Stress response or beneficial temperature acclimation: transcriptomic signatures in Antarctic fish ( Pachycara brachycephalum )

Stress response or beneficial temperature acclimation:transcriptomic signatures in Antarctic fish (Pachycarabrachycephalum)

H. S. WINDISCH,* S . FRICKENHAUS,*† U. JOHN,* R. KNUST,* H. -O. P €ORTNER* and

M. LUCASSEN*

*Alfred Wegener Institute, Helmholtz Center for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven,

Germany, †Hochschule Bremerhaven, Biotechnology, An der Karlstadt 8, 27568 Bremerhaven, Germany

Abstract

Research on the thermal biology of Antarctic marine organisms has increased aware-

ness of their vulnerability to climate change, as a flipside of their adaptation to life in

the permanent cold and their limited capacity to acclimate to variable temperatures.

Here, we employed a species-specific microarray of the Antarctic eelpout, Pachycarabrachycephalum, to identify long-term shifts in gene expression after 2 months of accli-

mation to six temperatures between �1 and 9 °C. Changes in cellular processes com-

prised signalling, post-translational modification, cytoskeleton remodelling, metabolic

shifts and alterations in the transcription as well as translation machinery. The magni-

tude of transcriptomic responses paralleled the change in whole animal performance.

Optimal growth at 3 °C occurred at a minimum in gene expression changes indicative

of a balanced steady state. The up-regulation of ribosomal transcripts at 5 °C and

above was accompanied by the transcriptomic activation of differential protein degra-

dation pathways, from proteasome-based degradation in the cold towards lysosomal

protein degradation in the warmth. From 7 °C upwards, increasing transcript levels

representing heat-shock proteins and an acute inflammatory response indicate cellular

stress. Such patterns may contribute to a warm-induced energy deficit and a strong

weight loss at temperatures above 6 °C. Together, cold or warm acclimation led to spe-

cific cellular rearrangements and the progressive development of functional imbalances

beyond the optimum temperature. The observed temperature-specific expression pro-

files reveal the molecular basis of thermal plasticity and refine present understanding

of the shape and positioning of the thermal performance curve of ectotherms on the

temperature scale.

Keywords: cDNA library, chronic thermal exposure, cold adaptation, ESTs, gene regulation,

microarray

Received 22 January 2014; revision received 13 May 2014; accepted 26 May 2014

Introduction

Temperature plays a crucial role for all poikilotherm

animals, because this factor is pervasive and affects vir-

tually all cellular components by altered viscosity of

media and body fluids, fluidity of membranes and

enzyme kinetics (Hochachka & Somero 2002). As a

consequence of trade-offs at all levels of biological

organization, molecular to systemic, animals specialize

in limited temperature ranges which mirror the degree

of ambient temperature variability and shape the range

of their biogeographical distribution (P€ortner 2002). The

degree of ambient temperature sensibility and thus

thermal specialization is highest in stenothermal polar

organisms (Somero et al. 1996). As a consequence, they

are thought to suffer most from ongoing ocean warm-

ing caused by the global climate change, which also

affects the waters around the Antarctic Peninsula (Gille

2002; Turner et al. 2005).Correspondence: H. S. Windisch, Fax: ++49 471 4831 1149;

E-mail: [email protected]

© 2014 John Wiley & Sons Ltd

Molecular Ecology (2014) 23, 3469–3482 doi: 10.1111/mec.12822

In response to altered temperatures, fish species from

temperate climates display extensive changes in expres-

sion profiles as shown by in-depth expression analyses

through microarrays (for review, see Douglas 2006).

However, comprehensive gene expression studies of

cold-adapted fish species are few. Some extant Antarctic

fish species have limited capacity to acclimate to higher

ambient water temperatures. Shifts of upper critical

temperatures to higher values after acclimation to 4 °Cin various Notothenioids and Zoarcid species indicate

