Table of Contents

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

In collaboration with the Seward Long-Term Observation Program (LTOP) (http://www.sfos.uaf.edu/sewardline/) and the NGA LTER Program (https://nga.lternet.edu/), we obtained Neocalanus flemingeri individuals during the annual April-May and September oceanographic cruises. During the spring cruises stage CV individuals were collected from four to six locations: spanning the inner shelf to outer shelf gradient along the Seward Line in the northern Gulf of Alaska and at least one station in adjoining Prince William Sound. Samples were collected using a CalVET net (53-µm mesh) towed vertically from 100 m depth to surface. Mixed plankton samples were immediately diluted with surface seawater, and maintained at ~5ºC prior to and during sorting. From each station actively swimming (healthy) N. flemingeri CVs were rapidly sorted under the microscope and preserved within 15 minutes to 1hr of the tow in RNALater Stabilization Reagent (QIAGEN). The rapid sorting of live plankton can lead to misidentifications given the presence of closely related congeners, thus species verification is recommended using the RNA-seq data to check the cytochrome c oxidase subunit 1 (mtCOI) sequence. In September, diapausing N. flemingeri adult females were sorted from collections obtained with a Midi Multinet towed vertically. Collections were diluted with seawater upon net retrieval. Individuals were sorted under the microscope from the 300-700 depth collections and either preserved immediately in RNALater or separated into holding containers for incubation experiments. In three years (2015, 2016, 2017) additional adult females were incubated for different lengths of time and preserved after a specified interval as indicated in the sample information. In July, 2019 we participated in a ship of opportunity cruise to the Gulf of Alaska Seamounts (SKQ201916S) and preserved diapausing adult females collected between 1000-2000 meters from two offshore stations (GAK19 and DeepQuinn).

RNA extraction, gene library preparation and RNA-seq

Total RNA was extracted from individual CV from each station using QIAGEN RNeasy Plus Mini Kit (catalog # 74134) in combination with a Qiashredder column (catalog # 79654) following the instructions of the manufacturer and stored at -80ºC. Total RNA concentration and quality were checked using an Agilent Model 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). For each station, total RNA from three of the ten individuals with high quality RNA yields were selected for RNA-seq and shipped on dry ice to the University of Georgia Genomics Facility (dna.uga.edu). There, double-stranded cDNA libraries were prepared from total RNA extracted using the Kapa Stranded mRNA-seq kit (KK8420) following manufacturer’s instructions. Briefly, RNA samples were first purified with two oligo-dT selection (polyA enrichment using oligodT beds), and then fragmented and reverse transcribed into double-stranded complementary DNA. Each sample was tagged with an indexed adapter and they were simultaneously paired-end sequenced (PE150 or PE75 bp) using an Illumina NextSeq 500 instrument using High-Output Flow Cell. Data in NCBI are the raw sequence reads.

Raw sequence reads have been deposited with links to BioProject accession numbers PRJNA324453, and PRJNA324453 in the NCBI (National Center for Biotechnology Information) BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).

To include BioProject, BioSample, and the SRA run accession to the submitted dataset, two tables from search results at NCBI were downloaded.

From a search at NCBI on the BioProject PRJNA324453 for the Adult Female dataset (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA324453), the results table of 175 rows was downloaded and named SraRunInfo_adult_females.csv for Adult Females. And results table of 204 rows for a search at NCBI on the BioProject PRJNA496596 for the CV dataset (https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA496596) was downloaded and named SraRunInfo_cv.csv.

The NCBI metadata table and submitted table were then processed with the BCO-DMO laminar tool.

Imported data from the submitted file CV-2015-2022-BCO-DMO.xlsx sheet name "Sheet 1 - sra_result" into laminar.

Imported data from the submitted file AdultFem-2015-2021-BCO-DMO.xlsx sheet name "Sheet 1 - sra_result (1)" into laminar.

Renamed parameter names according to BCO-DMO naming standards by replacing spaces with underscores.

A date column was added using the values of the existing year, month and day columns with the parameter name “Date” and format %Y-%m-%d.

Renamed the parameter Depth to Depth_Range to convey the parameter represents a range of depths.

Join the NCBI metadata file SraRunInfo_adult_females.csv onto the submitted adult female data file AdultFem-2015-2021-BCO-DMO.xlsx on the Experimental Accession SRX column.

Join the NCBI metadata file SraRunInfo_cv.csv onto the submitted CV data file CV-2015-2022-BCO-DMO.xlsx on the Experimental Accession SRX column.

These two joined tables were then combined together vertically to form the final dataset file named 922330_v1_rna_seq_flemingeri_cv_and_adult_female_2015-2022.csv.

Taxonomic names in the dataset were checked using the World Register of Marine Species (WoRMS) taxa match tool. All names matched accepted names exactly as of 2024-07-26. A supplemental file using taxonomy values from WoRMS was created and named species_list.csv and contains the following columns: ScientificName, AphiaID, LSID, Authority, Kingdom, Phylum, Class, Order, Family, Genus, Species.

NSF abstract:

The Gulf of Alaska supports a diverse and productive marine community that includes many commercially important fishes. Toward the base of this food web are small planktonic crustaceans that serve as the primary food source for many of these fish, as well as seabirds and marine mammals. The copepod Neocalanus flemingeri is one of these crustaceans, and it experiences rapid population growth during each spring's algal, or phytoplankton, bloom. An apparent mismatch between the presence of the youngest stages of the copepod, or nauplii, in early winter and the unpredictable timing of the spring phytoplankton bloom several months later raises important questions about when females reproduce and how this relates to survival and growth of nauplii. Two types of dormancy, diapause in adult females and physiological quiescence in nauplii, may be the key to the success of this copepod species. Timing and duration of the egg-laying period by adult females is linked to emergence from diapause. In addition, nauplii may enter a state of physiological quiescence while food resources are low, resuming growth after phytoplankton levels increase. This research will address a long-standing goal of biological oceanographers to understand dormancy and its role in controlling population cycles in marine copepods. It will use new technologies in molecular biology called transcriptomics to catalog the messages used by the cells to control copepod life processes, in this case those related to dormancy in adults and nauplii. Undergraduate students and a postdoctoral investigator will be trained in interdisciplinary research, and students from Native Hawaiian and Native Alaskan groups will be targeted for participation. Fishing is a major industry in the Gulf of Alaska, and outreach will focus on communicating the role copepods play in marine ecosystems. New content, including images, will be generated for existing websites: the Seward Line long-term observation program, the Alaska Ocean Observing System and the Gulf Watch Alaska Program.

Recruitment to the Neocalanus flemingeri spring population is dependent on successful emergence from diapause followed by reproduction, survival, and growth of the next generation. Individual-based models have made significant progress in predicting population growth in calanoid copepods using food, temperature, and advection as key environmental factors. Few of these models include predictors for naupliar recruitment, however, because little is known about this part of the life cycle given sampling difficulties and the lack of biomarkers to evaluate physiological state. This study will leverage existing monitoring efforts to track the N. flemingeri population during the winter and early spring. The research team will combine laboratory and field approaches to determine duration and synchronization of reproduction in emerging females and strategies for naupliar survival during low food conditions. Zooplankton samples will be processed to enumerate nauplii to species and to determine physiological condition of both nauplii and adult females. Gene expression studies will develop molecular markers for female dormancy and reproductive readiness and for naupliar growth and possible dormancy, which in turn will be used to evaluate field collected individuals. This will be the first comprehensive study to combine molecular and traditional tools to connect diapausing adults, naupliar production, and the resulting spring population of copepodites.

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.