Materials and Methods

Copepod collections, transfer to laboratory and preservation

Neocalanus flemingeri adult females were collected in Prince Williams Sound during the fall oceanographic cruises of the Seward Line Long-term Observation Program (LTOP) (http://www.sfos.uaf.edu/sewardline/) and northern Gulf of Alaska Long-term Ecological Research Program (NGA LTER). Samples were collected between 700 and 400 m, using an opening and closing multiple plankton sampler (0.5 m2 cross-sectional area; 153 µm mesh nets; Multinet, Hydro-Bios) towed vertically from 700 m depth. Plankton collections were immediately diluted with deep seawater, and a set of N. flemingeri adult females were sorted within 15 min (T0) or within 45 min (Wk1) of net retrieval and preserved individually in microcentrifuge tubes in RNAlater Stabilization Reagent (QIAGEN). Additional females were stored in the cold and dark (5°C) prior to sorting and placed in incubation flasks (Falcon flasks, 750 ml) and maintained up to 9.5 weeks at 5-6°C before microscopic examination and preservation in RNAlater.

Labidocera madurae were live sorted from mixed zooplankton samples collected in Kane‘ohe Bay, Oahu, Hawaii with a zooplankton net (30 cm diameter, 123 µm mesh) towed horizontally subsurface from a slowly moving boat. Collections were immediately diluted in seawater and transported to the laboratory. Adult females and mixed copepodid stages (CIII-CV) were live sorted within six hours of collection and either preserved in RNALater, or immediately processed for total RNA extraction. Each sample consisted of a group of individuals.

RNA extraction, gene library preparation and RNASeq

Total RNA was extracted from individuals 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 -80°C. Total RNA concentration and quality were checked using an Agilent Model 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Samples of extracted total RNA from individuals or group of copepods were shipped on dry ice to the University of Georgia Genomics and Bioinformatics Core (GGBC) 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 cDNA. Each sample was tagged with an indexed adapter and paired-end sequenced (PE150 bp or PE75 bp) using an Illumina NextSeq 500 instrument using a High or Medium Output Flow Cells. Short-sequence reads (RNA-Seq) were submitted to the short read archive (SRA) database at the National Center for Biotechnology Information (NCBI) for public access (see BioProjects PRJNA324453 and PRJNA324849) .

Additional cruise data can be found at https://portal.aoos.org/ and https://nga.lternet.edu/.

Station information:

Gulf Of Alaska Stations:

PWS2 (Lat: 60° 32.1′N; Long: 147° 48.2′W)
KIP2 (Lat: 60°17'N; Long: 147° 59'W)

Hawai’i:
Kane‘ohe Bay, Oahu (Hawaii) (Lat: 21 ̊4’N; Long: 157 ̊7’W)