an ability of warm hardening (Bilyk et al. 2012). Tran-

scriptomic studies are needed to understand which

molecular pathways contribute to shifting thermal lim-

its and shaping the thermal plasticity of species in their

specific habitats. To our knowledge, the transcriptomic

responses of broad sets of genes to warming have so

far been characterized in microarray studies of two Ant-

arctic fish species exposed to acute heat stress. The

expression of hundreds of genes involved in the cellular

stress response (CSR), as characterized by K€ultz (2005),

was altered in Trematomus bernacchii after exposure to

4 °C for 4 h and after recovery (Buckley & Somero

2009). This finding indicates remnant capacities to

respond to thermal stress, although this species appar-

ently lacks the classic heat-shock response (Hofmann

et al. 2000; Clark et al. 2008). Similarly, transcripts of

components of the acute inflammatory response were

found in Harpagifer antarcticus after exposure to 6 °Cover 24 h (Thorne et al. 2010). In addition, an acute

heat-shock response was detected as well as the induc-

tion of genes related to oxidative stress, which highly

resembles patterns known for eurythermal fish under

heat stress (Gracey et al. 2004; Podrabsky & Somero

2004; Logan & Somero 2011).

Besides these studies of acute thermal responses,

long-term experiments lasting at least 4 weeks (Peck

et al. 2014) are needed to characterize molecular under-

pinnings of the thermal window of ectothermal species

and their limited capacities to respond to changing tem-

peratures. Within this study, we develop a comprehen-

sive view of the thermal tolerance window and the

gene regulatory network behind for the Antarctic eel-

pout Pachycara brachycephalum (Pappenheim, 1912). This

species lives in Antarctic waters and inhabits the shelf

regions at depths from 200 to 1800 m (Anderson 1990).

The mean annual water temperature in the Southern

Ocean fluctuates between �1.5 and 2.0 °C due to the

constant conditions of the Antarctic circumpolar cur-

rent. Like other Antarctic species, P. brachycephalum is

able to acclimate to warmer temperatures (Windisch

et al. 2011; Bilyk et al. 2012) reaching the highest growth

rates around 4 °C (Brodte et al. 2006). Here, we studied

transcriptomic changes in P. brachycephalum after

2 months of exposure to six different temperatures

ranging from �1 °C up to 9 °C to further characterize

the thermal window of this species. We hypothesize

that unique expression patterns will indicate distinct

critical threshold temperatures/tipping points in the

thermal window. By linking whole animal performance

indicators to transcriptomic changes, we were able to

characterize the thermal window at the molecular level,

thereby deepening our understanding of the molecular

underpinning of observed physiological traits.

Material and methods

Animal collection and incubation

Specimens of Pachycara brachycephalum were caught

with baited traps around King George Island at posi-

tions 62°19.010S 58°35.490W; 62°16.860S 58°36.750W;

62°19.330S 58°33.800W; and 62°19.690S 58°33.680W during

expedition ANT-XXV/4 of RV ‘POLARSTERN’ in April

2009. Water conditions were monitored by a CTD sta-

tion close to all traps. The temperature at sampling

depths was about �1 °C. The animals were brought to

the AWI Bremerhaven and kept at 0 °C in recirculated

sea water at 34 PSU in one single tank. Fish were

allowed to acclimate to aquarium conditions for at least

6 months. Randomly chosen fish were transferred to

separate tanks (one for each temperature) and kept

individually in separate baskets to facilitate individual

monitoring and to reduce handling stress. Six different

temperatures (�1, 0, 3, 5, 7 and 9 °C) were applied to

groups of 12 fish per treatment for a total duration of

9 weeks. All fish were weighed under slight anaesthesia

(0.05 g/L MS222 in sea water) before experimentation.

Animal groups were warmed at 1 °C per day until they

reached their final temperature. Animals exposed to 7

and 9 °C were warmed in a stepwise procedure, first

being incubated for 1 week at 5 °C prior to their expo-

sure to 7 °C. Fish to be studied at 9 °C were held at

7 °C for another week before being exposed to the final

temperature. Control animals held at 0 °C were

exposed to the same handling procedures. Mortality

was monitored during experimentation, and dead ani-

mals were removed without being replaced.

Fish were fed ad libitum with Crangon crangon once a

week; feeding was terminated exactly 1 week before

sampling. For sampling, the fish were anaesthetized in

0.2 g/L MS222. Blood samples were removed with hep-

arinized syringes from the caudal vessel before killing

the fish. Liver samples were excised quickly, frozen

instantaneously in liquid nitrogen and stored at �80 °Cuntil further processing. Further tissue samples were

taken for follow-up studies. Handling and killing of the

fish was conducted in line with the recommendations of

the American Veterinary Medical Association (AVMA).


