Table of Contents

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

These data are further described in the following publications:

Hartline, D. K., Cieslak, M. C., Castelfranco, A. M., Lieberman, B., Roncalli, V., & Lenz, P. H. (2023). De novo transcriptomes of six calanoid copepods (Crustacea): a resource for the discovery of novel genes. Scientific Data, 10(1). https://doi.org/10.1038/s41597-023-02130-1

Roncalli, V., Niestroy, J., Cieslak, M. C., Castelfranco, A. M., Hopcroft, R. R., & Lenz, P. H. (2022). Physiological acclimatization in high‐latitude zooplankton. Molecular Ecology, 31(6), 1753–1765. Portico. https://doi.org/10.1111/mec.16354

Roncalli, V., Cieslak, M. C., Germano, M., Hopcroft, R. R., & Lenz, P. H. (2019). Regional heterogeneity impacts gene expression in the subarctic zooplankter Neocalanus flemingeri in the northern Gulf of Alaska. Communications Biology, 2(1). https://doi.org/10.1038/s42003-019-0565-5

Sample collection: Zooplankton were collected from depth (2015, 2017, 2018, and 2019) at two stations in Prince William Sound: “PWS2” (Lat: 60°32′. N, Long: -147°48.2′ W) and “PWS3” (Lat: 60°40.0′ N, Long: -147°40.0′ W,) and the Gulf station “GAK1” (Lat: 59º50.7′ N, Long: -149º28′ W). Collection date, station and depth stratum for each individual are given in Hartline et al. (2023) and Roncalli et al. (2019). Zooplankton collections were made using vertical net tows with either a QuadNet with two 150 µm and two 53 µm mesh nets (April and May collections), or a multiple opening and closing plankton net (0.25 m2 cross-sectional area; 150 μm mesh nets; Multinet-Midi, Hydro-Bios; September collections). Zooplankton samples were diluted, and copepods were sorted under a dissection microscope to select individuals from the target species. Briefly, live and undamaged individuals were identified and staged using morphological criteria and preserved in RNALater Stabilization Reagent. Preserved copepods were frozen first in -20ºC during the cruises, and then transferred to −80°C until further processing. Species identification were confirmed through the COI sequence in the assembled transcriptomes.

Total RNA extraction, library construction, RNA sequencing and quality control: For each target species, total RNA was extracted from individuals using QIAGEN RNeasy Plus Mini Kit (catalog # 74134) in combination with a Qiashredder column (catalog # 79654). Selection for sequencing was based on high RNA yields and purity of extraction (RIN>8). The final list included pre-adults (CV) for Neocalanus flemingeri (n=3), Neocalanus cristatus (n=1), Calanus marshallae (n=2), Eucalanus bungii (n=1), an adult male (developmental stage CVI) for Neocalanus plumchrus (n=1) and an adult female for Metridia pacifica (n=1). Total RNA was shipped on dry ice to the Georgia Genomics Bioinformatics Core (https://dna.uga.edu) for RNA-Seq. There, double-stranded cDNA libraries (KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads (cat #KK8421]) from each individual were multiplexed and sequenced using an Illumina Next-Seq 500 instrument (High-Output Flow Cell, 150 bp, paired end). Quality of each RNA-Seq library was reviewed with the FastQC software28. From each RNA-Seq library, low quality reads were removed using FASTQ Toolkit (v. 2.2.5 within BaseSpace). Illumina adaptors, reads <50 bp long, reads with an average Phred score <30 and the first 12 bp from each read, were removed from each library. The same workflow was applied to all nine datasets.

De novo assembly, mapping, core-gene statistics: Individual de novo transcriptomes were generated from each RNA-Seq dataset at the National Center for Genome Analysis Support's (NCGAS; Indiana University, Bloomington, IN, USA) Mason Linux cluster using Trinity software (v. 2.4.0, except N. plumchrus, v. 2.0.6). Initial evaluation involved self-mapping of reads against the respective de novo assembly using Bowtie2 software (v. 2.3.5.1). Completeness of each de novo assembly was evaluated using Benchmarking Universal Single-Copy Orthologs (BUSCO) software31 by searching each assembly for the presence of eukaryote “core” genes using the Arthropoda database as reference (BUSCO version 5.3.2, dataset: arthropoda_odb10 (2020-09-10, 90 genomes, 1,013 BUSCOs). RNA-Seq data and transcriptome shotgun assemblies (TSAs) have been deposited with links to BioProject accession numbers PRJNA496596, and PRJNA662858 in the NCBI (National Center for Biotechnology Information) BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/).

Functional annotation: Assemblies were functionally annotated against the NCBI Swiss-Prot protein and UniProt databases. Initial annotations were obtained by using the BLASTx algorithm on a local BLAST webserver with a Beowulf cluster using the Swiss-Prot protein database (downloaded February 2021) as reference and a threshold E-value of 10-5. Transcripts with BLAST annotations were then searched against the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases using UniProt.

Processing steps for submitted annotated individual transcriptome files and metadata

The following are the submitted annotated transcriptome files that are processed to include metadata information and then concatenated together.

