Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

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Journal of Experimental Marine Biology and Ecology

j ourna l homepage: www.e lsev ie r .com/ locate / jembe

Gene expression of the marine copepod Calanus finmarchicus: Responses tosmall-scale environmental variation in the Gulf of Maine (NW Atlantic Ocean)

E. Unal a,⁎, A. Bucklin a, P.H. Lenz b, D.W. Towle c,1

a Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Rd., Groton, CT 06340, USAb Pacific Biosciences Research Center, University of Hawaii at Manoa, Honolulu, HI 96822, USAc Center for Marine Functional Genomics, Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA

⁎ Corresponding author at: Department of Marine Scien1080 Shennecossett Rd., Groton, CT 06340, USA. Fax: +1

E-mail address: ebru.unal@uconn.edu (E. Unal).1 Deceased.

0022-0981/$ – see front matter. Published by Elsevier Bhttp://dx.doi.org/10.1016/j.jembe.2013.04.020

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 September 2012Received in revised form 26 April 2013Accepted 27 April 2013Available online 2 June 2013

Keywords:Calanus finmarchicusCopepodDNA microarrayFunctional genomicsGene expressionGulf of Maine

The pelagic copepod Calanus finmarchicus is one of the most important zooplankton species in the North At-lantic Ocean. Despite its ecological importance and pivotal role in the food chain, the molecular mechanismsunderlying this species' complex life history (ontogenetic development, reproduction, molting, and dia-pause) and physiology (digestion, neural processes, and membrane physiology) have remained poorly char-acterized. This study examined differential expression of nearly 1000 genes, selected based on physiologicalfunction and hypothesized ecological importance, for C. finmarchicus collected in the Gulf of Maine (NW At-lantic Ocean) using an oligonucleotide DNA microarray. Replicate analyses compared adult females andfinal-stage juveniles (Copepodite-V stages) collected from surface (0–30 m) and deep (130–170 m) layersduring April, 2008. Differentially expressed genes were identified by statistical analysis of multiple replicates,including a control for False Discovery Rate (FDR). Functional relationships of selected genes and/or proteinswere evaluated using the Gene Ontology Enrichment Analysis Software Toolkit (GOEAST). Genes involved inprotein synthesis, cell-cycle and tissue buildup were shown to be up-regulated (i.e., significantly higher ex-pression levels) in deep females and juveniles; genes related to protein turnover, cellular homeostasis, activ-ity, and stress/immunity responses were up-regulated in surface females. Additional functional analysesusing KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated up-regulation ofgenes encoding enzymes related to energy metabolism and osmoregulation in surface females, and citricacid cycle and anaerobic metabolism in deep females and juveniles. Overall, an active physiological statewas pronounced in surface females, and a number of processes related with emergence from diapausewere pronounced in deep females and juveniles.

Published by Elsevier B.V.

1. Introduction

1.1. Ecology and physiology of Calanus finmarchicus

The planktonic copepod C. finmarchicus (Crustacea, Copepoda) is anabundant and ecologically important species in the N Atlantic Oceanand the Barents Sea. The species plays a significant role in the pelagicfood web as a secondary producer transferring energy from primaryproducers to higher trophic levels. It is an important prey of the juvenilestages of ecologically important fish, including mackerel and herring(Prokopchuk and Sentyabov, 2006). The species follows a Subarctic bio-geographical distribution and is found from the New England shelf tothe Greenland and Barents Seas, with a latitudinal range from 40°N to80°N (Bryant et al., 1998).

ces, University of Connecticut,860 405 9153.

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C. finmarchicus is an especially important species in the pelagic as-semblage of the Gulf of Maine, where it may contribute up to 70% oftotal copepod biomass in spring and early summer and dominate zoo-plankton secondary production during spring in most years (Planqueand Batten, 2000). The Gulf of Maine is a semi-enclosed continentalshelf sea bounded by Cape Cod, Georges Bank, and southwesternNova Scotia (Fig. 1). The bathymetry of the Gulf of Maine is complex,with three deep basins, (Georges, Jordan, and Wilkinson) that exhibithighly-reduced exchange of waters deeper than 200 m (Townsendet al., 2005).

The complex life history of C. finmarchicus has been extensivelystudied in the Gulf of Maine region, most recently as a part of the USGLOBEC program (Wiebe et al., 2001). Individuals pass through six lar-val (naupliar) and six juvenile (copepodite) stages. Naupliar stages N1andN2do not feed, while later naupliar stages and all copepodite stagesfeed primarily on phytoplankton. In the southern Gulf of Maine,C. finmarchicus reproduction is closely tied to annual phytoplanktonproduction cycle, beginning in early January (Durbin et al., 2000).After reaching CV stage, a portion of the population descends to deepwaters in order to initiate diapause, while the remaining CV population

Fig. 1. Location of the Gulf of Maine in the Northwest Atlantic Ocean, showing the sampling station for the Calanus finmarchicus microarray analysis in Wilkinson Basin with a star.Vertically-stratified plankton tows were taken at station WB7 on April 17, 2008 (see Methods for more explanation).

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molt into adults, reproduce and spawn another generation (Hirche,1996). Diapause is an arrested state of development at the CV stagewith reduced metabolism during the late summer and autumn, whichmay serve as a means of surviving periods of low food abundance(Hirche, 1996). Copepodites accumulate lipid reserves throughout thesummer, which may enhance survival during diapause (Hirche, 1996;Miller and Ambrose, 2000) and provide energy to fuel gonad matura-tion, molting and reproduction in early spring (Rey-Rassat et al.,2002). More recently, a conceptual model of the possible role of lipids(in particular wax ester composition) in influencing diapause patterns,determining the overwintering depth, and population adaptations tothe hydrological conditions have been proposed (Irigoien, 2004; Pondet al., 2012).

C. finmarchicus appears to initiate diapause in both neritic and oce-anic waters of the North Atlantic, where diapausing populations canbe found within 50–100 m of the bottom in neritic environmentsand 500–1500 m in oceanic waters (Heath et al., 2004; Miller et al.,1991).

In the Gulf of Maine, C. finmarchicus populations emerge from dia-pause during winter and early spring, molt into adults at depth, and as-cend to surface waters. Two or three generations may be producedduring January–June in the Gulf of Maine, with a considerable overlapof generations (Durbin et al., 2000; Miller et al., 2000). The vertical dis-tribution of C. finmarchicus is known to be bimodal in the Gulf of Maineduring fall andwinter; the depth of highest abundance of diapausing in-dividuals has been reported to be below the cold intermediate layer,close to 150 m depth (Durbin et al., 1997; Johnson et al., 2006).