3470 H. S . WINDISCH ET AL.

These animal experiments were approved by the respon-

sible national authority [Freie Hansestadt Bremen,

reference number 522-27-11/02-00(93)].

Animal performance

Growth performance (GP) was calculated from the dif-

ference in weight between starting and end points of

the acclimation period. The hepatosomatic index (HSI)

was calculated according to Busacker et al. (1990). The

haematocrit was recorded in fresh blood samples by

means of a haematocrit centrifuge.

All performance parameters were analysed by apply-

ing one-way ANOVA at a significance level of P ≤ 0.05,

followed by a Student–Newman–Keuls post hoc test.

This was also applied to changes in GP, HSI and hae-

matocrit, when tested in pooled sets of ‘cold’ (�1°;0 °C), ‘intermediate’ (3°; 5 °C) and ‘warm’ exposures

(7°; 9 °C). Details of the statistical analyses are available

in the Appendix S1, Tables 1–3 (Supporting informa-

tion). Graphs showing GP, HSI and haematocrit (Fig. 1)

depict means � SEM calculated using SIGMAPLOT

(version 10; Systat Software).

Experimental design

Based on the quality (minimum ratios of absorptions at

260 nm/280 nm ≥2; 260 nm/230 nm ≥1.8) and integrity

(minimum ratio of 28S/18S rRNA between 2 and 3.5 as

well as a RNA integrity number (RIN) above 9.5 analy-

sed by capillary electrophoresis using a Bioanalyser:

Agilent Technologies, Waldbronn, Germany) of liver

RNA, five fish from each treatment and seven fish from

the group at 0 °C were selected for expression profiling.

Male specimens were preferably selected from each

group to reduce potential sex-specific differences. How-

ever, due to approximately 50% mortality at 9 °C, onlytwo males were available among the fish that survived

the experiment. Accordingly, two males and three

females were analysed in the expression analyses.

Selected fish had a mean body length of 21 � 0.94 cm

(�SEM) and a mean body weight of 35.61 � 5.06 g.

Liver RNA of all selected fish was pooled in equal

amounts to form the reference pool. RNA samples from

individual fish were hybridized against the reference

pool on single arrays.

Array design

The array design is based on a test array comprising

91 402 probes for 17 024 contigs of a cDNA library of

P. brachycephalum (SRA049761) that was constructed

from liver and heart tissue (Windisch et al. 2012).

Based on probe efficiency, a more compact array was

redesigned and produced by Agilent Technologies

encompassing 40 036 unique probes for 15 843 ESTs as

well as 1390 control probes for standardization pur-

poses (see below) involving synthetic RNA (Spike-In-

Kit, Agilent).

Sample preparation and labelling

Total RNA was extracted from 20 to 40 mg liver tissue

with RNeasy (Qiagen). Labelling reactions were started

with 200 ng total RNA and a 1:16 dilution of a positive

control RNA (Agilent RNA Spike-In Kit for two-colour

Hep

atos

omat

ic in

dex

(%)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

a

b,d,f

b,d

c

d,e

e

***

Cold Intermediate Warm

9Acclimation temperature (°C)

–1 0 3 5 7

Hae

mat

ocrit

(%)

0

5

10

15

20*



–1 0 3 5 7

(A) (B) (C)

Δ w

eigh

t in

%

–30

–20

–10

0

10

20

a

b,d,f,h,i

e

gc

b,d,f,h,j


***


–1 0 3 5 7

Fig. 1 Animal performance parameters. (A) Growth performance. (B) hepatosomatic index (HSI). (C) Haematocrit. Values are

means � SD, labelled with different letters to show significant differences (P ≤ 0.05) according to one-way ANOVA, followed by a Stu-

dent–Newman–Keuls post hoc test. Letters indicate significant difference between single temperature groups (a is different from b, c

from d, etc.; for further information, see Appendix S1 Table 1, Supporting information). Between the groups ‘cold’, ‘intermediate’

and ‘warm’ (means indicated as transparent bars), the warm group was significantly different in all performance parameters at dif-

ferent significance levels. These were indicated by asterisks: *for P ≤ 0.05; *** for P ≤ 0.001. Mortality was 0% in all groups, except

for those at �1 °C (8.3%) and at 9 °C (52.6%).