Filename: n-flem_CV2015_GAK1_AccAnnots_multispp_Aug23_q-fix-2.csv
Description: Neocalanus flemingeri 2015 stage CV annotated transcriptome

Filename: Nf2018_CV_Acc&Annot-single-rev_quoted.csv
Description: Neocalanus flemingeri 2018 stage CV annotated transcriptome

Filename: Nf2019_CV-PWS2_Acc&Annot.csv
Description: Neocalanus flemingeri 2019 stage CV annotated transcriptome

Filename: Np2015_maleR1_Acc&Annot.csv
Description: Neocalanus plumchrus 2015 stage Adult Male annotated transcriptome

Filename: Nc2017_CV_Acc&Annot.csv
Description: Neocalanus cristatus 2017 stage CV annotated transcriptome

Filename: Cm2017_CV_Acc&Annot.csv
Description: Calanus marshallae 2017 stage CV annotated transcriptome

Filename: Cm2018_CV_Acc&Annot.csv
Description: Calanus marshallae 2018 stage CV annotated transcriptome

Filename: Eb2017_CV_Acc&Annot.csv
Description: Eucalanus bungii 2017 stage CV annotated transcriptome

Filename: Mp2017_AF_Acc&Annot.csv
Description: Metridia pacifica 2017 stage Adult Female annotated transcriptome

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A metadata table with NCBI accession numbers was created using information from the submitter and by gathering accession numbers from NCBI for each BioProject.

Metadata from Submitter via email:
File Name: Summary Table for the Annotated Transcriptomes in BCO.docx
Title: Summary table from Lenz of metadata for transcriptome annotation files

Table columns
Species, Annot Filename, Stage/Collection information (which includes the Life Stage, Sex if indicated, Collection date, station, and collection depth range in meters), and NCBI TSA#

The accession number GHLB01000000 was changed to GHLB00000000 because the TSA project records accessions begin with a four-letter prefix of the TSA project followed by eight zeroes. A TSA project master accession (GenBank accession value) adds an extension of '.1' which indicates the version.

This explains it further:
See this 2013 article from NCBI on the definition of the TSA master accession number: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531190/

TSA projects will now contain a master record, in addition to records representing each of the assembled contigs. TSA will be using a similar accession number scheme to WGS as well. Like WGS accessions, the new TSA accessions have a four-letter prefix, representing the TSA project, followed by a two-digit version number and a six-digit contig number. For example, GAAA01000020 is contig 20 from the first version of TSA project GAAA. The TSA project master records have accessions that begin with the four-letter prefix followed by eight zeroes (e.g. GAAA00000000) and are indexed in the Nucleotide database.

A metadata table was created including metadata from the submitter:
File name: transcriptome_annotations_metadata_table.csv
Title: DM created metadata table of supporting information for each transcriptome annotation

Station lat and lon values from the 'Methods & Sampling' section of the submission were converted to decimal degrees with 6 digit precision.

A column named TSA_project_accession, with project accession numbers but without the version extension, was included to join the metadata table with the annotated transcriptome files later in processing, and it is removed in the final metadata table.

A column named TSA_Master_Accession which is the TSA_project_accession number with the version extension of '.1'.

From the National Center for Biotechnology Information (NCBI), various accession numbers and titles were added for each annotated transcriptome file.

Table columns:
Species, Station, Latitude, Longitude, Collection_date, Depth_range, Life_stage, Sex, BioProject, TSA_project_accession, TSA_Master_Accession, Study_Accession, Study_Title, Experiment_Accession, Experiment_Title, SRA_Accession, BioSample, Sample_Accession

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1) Used the BCO-DMO data processor laminar, to load in the submitted annotated transcriptomes files and the DM created metadata table. The fill value '#N/A' which stands for 'no result found' was kept.

2) Created a new column for each annotated transcriptome file, TSA_project_accession, to join on the metadata table using regular expressions to extract the first 4 letters of each TSA accession number and then concatenate 8 zeros. This is the NCBI format for a TSA project accession number.

3) Joined each annotated transcriptome file with the metadata table on the key TSA_project_accession.

4) Removed the parameter TSA_project_accession, since it was only used for the Join process. The TSA project accession number with the version will be recorded as the parameter TSA_master_accession.

5) Added a suffix ‘.1’ to each TSA accession number to indicate version 1.

6) Renamed Accession# to Genbank_accession which is the TSA accession value with a version extension.

7) Renamed parameters in each annotated transcriptome file by removing commas and parentheses, and replacing spaces with underscores to follow BCO-DMO parameter naming conventions.

8) Reordered the parameters so that experiment metadata parameters are at the beginning of each annotated transcriptome file and most NCBI accession numbers are at the end

9) Using a second laminar processor pipeline, loaded in the individual annotated transcriptomes files that were created by laminar processing above and concatenated all the individual annotated transcriptome files into the primary dataset file.

10) Saved the output of the laminar processing to the supplemental files of the metadata table and the individual transcriptome annotation files.

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Created a Species WoRMS taxonomy file species_list.csv by using the WoRMS website to create a table of the species name and their corresponding AphiaID and LSID values.

Checked that all the species names in the transcriptome annotation files matched those found in WoRMS.

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.