characterize the seasonal changes in the physiological profile of N. flemingeri across environmental gradients and across years; 3) compare physiological profiles across co-occurring calanid species (N. flemingeri, Neocalanus plumchrus, Neocalanus cristatus and Calanus marshallae); and 4) estimate the reproductive potential of the overwintering populations of N. flemingeri. The broader scientific significance includes the acquisition of new genomic data and molecular resources that will be made publicly available through established data repositories, and the development of new tools for routinely obtaining physiological profiles of copepods.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NOTE: Petra Lenz is a former Principal Investigator (PI) and Andrew Christie is a former Co-Principal Investigator (Co-PI) on this project (award #1756767). Daniel Hartline is the PI listed for the award #1756767 and is now a former Co-PI on this project.