Diapause includes a number of phases, with its initiation consistingof preparatory and induction phases, a refractory phase, and activationand termination phases (Hirche, 1996). In insects, the activation andtermination phases are characterized by a reactivation of the endo-crine system and its resultant increases in DNA/RNA synthesis andmetabolism (Denlinger, 2002). During diapause, copepods are behav-iorally inactive showing depressed respiration rates and limitedswimming (Hirche, 1996). Diapausing copepods are thought to havereduced transcriptional/and translational activity indicated by lowRNA/DNA ratios and reduced rates of protein synthesis (Wagner et al.,1998, 2001; Yebra et al., 2006). Much less is known about the physiolog-ical changes associated with the phases of diapause in C. finmarchicus,

although it has been proposed that it follows an insect-like trajectory(Hirche, 1996).

The early maturation processes including mitotic divisions, cellgrowth and early yolk synthesis take place upon being triggered by inter-nal lipid reserves, and the release of oocytes from the ovary occurs beforemoulting from CV to adult (Grigg and Bardwell, 1982; Niehoff et al.,2002). Gonad development of both diapausing and newly-moulted fe-males were reported to take place concurrently while the females wereresiding at depth >150 m (Madsen and Nielsen, 2001; Niehoff et al.,2002). Males mature earlier than females, and adults may mate as theymigrate to surface waters during which a spermatophore is depositedon the female and females begin reproducing in latewinter to early spring(Durbin et al., 1997; Hirche, 1996). During finalmaturation processes, oo-cytes complete maturation when females are ready to spawn, which in-volves chromosome modifications, incorporation of lipids and late yolksynthesis (Niehoff, 1998; Niehoff and Hirche, 1996; Niehoff et al., 2002).

1.2. Analysis of gene expression using oligonucleotide microarrays

A DNA microarray was designed for C. finmarchicus to investigategene expression consequences of major physiological functions (envi-ronmental stress, metabolism, molting, digestion, neural processes,and membrane physiology). The microarray has been used in a num-ber of experiments analyzing field-collected specimens (Lenz et al.,2012).

Microarrays are particularly useful for examination of gene ex-pression patterns in natural populations, which may exhibit sub-stantially greater variation in gene expression than laboratorymodel species, which in turn may show less variation in expressionof fewer genes (Colbourne et al., 2011). Differential gene expressionmay be used to infer variation in significant physiological processesaffecting longevity, reproductive fitness and probability of survival(Oleksiak et al., 2002). However, high levels of variation among in-dividuals may interfere with or prevent resolution of differencesamong populations or life stages.

In this study, we examined expression levels of ~1000 functionallyimportant genes for C. finmarchicus collected from surface and deepwa-ters of the Gulf of Maine using a custom-designed “Calanus physiologymicroarray”. We hypothesized that surface and deep copepods are

78 E. Unal et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

physiologically distinct as a result of different environmental pressuresand life-history parameters; and that these differences are reflected insignificant differences in gene expression. The significance of thisstudy lies in unraveling the molecular mechanisms underlying thephysiological processes and life history parameters necessary to com-plete the species' complex life history in a region ofmarked seasonal en-vironmental variation.

2. Methods

2.1. Sample collection

Samples were collected in April 17, 2008 fromWilkinson Basin inthe Gulf of Maine (Fig. 1) as part of a multi-year time-series study bythe Center for Coastal Ocean Observation and Analysis (COOA) at theUniversity of New Hampshire, USA. Vertically-stratified planktontows were taken using a 1/4-m MOCNESS (Wiebe et al., 1985)equipped with 150 μm mesh nets. Physical (temperature, salinity)and biological (fluorescence) data were collected for the sampleddepth region (see supplementary data). Zooplankton samples fromtwo depth strata, surface (0–30 m) and deep (130–170 m) were se-lected for analysis. The surface samples targeted the chlorophyllmaximum where the copepods feed and reproduce, and deep sam-ples targeted copepods that were located in warmer water belowthe cold intermediate layer of the Gulf of Maine, where dense con-centrations of diapausing C. finmarchicus are typically found. Thelive copepods were immediately transferred to jars filled with fil-tered seawater at ambient water temperature, transported to thelaboratory, and then allowed to acclimate at their in situ tempera-ture for 24–48 h in the dark to minimize the effects of capture stress,as suggested by Saumweber (2005). Live copepodswere then individu-ally frozen in cryovials in liquid nitrogen. Although this incubationmight have resulted in transcriptional artifacts, the post-collection peri-od of rest was shown to be critical to eliminate collection artifacts forphysiological studies of C. finmarchicus by Saumweber (2005).

2.2. Design of the microarray experiment

For this study, two comparisons were performed: deep females vs.surface females and deep CVs vs. surface females. Five biological repli-cates, each with 9–12 pooled C. finmarchicus individuals, were doneper comparison. Dual-color microarray hybridizations were performedwith two Alexa-fluor fluorescent labels (red emission at 647 nm;green emission at 555 nm). Themicroarray platform and the data serieswere submitted to The Gene Expression Omnibus (GEO) (Edgar et al.,2002) with the series accession number GSE33086 and the microarrayplatform accession number GPL14742 (http://www.ncbi.nlm.nih.gov/geo/).

2.3. Oligonucleotide microarray design

Analysis focused on ~11,000 Expressed Sequence Tags (ESTs) iden-tified from a normalized cDNA library of C. finmarchicus created byDavid Towle (Mount Desert Island Biological Laboratory, Maine) as de-scribed in Towle and Smith (2006) and Lenz et al. (2012). The EST se-quences are available on the National Center for Biotechnologyinformation (NCBI) database (www.ncbi.nlm.nih.gov). Functional anal-ysis was done to sort the ESTs by putative physiological function and toidentify transcripts that encode proteins involved in the generation/regulation of population dynamics. A total of 995 contigs assembledby Partigene that matched genes of known function were selected forinclusion in a custom “Calanus physiology microarray” printed withunique 50 bp oligonucleotide probes (Lenz et al., 2012).

2.4. RNA extraction and cDNA labeling

Liquid-nitrogen frozen C. finmarchicus individuals were used fortotal RNA extraction of pools of 9–10 female and 10–12 CV individ-uals using RNeasy Mini Kit (Qiagen) following the instructions ofthe manufacturer, with a final elution volume of 30 μl. Based onthe quantification of total RNA by the Agilent 2100 Bioanalyzer, 4–10 μg of total RNA was used for first strand synthesis reactions.Fluorescently-labeled cDNA was generated using SuperScript™Plus Direct cDNA Labeling System (Invitrogen). First-strand cDNAsynthesis was performed on samples using SuperScript III RT and la-beled using either Alexa Fluor 555-aha-dUTP or Alexa Fluor647-aha-dUTP nucleotides, with green and red fluorescent emis-sions, respectively.