GENE EXPRESSION PATTERNS IN COLD- ADAPTED FISH 3471

arrays) to monitor the procedure of sample amplification

and microarray workflow. Reference RNA-containing

spike-A-mix was labelled with cyanine-3; samples con-

taining spike-B-mix were labelled with cyanine-5.

Labelled and amplified cRNA was purified with the

RNeasy kit (Qiagen). The labelling products were quan-

tified using the NanoDrop ND 10000s microarray

measurement protocol.

Hybridization and feature extraction

All reactions were standardized (volume and yield)

according to manufacturer’s recommendations for

8 9 60 K array formats. Dye-labelled samples contain-

ing 300 ng sample and reference each were hybridized

for 17 h at 65 °C. After washing (GE Wash Buffer 1+2;Agilent) and dye stabilization, slides were directly

scanned with an Agilent G2565AA scanner under utili-

zation of the AgilentHD_GX_2Color protocol.

Data processing and interpretation

Data were extracted with the Feature Extraction Soft-

ware version 9.0 (Agilent) applying the GE2_107_Sep09

protocol. Normalization within (loess method) and

between arrays (aquantile method) was performed after

background correction (movingmin method) as imple-

mented in Limma (Smyth & Speed 2003) in R (R-Devel-

opment-Core-Team 2011) to smooth the data and

exclude error-prone weak signals.

We conducted Significance Analysis for Microarray

data (SAM, after Tusher et al. 2001) within the MeV

software environment (Saeed et al. 2006). For the multi-

class comparisons of all six groups, the delta value was

adjusted to 0.082 for a median false-positive rate

q ≤ 0.001% and a 90th percentile FDR ≤ 0.2%. The

resulting data, comprised of 1120 responsive probes

representing 664 contigs (approximately 4.3% of all

ESTs on the array), were further analysed to character-

ize gene expression patterns. Sequence annotations

were obtained by BLASTx searches (Altschul et al. 1990)

with an e-value cut-off 10�3 against the SwissProt and

the nonredundant database. Furthermore, contigs were

assigned to gene models based on a rpstBLASTn

against the fish-specific orthologous sequences, that is,

fiNOG, with an e-value cut-off of 10�20 (see Windisch

et al. 2012) for the assignment of transcripts to ortholo-

gous sequences.

Responsive contigs determined by SAM were filtered

within each temperature group for an absolute fold

change FC ≥ 1.5 to examine imbalances in gene regula-

tion (see Fig. 2). In addition, super-ordinated categories

of orthologous groups (COG) of fiNOGs were used for

a colour-scale diagram of affected functions in three

temperature groups (Fig. 3).

Due to the lack of functional information, responsive

transcripts inside the categories R (general function pre-

diction only), S (function unknown) and X (no result)

were postponed.

For all ESTs with an absolute FC ≥ 1.5, GO

enrichments were performed at an alpha level of 0.05,

followed by a correction for multiple testing by

FDR ≤ 0.05 (Benjamini and Hochberg) with BLAST2GO

(Conesa et al. 2005). To depict the changes of all SAM-

identified transcripts with temperature, all COG-

assigned transcripts (besides from COG categories R, S

and X) were arranged by hierarchical clustering with

Pearson’s correlation-based distance metrics and

(B)Warm

Inter-mediateCold 51

45716

0

134209

135122

00

12020

(A)

9

Up - regulated

Down - regulated–200

–100

0

100

200Cold Intermediate Warm

Acclimation temperature (°C)–1 0 3 5 7

Res

pons

ive

tran

scrip

ts

Fig. 2 Expression of regulated genes. (A) Transcript levels of 664 genes responsive to temperature (determined by SAM) were filtered

within each temperature group by a minimum 1.5-fold change to generate a semi-quantitative overview and determine the relation-

ship between thermal exposure and regulatory effort. (B) Using absolute counts of unique transcripts from the left panel, we analy-

sed unique as well as shared temperature-dependent regulated transcripts in a Venn diagram for groups of cold, intermediate and

warm exposures.



complete linkage clustering in MeV (see Appendix S2

clusters 1–3, Supporting information).

