A transcriptome resource for the copepod Calanus glacialis across a range of culture temperatures

The copepod Calanus glacialis plays a key role in the Arctic pelagic ecosystem. Despite its ecological importance and ongoing climate changes, limited knowledge at the genomic level has hindered the understanding of the molecular processes underlying environmental stress responses and ecological adaptation. Transcriptome data was generated from an experiment with C. glacialis copepodite (CV) subjected to five different temperatures. We obtained a total of 512,352 high-quality 454 pyrosequencing reads, which were assembled into 55,562 contigs distributed in 128 KEGG pathways. Functional analysis revealed numerous genes related to diverse biological functions and processes, including members of all major conserved signaling pathways. Comparative analysis of acclimated individuals to experimental temperatures has provided information about gene variations observed in several pathways (e.g. genes involved in energy, lipid and amino acid metabolism were shown to be down-regulated with increasing temperatures). These mRNA sequence resources will facilitate further studies on genomics and physiology-driven molecular processes in C. glacialis and related species.


Introduction
Climate change is dramatically affecting Arctic ecosystems, causing changes in oceanic circulation, sea ice loss and temperature increases that may alter marine community structure (e.g., demographic traits, spatial range, biological interactions) and ecosystem function (Post et al., 2009;Slagstad et al., 2011). The calanoid copepode Calanus glacialis plays a major role in the trophodynamics of Arctic pelagic ecosystems and is the dominant species of the genus in the northern Barents Sea (Tande, 1991). Warming of the Arctic is predicted to induce a possible replacement of C. glacialis by its boreal sibling Calanus finmarchicus (Reygondeau and Beaugrand, 2011;Weydmann et al., 2014a). Consequently, it is essential to understand how climate change might affect the biogeography and population dynamics of C. glacialis, and to predict the response and adaptability of the species to environmental fluctuations . In an effort to provide comprehensive genomic resources for C. glacialis and a baseline for future physiological studies, we have used Roche 454 pyrosequencing technology to characterize the temperature responsive transcriptome of this species.

Sample collection and temperature experiment
Mesozooplankton samples were collected in the Barents Sea, NE of the Hopen Island (77°08.6′N 28°11.0′E; average water temperature −0.6°C), with vertical tows using a WP-2 net (0.25 m-2 opening; 0.2 mm mesh size; with a large non-filtrating cod end) in June 2009. Sixty C. glacialis copepodites at the 5th stage (CV) were gently picked and randomly assigned to 6 groups of 10 individuals. One of these, representing natural conditions (NAT), was immediately frozen in liquid nitrogen and stored at −80°C. The other 5 groups were placed in flasks (200 ml) filled with filtered seawater and placed in a laboratory cooler (type CHL 1 B) at 0°C. After 36 h of incubation all but one of the groups were transferred to a second cooler at 2.5°C. This process was repeated with 2.5°C increments every 36 h. At the end of the experiment (204 h) the individuals incubated at 0°C (T0), 2.5°C (T2.5), 5°C (T5), 7.5°C (T7.5) and 10°C (T10) were flash frozen in liquid nitrogen and stored at − 80°C. See Supplementary methods for RNA preparation, cDNA synthesis and pyrosequencing.

Bioinformatic analysis
Sequence quality-filtering, assembly and annotation were performed essentially as described in Martins et al. (2013). An overview Marine Genomics 23 (2015)  . Highly represented domains, as determined by the total number of reads (N1000) mapping to the domain, were associated with cytoskeletal-related proteins and essential cell functions including energy production (glyceraldehyde 3-phosphate dehydrogenase, ATP synthase, and NADH dehydrogenase), metabolite transport (mitochondrial carrier, sugar transport and lipocalin), fatty acid biosynthesis (fatty acid desaturase), lipid catabolism (Acyl-coA dehydrogenase), cell differentiation (Ras family), protein synthesis (ribosomal genes), and signal transduction and transcription regulation (protein kinases, protein tyrosine kinases, WD40). Additionally, numerous abundant transcripts were involved in the cellular stress response; redox, antioxidant reactions and stress-related processes (cytochrome P450, glutathione S-transferase, NADH ubiquitone, thioredoxin and heat shock proteins-HSP70, HSP90, HSP40). Several potential homologues belonging to the major conserved animal signaling pathways were also identified (e.g. Wnt, Notch, Hedgehog, TGF-, JAK-STAT and MAPK; Pires-da Silva and Sommer, 2003). Overall response to temperature of metabolic and regulatory pathways (R statistics using IDEG6, significant threshold of 0.05, corrected for multiple tests using the False Discovery Rate, FDR b 0.1; Romualdi et al., 2003;Stekel et al., 2000) showed different regulation mechanisms and a patchwork of up-and downregulated steps in some KEGG pathways was observed (Table 2). Furthermore, we tested a subset of simple sequence repeat (SSR) types and nine polymorphic microsatellites were suitable for population genetic studies as described in Weydmann et al., 2014b. In conclusion, we performed de novo transcriptome sequencing of C. glacialis incubated at increasing temperatures representing realistic warming scenarios. This pyrosequencing effort provides clues to the identification of genes potentially involved in temperature responses and generates essential molecular tools that will be useful in further genetic and genomic studies of this species.

Data deposition
The 454 sequence reads of C. glacialis were submitted to NCBI Short Read Archive (SRA) under the accession number SRP053198. The assembled transcriptome data were deposited in the European Nucleotide Archive (accession numbers HACJ01000001-HACJ01054344). a Median length (N50)-620. b E-value ≤ 1e −6 .

Table 2
Selected list of KEGG biochemical mappings for C. glacialis transcriptome data and functional annotation of potential up-and down-regulated genes showing significant differential expression in the temperature experiment.
KEGG pathway Pathway ID No of annotated enzymes Stress regulation a