2.5. Microarray hybridization and scanning

Printed slides were pre-hybridized following the manufacturer'sinstructions (Pronto Microarray Hybridization Kit, Corning). The in-cubation of samples were carried out in a Micro Array User Interface(MAUI) Hybridization Chamber (BioMicro Systems) overnight at42 °C for ~15 h. The slides were scanned by using Genepix Pro Ver.4.1 (Molecular Devices, Union City, CA) scanner with AutoPMT set-ting. At the completion of each scan, images were visually checkedto align the features in a specific block to their appropriate locationon a deconvolution file. Microarray data were then uploaded to Acu-ity 4.0 Microarray Informatics Software (Molecular Devices, UnionCity, CA) for comprehensive analysis.

2.6. Data normalization and screening

The normalization, quality control and gene filtering of the mi-croarray data were performed by using Acuity 4.0 Microarray Infor-matics Software (Molecular Devices, Inc.). Lowess normalization(with data centering) was performed for individual slides usingprint-tip (block-by-block) normalization, with a smoothing factorof 0.4 for 4 iterations (Yang et al., 2002). The Lowess normalizedmedian Log2 ratios of the individual dye intensities were used forsubsequent analysis. Following normalization of each slide, pre-processing of the microarrays was performed, including the spotquality analysis and array scaling. Unreliable data were removedfrom analyses by applying quality control criteria, set to includeonly those spots with a small percentage (b3%) of saturated pixelsthat were not flagged or absent, of relatively uniform intensity andbackground, and detectable above background levels. Following remov-al of unreliable data, an average of 72–77% of the genes was used for thesubsequent differential expression analysis. In order to adjust for differ-ent sample variances in log ratios across the slides, the scale normaliza-tion was performed across the replicate arrays for each comparison(Yang et al., 2002).

2.7. Differential gene expression analysis

In order to identify the significantly differentially expressed genes,multiple statistical analyses were performed with a control of FDR.First, a volcano plot was generated for each comparison in Acuity soft-ware by using a fold-change filter (a minimum of two-fold expressionchange present in at least 70% replicate arrays) and t-test (p b 0.05)along with Principle Component Analysis (PCA). Second, SignificanceAnalysis of Microarrays (SAM; Tusher et al., 2001) was performedusing a one-class data setting. The genes that were significantly differ-entially expressed (FDR b 0.01) were identified; only those genesdeemed significant by both methods were included in the final lists.In general, most transcripts showed less than four-fold expression dif-ferences, with a few exceptions.

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2.8. Functional analysis

Gene ontology (GO) analysis was carried out by using Blast2GOSuite (Conesa et al., 2005; Götz et al., 2008) for the selected EST contigs.Following annotation, InterPro Scan (Mulder and Apweiler, 2007) andAnnex (annotation augmentation) were carried out successively inorder to compare different databases and validate, add or remove GOterms (Conesa andGötz, 2008). Enzyme codemapping and Kyoto Ency-clopedia of Genes and Genomes (KEGG) pathway map analysis werealso carried out (Kanehisa and Goto, 2000).

In order to identify statistically over-represented GO terms, gene en-richment analysis was carried out for the selected gene lists using GeneOntology Enrichment Analysis Software Toolkit (GOEAST; Zheng andWang, 2008), aweb-based software toolkit used to identify significantlyenriched GO terms among a given list of genes (http://omicslab.genetics.ac.cn/GOEAST/). A hypergeometric test was used as the defaultstatistical method to test these associations. The enrichment p-valueswere adjusted for multiple tests by using Benjamini–Yekutieli method(Benjamini and Yekutieli, 2001). The Multi-GOEAST feature was usedto allow cross-comparison of the GO enrichment status of selected upand down-regulated genes in order to identify functional correlationsamong them. Area-proportional Venn diagrams were generated inorder to compare and visualize selected lists by using BioVenn software(Hulsen et al., 2008).

Following subsequent functional annotation analyses, significantlyregulated genes were organized by general functional categories, alsoindicating their more specific functions (Tables 1–5). It is importantto note that multiple functions can potentially be assigned to manyof these genes; these functions should not be regarded as exclusive.

3. Results

3.1. Characterization of C. finmarchicus populations in Wilkinson Basin

During the sampling period, the vertical distribution ofC. finmarchicus was characterized by the presence of a large num-ber of adult females with mature gonads, along with many naupliiand early copepodite stages. No stage CV individuals were collectedfrom the surface waters. At the time of sampling (April 17, 2008),the lower limit of the cold intermediate layer (4.6 °C) was locatedat ~118 m depth (see supplementary data). The temperature ofsurface (0–30 m) and deep water (130–170 m) was similar, rangingbetween 4.2–6.4 °C. Salinity was higher in deep water (33.1–33.8 ppt)compared to surface water (32.4–32.8 ppt). Fluorescence was high atthe surface, with the highest value at 11 m, indicative of an early springbloom that was observed on April 6, 2008 (see http://www.nefsc.noaa.gov/omes/OMES/fall2008/adv7.html).

3.2. Differential regulation

3.2.1. Deep females vs. surface femalesA total of 43 significantly up-regulated transcripts (FDR b 0.01)

was identified in deep females compared to surface females(Fig. 2A, Table 1). The most differentially-expressed functional cat-egories among up-regulated genes involved: protein synthesis [in-cluding transcription and translation factors, a ribosomal protein, agrowth factor regulator (parafibromin), and amino acid metabo-lism]; cell-cycle (including mitosis and cytokinesis); and develop-ment (including neurogenesis, synapsis, axon myelination, andembryogenesis; Table 1). Specifically, cyclin B1 and B2 are involvedin mitosis and cytokinesis, while septins appear to be involved invarious other aspects of the organization of cell surface, exhibitingcell-cycle-coordinated rearrangements within Drosophila embryos(Fares et al., 1995; Shih et al., 2002). Also represented were tran-scripts involved in carbohydrate (citric acid cycle and glycolysis),energy (oxidative phosphorylation) and lipid metabolism (fatty

acid catabolism), as well as genes coding for membrane transport, a cir-cadian clock protein (timeless) and a heat-shock protein (Table 1).

A total of 48 significantly down-regulated transcripts (FDR b 0.01)was identified in deep females vs. surface females (Fig. 2B, Table 2).The major functional categories for down-regulated genes were:protein catabolism (including apoptosis and cell differentiation);carbohydrate catabolism/transport; energy metabolism; cellularion homeostasis; osmoregulation; neuronal morphogenesis (in-cluding, dendrite morphogenesis, neural differentiation, eye pig-ment development, and phototransduction); and innate immunity(including cellular defense and detoxification; Table 2).