To evaluate sex-related biases in the 9 °C group, we

compared the expression profiles of females and males

by means of an unpaired t-test at a significance level of

P ≤ 0.05 (see Appendix S3, Supporting information)

using MeV. In total, we identified 20 contigs among all

664 temperature-sensitive transcripts with differing

expression levels between males and females.

Results and discussion

Whole animal performance

The concept of oxygen and capacity-limited thermal tol-

erance (OCLTT) (P€ortner 2010) serves as framework to

address thermal thresholds and the physiological per-

formance of a species within its thermal tolerance

window. Indicators of performance as well as thermal

limitation such as growth, arterial blood flow, heart

rate, aerobic and anaerobic metabolites reflect the aero-

bic scope and shape of the temperature-dependent per-

formance window (P€ortner & Knust 2007). For the

Antarctic eelpout, acute temperature ramp experiments

identified an upper critical thermal limit between 9 and

10 °C indicated by shifts in pHi, increased oxygen con-

sumption rates and the onset of anaerobic metabolism

in various tissues (van Dijk et al. 1999; Mark et al. 2002).

In the present long-term study, similar critical limits

were detected by assessing mortality rates during

experimentation. All animals of incubations 0, 3 and

5 °C survived the exposure for 2 months. At �1 and

7 °C, one of 12 animals died in each group, whereas at

9 °C, approximately 50% of the incubated animals died,

indicating that this temperature is beyond the long-term

upper thermal tolerance limit.

J Translation, ribosomal structure and biogenesis

A RNA processing and modification

K Transcription

L Replication, recombination and repair

B Chromatin structure and dynamics

D Cell cycle control, cell division, chromosome partitioning

Y Nuclear structure

V Defense mechanisms

T Signal transduction mechanisms

M Cell wall/membrane/envelope biogenesis

N Cell motility

Z Cytoskeleton

W Extracellular structures

U Intracellular trafficking, secretion, and vesicular transport

O Posttranslational modification, protein turnover, chaperones

C Energy production and conversion

G Carbohydrate transport and metabolism

E Amino acid transport and metabolism

F Nucleotide transport and metabolism

H Coenzyme transport and metabolism

I Lipid transport and metabolism

P Inorganic ion transport and metabolism

Q Secondary metabolites biosynthesis, transport and catabolism

R General function prediction only

S Function unknown

X No result

Up Down

War

m

Inte

r-m

edia

te

Col

d

War

m

Col

d

Inte

r-m

edia

te

1%5%10% <1%

1%5%10% <1%

Info

rmat

ion

stor

age

and

proc

essi

ngC

ellu

lar p

roce

ss a

nd s

igna

ling

Met

abol

ism

Poo

rlych

arac

teriz

edFig. 3 Functional overview of regulated

genes. Responsive transcripts determined

by SAM with a minimum ≤1.5-fold change

were included in a functional overview of

regulated genes in COG/KOG categories.

The overview shows percentage frequen-

cies of functions within a treatment across

categories.



Growth performance (Fig. 1A) reflects the long-term

availability of aerobic scope, as excess aerobic energy is

available for productivity (P€ortner 2010). At low incuba-

tion temperatures (‘cold’), animals displayed a gain in

total body mass of 3.87% � 2.35% at �1 °C and

7.23% � 0.83 at 0 °C. Although these temperatures rep-

resent the thermal range of the eelpout’s habitat, the

highest weight gain (11.17% � 4.73%) was found at

3 °C. At 5 °C, the weight gain of 8.31% � 2.14% was

still higher than that at cold temperatures (both temper-

atures are referred to as ‘intermediate’). In contrast, the

animals exposed to the two highest temperatures

(assigned as ‘warm’) showed weight losses of

�6.23% � 2.95% at 7 °C turning into a dramatic decline

of body weight by �26.03% � 4.28% at 9 °C. Animals

exposed to 9 °C differed significantly from all remain-

ing groups in terms of their large weight loss

(P ≤ 0.001). The same still applies to animals exposed to

7 °C, although P-values were different from those at

9 °C (P = 0.002–0.027); for a detailed statistical

summary, see Appendix S1, Table 1 (Supporting

information).