3.2.2. Deep CVs vs. surface femalesFewer transcripts were differentially expressed in deep CVs vs. deep

females (based on comparison of both to surface females), but were an-notated to more functional categories. A total of 10 transcripts was sig-nificantly up-regulated (FDR b 0.01) in deep CVs vs. surface females(Fig. 2A) in the categories of protein synthesis (a transcription factor,translation factor, and a ribosomal protein); oxygen and protein trans-port across membranes; a neuronal transcript (ras-related protein);cell-cycle (G2/mitotic-specific cyclin-B2) and DNA synthesis (Ribonu-cleotide reductase M2 polypeptide); a multifunctional transmembraneprotein (surfeit locus protein, involved in protein trafficking larval de-velopment, sex differentiation and locomotion); and a circadian clockprotein (timeless, involved in rhythmic behavior and response to lightstimulus; Table 3).

A total of 16 transcripts was identified to be significantly down-regulated (FDR b 0.01) in deep CVs vs. surface females (Fig. 2B) in thecategories of protein catabolism, cellular homeostasis, and stress/im-munity responses (Table 4). Additional transcripts were associatedwith neuronal development [including neuronal outgrowth and behav-ioral regulation (learning and memory)] and were indicative of late de-velopmental stages.

3.2.3. Transcripts common to both comparisonsOf 47 up-regulated transcripts in deep females or CVs vs. surface

females, six were common to both comparisons (Fig. 2A), includingthose involved in translation, cell-cycle, and oxygen/protein transport,as well as timeless, the circadian clock protein (Tables 1 and 3). Of 52down-regulated transcripts, 12 were common to both comparisons(Fig. 2B), including those involved in protein degradation, cellularion homeostasis, neural differentiation, muscle development and oo-genesis, nutrient/ion and oxygen transport, and stress/immunity re-sponses (Tables 2 and 4).

3.3. KEGG pathway map analysis

KEGG pathway analysis was used to identify the enzymatic func-tions of 12 up-regulated and five down-regulated genes in the con-text of metabolic pathways for comparisons between deep femalesor CVs and surface females (Table 5). Up-regulated enzymes werecategorized in citric acid cycle and glycolysis/gluconeogenesis, oxi-dative phosphorylation, nucleotide and amino acid metabolisms,and synthesis and degradation of ketone bodies. Down-regulated en-zymes were involved in starch and sucrose metabolism, nitrogenmetabolism, arginine and proline metabolism, and metabolism.Two up-regulated enzymes (ribonucleoside diphosphate reductaseandmonophenol monooxygenase) and one down-regulated enzyme(carbonate dehydratase, or carbonic anhydrase) were common toboth comparisons (Table 5).

4. Discussion

The goal of this study was to characterize patterns of gene expres-sion in the copepod C. finmarchicus associated with key life historyevents and processes, including emergence from diapause and

Table 1Functional annotation of up-regulated genes showing significant differential expression for deep females vs. surface females (Significance Analysis of Microarrays, FDR b 0.01). Intotal, 43 genes were significantly up-regulated in deep females. The genes that were up-regulated in both deep females and deep CVs are indicated with an asterisk (*). Score (d):t-statistic value calculated by SAM, q-value: the lowest false discovery rate at which the gene is called significant (see Methods for more explanation), EC No: Enzyme CommissionNumber.

Probe I.D. Annotation Score (d) q-value EC No Specific function

Protein synthesisCFX00418 DNA-directed RNA polymerase II largest subunit, RPB1 2.89 0.0023 EC:2.7.7.6 TranscriptionCFX00927 Transcription initiation factor IIA gamma chain 2.36 0.0051 – TranscriptionCFX00131 Eukaryotic translation initiation factor 3, subunit 6

interacting protein4.56 0.0000 – Translation

CFX00278 Translation elongation factor-1 gamma 4.52 0.0000 – Translation, nuclear export of proteinsCFX00436* Eukaryotic translation initiation factor 4E 4.19 0.0000 – TranslationCFX01829 40S ribosomal protein S21 (rps21) 2.71 0.0037 – Processing and maturation of 18S ribosomesCFX02600 Parafibromin 2.25 0.0054 – Transcriptional regulation, embryonic developmentCFX00563 Aspartate aminotransferase (AST) 2.46 0.0037 EC:2.6.1.1 Amino acid metabolismCFX02585 Selenophosphate synthetase 1 4.99 0.0000 EC:2.7.9.3 Amino acid and vitamin B6 metabolism

Cell-cycle, DNA synthesis and developmentCFX01420* G2/mitotic-specific cyclin-B2 3.83 0.0000 – Mitotic cell-cycle regulation, growthCFX02293 Cyclin B1 4.16 0.0000 – MitosisCFX00787 40S ribosomal protein SA (Laminin receptor) 3.57 0.0000 EC:3.6.5.3 Mitotic spindle elongationCFX00362 Alpha tubulin 2.25 0.0054 EC:3.6.5.1–4 Microtubule structure, mitosisCFX00675 Septin-1 4.69 0.0000 EC:3.6.5.1 Cell division (cytokinesis)CFX00423* Ribonucleotide reductase M2 polypeptide 3.60 0.0000 EC:1.17.4.1 DNA synthesis, nucleotide metabolismCFX00142 Ribonucleoside reductase large chain 3.41 0.0000 – DNA synthesisCFX00383 Flap endonuclease-1 2.80 0.0023 – DNA repairCFX02540 Target of EGR1, member 1 (nuclear) 3.04 0.0000 – Inhibits cell growth rate and cell-cycleCFX01186 Adhesion regulating molecule 1 (arm-1) 3.11 0.0000 – Embryogenesis, female reproduction, cell

development/differentiationCFX00811 D-Titin (Kettin) 3.01 0.0023 – Striated muscle development

Energy, lipid and carbohydrate metabolismCFX00397 ATP synthase, H + transporting, cardiac muscle 2.85 0.0023 EC: 3.6.3.14

EC: 3.6.3.6Oxidative phosphorylation

CFX00799 NADH:ubiquinone oxidoreductase 1 kDa subunit(flavoprotein 1)

2.95 0.0023 EC:1.6.5.3 Oxidative phosphorylation

CFX03325 2-hydroxyacyl-CoA lyase 1 3.77 0.0000 – Fatty acid catabolismCFX00330 Protein phosphatase 1, catalytic subunit, beta isoform 1 3.35 0.0000 EC:3.1.3.16 Glycogen metabolism, circadian clockCFX02052 Fumarate hydratase, mitochondrial precursor (Fumarase) 2.98 0.0023 EC:4.2.1.2 Carbohydrate metabolism, citric acid cycleCFX01354 Dihydrolipoamide acetyltransferase 2.61 0.0037 EC:2.3.1.12 Glycolysis, citric acid cycleCFX01451 Ribophorin I 2.82 0.0023 EC:2.4.1.119 Glycosylation of polypeptides

Neuronal involvementCFX01739 Protein 60A precursor (Protein glass bottom boat) 5.35 0.0000 – Synaptic homeostasisCFX03318 Protein spinster (benchwarmer) 2.91 0.0023 – Central Nervous System, synaptic growthCFX01375 HMG Coenzyme A synthase, isoform a 4.08 0.0000 EC:2.3.3.10 Cholesterol synthesis, axon myelinationCFX02403 Glutamate carboxypeptidase 3.12 0.0000 EC:3.4.17.21 Glutamate pathwayCFX00527 N-methyl-D-aspartate receptor-associated protein 3.00 0.0023 – Glutamate pathway, synapse formationCFX02215 Cytoplasmic polyadenylation element binding protein

1 isoform 35.39 0.0000 – mRNA translation in motor neurons

CFX01761 Crooked neck protein 3.06 0.0000 – mRNA processing, neurogenesisCFX02413 WD40-repeat containing protein (notchless) 2.44 0.0037 – Notch signaling pathway, neurogenesisCFX02013 Porcupine like protein 3.19 0.0000 – Development of neural tube, Wnt signaling

Membrane and transport proteinsCFX00147* Hemocyanin subunit A precursor 3.40 0.0000 EC:1.14.18.1 Oxygen transportCFX01167 Hemocyanin D chain 3.46 0.0000 – Oxygen transportCFX01168* Karyopherin alpha 2 (importin) 4.94 0.0000 – Protein transport, cell-cycleCFX02096 Cation efflux protein/zinc transporter (solute carrier

family 30)4.59 0.0000 – Intracellular zinc transport

CFX01294 Lantibiotic synthetase component c-like 3 (LanC like) 3.64 0.0000 – Membrane protein, binds glutathione

OtherCFX04266* Timeless (circadian clock protein) 3.81 0.0000 – Circadian rhythm, mating behaviorCFX02151 Heat shock protein 75 kDa (TRAP1) 3.34 0.0000 – Mitochondrial hsp90 protein, signal transduction,

protein folding/degradation

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resumption of an active physiological state (e.g., feeding and repro-duction). Based on analysis of expression patterns of the 995-geneDNAmicroarray, there were clear patterns of differential gene expres-sion between C. finmarchicus collected from deep (130–150 m) andshallow (0–30 m) depth strata in Wilkinson Basin in the Gulf ofMaine during April, 2008. Surface females showed up-regulation oftranscripts involved in metabolic activity, protein catabolism, latestages of neuronal and embryonic development, cellular homeostasis,

and stress/immunity responses (Tables 2 and 4). Deep females (and tosome extent deep CVs) showed up-regulation of protein synthesis, ac-tivation of cell-cycle, rebuilding of the nervous system and tissue, oo-genesis and early embryogenesis, which are consistent withprocesses associated with emergence from diapause (Tables 1 and3). Deep CVs showed more variable gene expression patterns andfewer genes that appeared to be significantly regulated, likelyreflecting a more variable physiological state in this stage.

Table 2Functional annotation of down-regulated genes showing significant differential expression for deep females vs. surface females (Significance Analysis of Microarrays, FDR b 0.01).In total, 48 genes were significantly down-regulated in deep females, which also indicate their corresponding up-regulation in surface females. The genes that were down-regulatedin both deep females and CVs are indicated with an asterix (*). Score (d): t-statistic value calculated by SAM, q-value: the lowest false discovery rate at which the gene is calledsignificant (see Methods for more explanation), EC No: Enzyme Commission Number. The genes that could not be reliably assigned a specific function are indicated with a questionmark.

Probe I.D. Annotation Score (d) q-value EC No Specific function

Protein synthesisCFX00977 Eukaryotic peptide chain release factor subunit 1 −5.18 0.0000 – Translation terminationCFX00564 Methyltransferase like protein 2 −2.43 0.0051 – Silencing/activation of genes, alternate splicing

Protein catabolismCFX03555* Lysosomal aspartic protease −4.37 0.0000 EC:3.4.23.0 Apoptosis, protein degradation, termination of vitellogenin

synthesisCFX00720 Matrix metalloproteinase −4.84 0.0000 EC:3.4.24.0 Cell proliferation, migration, differentiation, apoptosisCFX00697 Agmatinase −3.90 0.0000 EC:3.5.3.11 Hydrolase enzyme, arginine/proline metabolismCFX00553 Caspase 7 (apoptosis-related cysteine peptidase) −2.94 0.0027 – Cell death, apoptosis

DevelopmentCFX01655* M-spondin CG10145-PA −3.76 0.0000 Muscle cell attachment, cytoskeletal organizationCFX02323* Vitellogenin receptor −3.06 0.0000 – OogenesisCFX00136 OMA1 homolog, zinc metallopeptidase −3.51 0.0000 – Oocyte maturationCFX00576 Limkain b1 −3.21 0.0000 – Essential regulator of oogenesis

Energy and carbohydrate metabolism, cellular homeostasisCFX01897 FAD linked oxidase domain protein −2.91 0.0027 EC:1.5.3.6 Energy production and conversionCFX00634 NADH dehydrogenase iron–sulfur protein 2,

mitochondrial precursor−3.00 0.0000 EC:1.6.99.3 Oxidative phosphorylation

CFX00588 NADH dehydrogenase subunit 5 −3.50 0.0000 – Oxidative phosphorylationCFX01954 Arginine kinase −3.47 0.0000 – Maintenance of ATP levelsCFX02793 Trehalase (brush-border membrane glycoprotein) −5.48 0.0000 EC:3.2.1.28 Hydrolysis and breakdown of carbohydratesCFX00306 UDP-sugar transporter UST74c (fringe connection protein) −3.50 0.0000 – Carbohydrate transportCFX04127* Carbonic anhydrase (GPL-linked) −2.95 0.0027 EC:4.2.1.1 Osmoregulation, acid–base balanceCFX02201* Mucolipin 3 −2.44 0.0051 – Organellar ion homeostasis (endosome/lysosome), signal

trunsductionCFX03405 Multiple PDZ domain protein 1 −3.16 0.0000 – Cellular ion homeostasis, regulation of membrane potential