Overall, the present growth data result in a bell-

shaped GP curve, similar to the one found in a former

study of this species, where maximum growth was

found at 4 °C and growth rates were similar at 6 and

0 °C (Brodte et al. 2006). The point where GP turns

negative between 6 and 7 °C resembles a distinct

thermal threshold, beyond which long-term survival of

Pachycara brachycephalum becomes impossible. Results

obtained during all individual treatments below this

threshold are significantly different to those obtained at

temperatures above (P ≤ 0.05). Notably, this tempera-

ture corresponds to the upper pejus temperature (sensu

OCLTT concept), at which aerobic performance

becomes constrained (P€ortner 2010).

The hepatosomatic index represents an important

physiological fitness parameter in fish (Busacker et al.

1990) because the liver is a central hub for the storage

and conversion of high-energy substrates. In addition, it

also plays an important role in detoxification and

humoral control. Hence, this tissue constitutes an excel-

lent target to characterize key mechanisms of thermal

acclimation using transcriptomics. We want to stress

that the subsequent analyses of hepatic gene expression

data are used as a proxy for mechanisms that may also

affect the whole animal.

When analysing liver mass, a decrease of the mean

HSI to 1.55 � 0.11 in the warm-exposed group indicates

a massive loss of total liver weight compared with the

cold group with a HSI of 2.7 � 0.16 and the intermedi-

ate one with a HSI of 2.41 � 0.15 (Fig. 1B). As feeding

was successful in all groups, GP and HSI data indicate

elevated energy demand at 7 and 9 °C, which was not

covered by normal food intake. The deficiencies caused

through warming have been balanced by the mobiliza-

tion of body-own reserves from the liver and possibly

elsewhere. Moreover, oxygen supply becomes limiting

at these temperatures and is also limiting energy con-

version (van Dijk et al. 1999). We actually observed a

rise in haematocrit levels in the warm-exposed group,

in line with oxygen limitations and a certain threshold

(Fig. 1C), from 12.15% � 1.11% to 12.58% � 1.1% at

cold and intermediate temperatures, respectively, to

17.04% � 1.10% after chronic warm exposure. So far,

only modest, if any, changes in haematocrit have been

reported in response to higher temperatures in Antarc-

tic fishes (Tetens et al. 1984; Lowe & Davison 2005;

Hudson et al. 2008). Elevated haematocrit levels are

suitable to compensate for functional hypoxaemia

resulting from increased oxygen demand in the

warmth, further contributing to a ‘warm hardiness’

(Windisch et al. 2011). The existing knowledge on aero-

bic scope together with physiological fitness parameters

at whole animal and tissue levels provides evidence for

thermal constraints setting in below �1 °C and above

6 °C (van Dijk et al. 1999; Mark et al. 2002), as well as

for a thermal optimum at around 3–4 °C (Brodte et al.

2006).

Functional characteristics of temperature-conditionedtranscriptomes

The monitored performance patterns indicate successful

thermal acclimation within the thermal optimum range

but also capacity limits at temperatures beyond. Con-

straints in performance may be paralleled by a shift

from a homeostatic response into a CSR (K€ultz 2005) at

certain threshold temperatures. When analysing the

expression profiles by SAM, 1120 responsive probes

representing 664 contigs were found to be temperature

sensitive, corresponding to 4.3% of all contigs repre-

sented on the array. An imbalance of up- and down-

regulation became visible (Fig. 2A), when examining

the median expression of transcripts among the applied

temperatures with a minimum absolute fold change of

FC ≥ 1.5. The extent of up- and down-regulation is sim-

ilar at cold and warm temperatures, whereas the

response is clearly reduced in the intermediate group.

In this group, only seven unique transcripts are up-reg-

ulated and five are repressed. The majority of respon-

sive genes in the intermediate group are subsets of the

cold group and warm group likely representing genes

playing a role in adjustments to the respective condi-

tions (Fig. 2B). Most of the thermally sensitive genes

have different expression profiles outside of the opti-

mum growth range (Figs 1A and 2A). The appearance

of unique cold- and warm-specific transcripts indicates



distinct cellular rearrangements at both edges of the

thermal window. Under more extreme conditions, the

induction of differing gene products may support meta-

bolic maintenance.