Neuronal involvementCFX00814 Alpha 5 type IV collagen isoform 1 −2.85 0.0027 – Axon guidanceCFX01880 ATP-sensitive inward rectifier potassium channel −2.10 0.0076 – Regulation of tube sizeCFX02779 DOMON domain-containing protein (knickkopf) −3.56 0.0000 – Regulation of tube sizeCFX04633 Prepro-A-type allatostatin −5.32 0.0000 – Regulation of juvenile hormoneCFX00063 Semaphorin-1A (Fasciclin IV) −3.37 0.0000 – Dendrite morphogenesisCFX01077 Microtubule-associated protein (futsch) −2.66 0.0043 – Dendrite morphogenesisCFX00463 Brg-1 associated factor −2.65 0.0043 – Dendrite morphogenesisCFX01842 Klingon CG6669-PA −2.61 0.0043 – Limbic system-associated membrane proteinCFX01616 * Gelsolin precursor −2.22 0.0061 – Actin-modulating protein, regulation of neural

differentiationCFX01426 Rhodopsin −3.20 0.0000 – PhototransductionCFX00608 Karmoisin CG12286-PA −3.34 0.0000 – Eye pigment development

Membrane and transport proteinsCFX00422* Permease, Major Facilitator Superfamily transporter −6.21 0.0000 – Nutrient and ion transportCFX01543* Major facilitator transporter −3.95 0.0000 – Nutrient and ion transportCFX01757 Sodium–potassium-transporting ATPase subunit beta 2 −3.74 0.0000 EC:3.6.3.9 Ion transportCFX00652 Organic cation transporter −4.65 0.0000 – Cation transportCFX00178 Chloride channel protein 3 −3.03 0.0000 – Cation transportCFX01033* Globin x −3.79 0.0000 – Oxygen transport

Stress response and innate immunityCFX04122 Hypoxia indicuble factor 1 alpha −4.45 0.0000 – Oxygen homeostasis, oxidative stressCFX02526* DNA-damage inducible protein −3.09 0.0226 EC:3.4.23.0 DNA repair, cell-cycle control, immune responseCFX02181 Mitogen-activated protein kinase (MAP kinase) −2.42 0.0051 EC:2.7.11.0 Cellular responses to external stimuliCFX00417 Aldehyde dehydrogenase −2.40 0.0051 – Cellular defense against hyperosmotic stressCFX01818 Alpha-2 macroglobulin −6.41 0.0000 – Protease binding proteinCFX01394* Integrin alpha 1 −2.98 0.0000 – Cell attachment to ECM, signal transduction, innate immunityCFX00103* Cytochrome P450, 2 k6 (CYP2K6) −4.45 0.0000 EC:1.14.14 DetoxificationCFX03613 Cytochrome P450, 6a14 (CYP6A14) −3.07 0.0000 – Detoxification, metabolism of insect hormonesCFX00331 Cytochrome P450, 330a1 (CYP330A1) −2.30 0.0061 – Detoxification, ecdysteroid regulation

OtherCFX01702 Hypothetical protein (Williams–Beuren syndrome

region 27-like)−3.00 0.0000 EC:2.1.1.0 ?

CFX00284 Copper resistance protein A (son DNA-binding proteinisoform-2)

−4.35 0.0000 – ?

CFX00528 Kelch domain containing protein 2 −2.08 0.0076 – ?

81E. Unal et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

Table 3Functional annotation of up-regulated genes showing significant differential expression for deep CVs vs. surface females (SAM, FDR b 0.01). In total, 10 genes were significantlyup-regulated in deep CVs. The genes that were up-regulated in both deep females and CVs are indicated with an asterisk (*). Score (d): t-statistic value calculated by SAM,q-value: the lowest false discovery rate at which the gene is called significant (see Methods for more explanation), EC No: Enzyme Commission Number.

Probe I.D. Annotation Score (d) q-value EC No Specific function

Protein synthesisCFX00565 SPT transcription factor family 3.67 0.0000 – Transcription, embryonic developmentCFX00436* Eukaryotic translation initiation factor 4E 3.91 0.0000 – TranslationCFX00141 Ribosomal protein L4 3.40 0.0193 – Component of 60S subunit, development

Cell-cycle and DNA synthesisCFX01420* G2/mitotic-specific cyclin-B2 2.80 0.0397 – Mitotic cell-cycle regulation, growthCFX00423* Ribonucleotide reductase M2 polypeptide 3.30 0.0298 EC:1.17.4.1 DNA replication, nucleotide metabolism

Neuronal involvementCFX02001 Ras-related protein Rab-10 4.08 0.0000 EC:3.6.5.1 Signal transduction, neurotransmitter release,

protein transport and localizationMembrane and transport proteinsCFX00147* Hemocyanin subunit A precursor 2.84 0.0397 EC:1.14.18.1 Oxygen transport, alkaloid biosynthesisCFX01168* Karyopherin alpha 2 (importin) 2.74 0.0397 – Protein transport, response to nutrientCFX02724 Surfeit locus protein 4 homolog 2.61 0.0397 – Transmembrane trafficking (Endoplasmis reticulum),

larval development, sex differentiation, locomotionOtherCFX04266* Timeless (circadian clock protein) 3.76 0.0000 – Circadian rhythm, rhythmic behavior, response to

light stimulus

82 E. Unal et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

4.1. Life history events of C. finmarchicus in Wilkinson Basin

The vertical distribution of C. finmarchicus is known to be bimodal inGulf of Maine during fall and winter; the depth of highest abundance ofdiapausing individuals has been reported to be below the cold inter-mediate layer, close to 150 m depth (Durbin et al., 1997; Johnsonet al., 2006). In the spring, deep CV individuals emerge from dia-pause, molt into adults and migrate to the surface. These surface fe-males are behaviorally active and they are feeding on the springphytoplankton bloom (roughly covering the period between mid-

Table 4Functional annotation of down-regulated transcripts showing significant differential expresIn total, 16 genes were significantly down-regulated in deep CVs, which also indicate their cboth deep females and CVs are indicated with an asterisk (*). Score (d): t-statistic value calcnificant (see Methods for more explanation), EC No: Enzyme Commission Number.

Probe I.D. Annotation Score (d) q-value

Protein catabolismCFX03555* Lysosomal aspartic protease −4.37 0.0000CFX02067 26S proteasome regulatory subunit −3.03 0.0226

DevelopmentCFX02323* Vitellogenin receptor −3.15 0.0226CFX01655* M-spondin CG10145-PA −3.76 0.0000

Cellular homeostasisCFX04127* Carbonic anhydrase (GPL-linked) −2.72 0.0285CFX02201* Mucolipin 3 −3.40 0.0000

Neuronal involvementCFX01235 ADP-ribosylation factor-like protein −3.45 0.0000CFX03267 Neurofibromin −2.95 0.0226

CFX01616 * Gelsolin precursor −2.63 0.0285

Membrane and transport proteinsCFX00422* Permease, Major Facilitator Superfamily

(MFS) transporter−3.87 0.0000

CFX01543* Major facilitator transporter −3.95 0.0000CFX01487 Nucleoporin 50 kDa −2.79 0.0285CFX01033* Globin x −6.09 0.0000

Stress response and innate immunityCFX02526* DNA-damage inducible protein −3.09 0.0226CFX01394* Integrin alpha 1 −2.73 0.0285CFX00103* Cytochrome P450, 2 kD (CYP2K6) −3.15 0.0226

March and late April), reproducing and presumably avoiding preda-tors. Consistent with results of extensive studies of the life history ofthis species, (e.g., Durbin et al., 2000; Hirche, 1996), the surface adultfemales collected in mid-April most probably belonged to the G0generation. Individuals from the G1 regeneration were present insurface waters, but they were limited to early developmental stagesof nauplii and early copepodites (Jeffrey Runge, pers. comm.). Adultcopepods collected from deepwater (130–170 m) are alsomost like-ly individuals belonging to the G0 generation; they presumably rep-resent individuals that have emerged from diapause more recently,

sion for deep CVs vs. surface females (Significance Analysis of Microarrays, FDR b 0.01).orresponding up-regulation in surface females. The genes that were down-regulated inulated by SAM, q-value: the lowest false discovery rate at which the gene is called sig-

EC No Specific function

EC:3.4.23.0 Apoptosis, termination of vitellogenin synthesis– Active degradation of short-lived proteins

– OogenesisMuscle cell attachment, cytoskeletal organization, adhesionand outgrowth of embryonic neurons, innate immunity

EC:4.2.1.1 Osmoregulation, acid–base balance– Organellar ion homeostasis (endosome/lysosome),

signal transduction

– Signal transduction, vesicular trafficking– Learning and memory, oxidative stress response, rhythmic

behavior– Actin-modulating protein, regulation of neural

differentiation, spermatid development

– Nutrient and ion transport

– Nutrient and ion transport– Macromolecule transport between nucleus and cytoplasm– Oxygen transport

EC:3.4.23.0 DNA repair, cell-cycle control, immune response– Cell attachment to ECM, signal transduction, innate immunityEC:1.14.14 Detoxification

Table 5KEGG pathway map analysis of the differentially regulated genes obtained for Daphnia pulexa. 12 up-regulated and five down-regulated genes for deep females vs. surface females,two up-regulated and only one down-regulated gene for deep CVs vs. surface females were identified to take place in KEGG metabolic pathways. The KEGG pathway map numbers(Map No), probe IDs, enzyme names and enzyme commission numbers (EC No) are also listed for each pathway. The up- and down-regulation of the corresponding genes indicatedin the last column refers to deep females vs. surface females, and deep CVs vs. surface females.

KEGG pathway Map no. Probe ID Enzyme name EC no. Deep f up/down Deep CV up/down

Carbohydrate metabolismGlycolysis/Gluconeogenesis 00010 CFX01354 Dihydrolipoyllysine-residue acetyltransferase 2.3.1.12 Up _Citrate cycle (TCA cycle) 00020 CFX02052 Fumarate hydratase 4.2.1.2 Up _

CFX01354 Dihydrolipoyllysine-residue acetyltransferase 2.3.1.12 Up _Pyruvate metabolism 00620 CFX01354 Dihydrolipoyllysine-residue acetyltransferase 2.3.1.12 Up _Butanoate metabolism 00650 CFX01375 Hydroxymethylglutaryl-CoA synthase 2.3.3.10 Up _Starch and sucrose metabolism 00500 CFX02793 Alpha, alpha-trehalase 3.2.1.28 Down _

Energy metabolismOxidative phosphorylation 00190 CFX00634 NADH dehydrogenase 1.6.99.3 Down _

CFX00799 NADH dehydrogenase (ubiquinone) 1.6.5.3 Up _CFX00397 H +-exporting ATPase 3.6.3.6 Up _CFX00397 H +-transporting two-sector ATPase 3.6.3.14 Up _

Nitrogen metabolism 00910 CFX04127 Carbonate dehydratase 4.2.1.1 Down DownLipid metabolism

Synthesis and degradation of ketone bodies 00072 CFX01375 Hydroxymethylglutaryl-CoA synthase 2.3.3.10 Up _Nucleotide metabolism

Purine metabolism 00230 CFX00418 DNA-directed RNA polymerase 2.7.7.6 Up _CFX00423 Ribonucleoside-diphosphate reductase 1.17.4.1 Up Up

Pyrimidine metabolism 00240 CFX00418 DNA-directed RNA polymerase 2.7.7.6 Up _CFX00423 Ribonucleoside-diphosphate reductase 1.17.4.1 Up Up

Amino acid metabolismPhenylalanine metabolism 00360 CFX00563 Aspartate transaminase 2.6.1.1 Up _Alanine, aspartate and glutamate metabolism 00250 CFX00563 Aspartate transaminase 2.6.1.1 Up _Phenylalanine, tyrosine and tryptophan biosynthesis 00400 CFX00563 Aspartate transaminase 2.6.1.1 Up _Cysteine and methionine metabolism 00270 CFX00563 Aspartate transaminase 2.6.1.1 Up _Valine, leucine and isoleucine degradation 00280 CFX01375 Hydroxymethylglutaryl-CoA synthase 2.3.3.10 Up _Arginine and proline metabolism 00330 CFX00563 Aspartate transaminase 2.6.1.1 Up _

CFX00697 Agmatinase 3.5.3.11 Down _Tyrosine metabolism 00350 CFX00563 Aspartate transaminase 2.6.1.1 Up _

CFX00147 Monophenol monooxygenase 1.14.18.1 Up UpMetabolism of other amino acid s

Selenocompound metabolism 00450 CFX02585 Selenide, water dikinase 2.7.9.3 Up _Glutathione metabolism 00480 CFX00423 Ribonucleoside-diphosphate reductase 1.17.4.1 Up Up

Glycan biosythesis and metabolismN-Glycan biosynthesis 00510 CFX01451 Dolichyl-diphosphooligosaccharide-protein

glycotransferase2.4.1.119 Up _

Metabolism of cofactors and vitaminsRiboflavin metabolism 00740 CFX00147 Monophenol monooxygenase 1.14.18.1 Up UpNicotinate and nicotinamide metabolism 00760 CFX01897 (R)-6-hydroxynicotine oxidase 1.5.3.6 Down _

a KEGG Pathway maps for D. pulex obtained from http://www.genome.jp/kegg-bin/show_organism?menu_type=pathway_maps&org=ddpu.