In liver, expression levels change generally in the

same direction (i.e. up or down) in both sexes, but seem

to be only slightly more pronounced in females com-

pared with males in the 9 °C group. Further studies

need to characterize and compare the acclimation

potentials of males and females, which was beyond the

scope of the present study, but the higher responsive-

ness in females may reflect a higher robustness and

potential to acclimate and survive high temperatures.

As the same patterns prevail, we can exclude a sex-

induced bias in the interpretation of data at 9 °C.The following sections provide an overview of pro-

cesses affected the most. To identify the prevailing

molecular mechanisms, we attributed the responsive

transcript sets to fish-specific orthologies (fiNOGs)

classified in functional COG (Fig. 3). In addition, GO

enrichments were performed to specify and emphasize

regulated processes and functions within COG/KOG

categories (cf. Tables 1 and 2), as discussed in the

following sections.

Transcription machinery

Several mediators of transcription (e.g. transcription ini-

tiation factor 2b, or TATA box-binding protein) display

opposing temperature-dependent expression profiles

among treatments (Fig. 3 category K; Appendix S2 clus-

ter 1, Supporting information). Cold-induced genes are

ones directly involved in transcription, whereas those

expressed in the warmth are rather related to signal

transduction (Appendix S2 cluster 1, cat. A&K, Support-

ing information). The higher expression levels of

transcription components and target protein RNAs in

the cold may compensate kinetic constraints at low

Table 1 GO enrichments of up-regulated transcripts. Results were generated by means of a two-tailed Fischer´s exact test in BLAST2GO

at a significance level of P ≤ 0.05. Data were filtered with a maximum FDR ≤ 0.05 to exclude false discovery. All terms were

overrepresented. The last column indicates a hand-curated cross-reference to COG/KOG categories

GO Term Name Type Cold Intermediate Warm CR

GO:0035770 Ribonucleoprotein granule C 4.90E-05 A

GO:0005839 Proteasome core complex C 2.00E-04 O

GO:0005960 Glycine cleavage complex C 2.50E-05 E

GO:0005852 Eukaryotic translation initiation factor 3 complex C 4.00E-05 J

GO:0022625 Cytosolic large ribosomal subunit C 2.90E-06 2.90E-08 J

GO:0004298 Threonine-type endopeptidase activity F 1.70E-04 O

GO:0003735 Structural constituent of ribosome F 1.50E-07 6.20E-10 J

GO:0019843 rRNA binding F 1.40E-04 J

GO:0003723 RNA binding F 9.20E-05 J

GO:0031435 Mitogen-activated protein kinase kinase kinase binding F 8.50E-05 T

GO:0008168 Methyltransferase activity F 1.30E-05 JLQ

GO:0004450 Isocitrate dehydrogenase (NADP+) activity F 2.00E-04 CG

GO:0030742 GTP-dependent protein binding F 2.00E-04 TO

GO:0006414 Translational elongation P 1.80E-05 8.10E-11 J

GO:0006949 Syncytium formation P 5.60E-05 ZU

GO:0042254 Ribosome biogenesis P 3.60E-07 4.20E-09 J

GO:0000079 Regulation of cyclin-dependent protein kinase activity P 8.80E-06 D

GO:0019643 Reductive tricarboxylic acid cycle P 2.40E-04 C

GO:0051603 Proteolysis involved in cellular protein catabolic process P 8.30E-05 E

GO:0031274 Positive regulation of pseudopodium assembly P 8.50E-05 Z

GO:0043552 Positive regulation of phosphatidylinositol 3-kinase activity P 4.90E-05 I

GO:0046330 Positive regulation of JNK cascade P 4.90E-05 T

GO:0045740 Positive regulation of DNA replication P 2.80E-04 L

GO:0007097 Nuclear migration P 1.10E-06 L

GO:0048664 Neuron fate determination P 1.00E-05 X

GO:0007095 Mitotic cell cycle G2/M transition DNA damage checkpoint P 1.70E-06 D

GO:0030225 Macrophage differentiation P 2.80E-04 X

GO:0006102 Isocitrate metabolic process P 2.00E-04 C

GO:0019464 Glycine decarboxylation via glycine cleavage system P 2.50E-05 E

GO:0046847 Filopodium assembly P 1.30E-04 U

GO:0034332 Adherens junction organization P 2.50E-05 U



temperatures. This is in line with higher RNA:protein

ratios observed for various species in a meta-analysis by

Fraser et al. (2002). Also, ribonucleoproteins, such as u6/

u4 snRNA proteins, are induced in the cold (Table 1,

GO:0035770; Appendix S2 cluster 1, cat. A, Supporting

information) and are involved in splicing, indicating a

different way of RNA processing in the cold.