83E. Unal et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

but had yet to initiate their ascent to surface waters. Similarly, thedeep CV individuals may have been in the late stages of diapause ei-ther from a resident or an advected population; although not docu-mented, diel vertical migration among stage CV copepodites cannotbe ruled out.

The current study focuses on emergence from diapause and devel-opment in April, and therefore cannot provide information about therecently proposed hypothesis emphasizing the role of wax esters indiapause (Pond et al., 2012). However, it is also important to notethat samples collected in April might have introduced some degreeof overlap in the results of comparisons. An ideal sampling regimenwould therefore include multiple samplings of spring surface CVsand fall deep CVs in order to effectively compare diapausing andnon-diapausing individuals.

4.2. Patterns of gene expression associated with life history events

4.2.1. Surface femalesThe up-regulation observed in transcripts involved in late stages of

development, cellular homeostasis, and stress/immunity responsesmay be an indication of the active metabolic status of surface females,including exposure to environmental stressors (Tables 2 and 4). Severaltranscripts were involved in defense mechanisms, which are known toplay a role in innate immunity in insects (Peretz et al., 2007; Zhuanget al., 2008). Other transcripts withwell studied functions in arthropods

that showed up-regulation in surface females included those involvedin neuronal regulation (Xia and Chiang, 2009), acid–base homeostasis(Henry, 1996; Towle and Weihrauch, 2001), and signal transduction(Cheng et al., 2010).

Up-regulation of rhodopsin and karmoisin (Table 2), which areinvolved in phototransduction and eye pigment development, re-spectively, is consistent with the importance of light in the modu-lation of behavior in surface females. Surface females were alsocharacterized by a number of transcripts involved in protein turn-over including programmed cell death (apoptosis; Table 2). For ex-ample, caspase 7 is a well-known executioner protein of apoptosisin a wide variety of organisms (Cohen, 1997). The 26S proteasomeregulatory subunit catalyzes protein degradation, and is actively in-volved in degradation of short-lived proteins (Voges et al., 1999).

Up-regulated transcripts in surface females also provided evidenceof late-stage embryogenesis, consistent with reports by Niehoff et al.(2002), who found that embryogenesis in C. finmarchicus females iscompleted in surface waters. The up-regulated transcripts includedM-spondin (Umemiya et al., 1997) and a DOMON-domain containingprotein (Moussian et al., 2006) known from Drosophila embryos(Table 2). Microtubule-associated proteins, which are known to stabi-lize microtubules and play a major role in maintenance of nervous sys-tem through adulthood in Drosophila (da Cruz et al., 2005), were one ofthe most differentially expressed transcripts in this study (more thanfour-fold up-regulation in surface females).

Fig. 2. Area-proportional Venn diagram showing the number of significantly differentially expressed genes identified for each comparison along with the genes that overlap be-tween comparisons. Probe IDs for both comparisons are indicated for A) Up-regulated genes B) Down-regulated genes (See Tables 1–4 for gene annotations).

84 E. Unal et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 76–85

4.2.2. Deep females and CVsUp-regulated transcripts in deep females (and to some extent deep

CVs) were generally indicative of multiple emergent processes (seeHirche, 1996), including protein synthesis, activation of cell-cycle, re-building of the nervous system, rebuilding of tissue, and early embryo-genesis. The specific genes (see Table 1) are known to be involved intranscriptional regulation of embryonic development (Wang et al.,2008), which is consistent with early embryonic development occur-ring in deep females, as reported byNiehoff et al. (2002). The significantup-regulation of these genes in deep CVs and adult females vs. surfaceadult females (Tables 1 and 3) suggests that protein synthesis mayalso be up-regulated as part of the biological processes associatedwith emergence fromdiapause. Diapausing individuals in the refractoryperiod are characterized by down-regulated transcriptional and trans-lational activity (low RNA/DNA ratios) in C. finmarchicus (Wagneret al., 1998; Yebra et al., 2006).

Both deep females and CVs showed up-regulation of genes asso-ciated with control of developmental processes like chromatin struc-ture and cell-cycle progression, DNA replication and nucleartransport (Tables 1 and 3). These proteins are thought to play essen-tial roles in oogenesis in Drosophila (Goldfarb et al., 2004; Gorjánáczet al., 2002), and may have similar functions in C. finmarchicus.

Deep females also showed up-regulation of transcripts involved inDNA replication, mitosis and other cell-cycle processes, consistentwith increased cell division occurring during early embryogenesisand tissue rebuilding in adults that developed from diapausing indi-viduals (Table 1). Since the majority of cell division occurs in embryo-genesis, the up-regulation of these transcripts in deep females mayindicate active gonad maturation process (early embryogenesis).

Genes known to be involved in structural, muscle and nervoussystem development in Caenorhabditis elegans (Hutter et al., 2000)and Drosophila (Martin et al., 1999) were also up-regulated in deepfemales, suggesting active biosynthesis of cellular membranes andmicrotubule structures associated with growth.

5. Conclusion

In the Gulf of Maine ecosystem, C. finmarchicus shows distinctgene expression patterns between deep and surface water layersthat are in consistent with its life history trajectory and associatedphysiological status. Overall, surface females showed evidence ofan active metabolic state with greater need of maintaining cellularhomeostasis, activation in stress/immunity responses, and late

embryogenesis. Deep females (and to some extent deep CVs)showed evidence of activation in metabolic machinery, indicativeof emergence from diapause, oogenesis, early embryogenesis andtissue buildup with increased rates of cell division. Our results dem-onstrate that targeted gene expression studies applied to naturalpopulations can successfully resolve distinct gene expression pat-terns, despite natural variability in field populations. The genes iden-tified in this study are promising candidates for further investigation inlaboratory studies to understand their roles in metabolic pathways andtheir regulatory mechanisms in C. finmarchicus.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jembe.2013.04.020.

Acknowledgements

Wewould like to thank to Christine Smith (MDIBL) for her gener-ous help during microarray hybridizations. We are particularlygrateful to Patrick Hassett and Andrew Christie for their valuablecontributions regarding physiological interpretations of the genes,and for their helpful comments.Wewould like to offer special thanksto Helen Poynton, who provided useful advice and comments on themanuscript. Assistance provided by Christopher Manning and JasonBeaudet regarding sampling for this study was greatly appreciated.We also would like to thank to Peter Wiebe for his help with themaps and profiles, and to Jeffrey Runge for communications aboutthe life history of C. finmarchicus. Funding was provided by the NSFBiological Oceanography Program (Awards OCE-0815047 to AnnBucklin and OCE-1040597 to Petra H. Lenz), and NIH IDeA Networksof Biomedical Research Excellence (INBRE grant P20RR016463 toPatricia Hand).[SS]

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