At warm temperatures, transcriptomic reprogram-

ming involves the up-regulation of transcripts of differ-

ent helicases and histones (Appendix S2 cluster 1, cat.

B+L, Supporting information). Both have a large impact

on the condensation state of DNA and the provision of

methylation sites to trigger the expression and silencing

of genes. However, it is conceivable that damaged

proteins involved in DNA and RNA maintenance and

processing display elevated turnover rates. Instability

of folding may impair the function of large protein

complexes, as their functionality depends on the correct

assembly of single components (Feller & Gerday 2003).

Further evidence for impaired protein stability stems

from the induction of chaperones at intermediate tem-

peratures and above. Proteins containing WD-repeat

domains (tryptophan–aspartate repeats) are important

as scaffolds for many protein–protein interactions

(Appendix S2 cluster 1, cat. A, Supporting information).

Their contribution to the formation of the transcriptional

initiation complex has been described in detail (Smith

2008), and their induction in P. brachycephalum seems to

be important for the translation process in the warmth.

Protein biosynthesis

The strongest changes occur in the translation process

due to altered expression levels of ribosomal

Table 2 GO enrichments of down-regulated transcripts. Results were generated by means of a two-tailed Fischer´s exact test in

BLAST2GO at a significance level of P ≤ 0.05. Data were filtered with a maximum FDR ≤ 0.05 to exclude false discovery. All terms were

overrepresented. The last column indicates a hand-curated cross-reference to COG/KOG categories

GO Term Name Type Cold Intermediate Warm CR

GO:0005852 Eukaryotic translation initiation factor 3 complex C 2.90E-06 J

GO:0001650 Fibrillar centre C 5.50E-05 A

GO:0005960 Glycine cleavage complex C 4.90E-05 E

GO:0005874 Microtubule C 1.60E-04 2.00E-04 Z

GO:0005839 Proteasome core complex C 5.40E-05 O

GO:0015171 Amino acid transmembrane transporter activity F 6.90E-05 E

GO:0004364 Glutathione transferase activity F 2.10E-05 9.60E-05 O

GO:0005525 GTP binding F 5.00E-05 T

GO:0030742 GTP-dependent protein binding F 8.80E-05 T

GO:0003924 GTPase activity F 3.50E-06 2.10E-05 T

GO:0031435 Mitogen-activated protein kinase kinase kinase binding F 3.70E-05 1.70E-04 T

GO:0016903 Oxidoreductase activity, acting on the aldehyde

or oxo group of donors

F 2.30E-05 X

GO:0004298 Threonine-type endopeptidase activity F 4.70E-05 O

GO:0034332 Adherens junction organization P 1.10E-05 4.90E-05 U

GO:0022402 Cell cycle process P 7.00E-05 D

GO:0035088 Establishment or maintenance of apical/basal cell polarity P 2.20E-04 U

GO:0046847 Filopodium assembly P 5.90E-05 2.60E-04 U

GO:0006803 Glutathione conjugation reaction P 2.10E-05 9.60E-05 X

GO:0019464 Glycine decarboxylation via glycine cleavage system P 4.90E-05 E

GO:0015807 L-amino acid transport P 1.70E-04 E

GO:0030225 Macrophage differentiation P 1.20E-04 X

GO:0007018 Microtubule-based movement P 2.40E-06 2.90E-05 U

GO:0000278 Mitotic cell cycle P 1.50E-05 D

GO:0007095 Mitotic cell cycle G2/M transition DNA damage checkpoint P 2.90E-06 D

GO:0048664 Neuron fate determination P 4.30E-06 2.00E-05 X

GO:0007097 Nuclear migration P 3.70E-05 1.70E-04 L

GO:0045740 Positive regulation of DNA replication P 1.20E-04 L

GO:0046330 Positive regulation of JNK cascade P 2.10E-05 9.60E-05 T

GO:0043552 Positive regulation of phosphatidylinositol 3-kinase activity P 2.10E-05 9.60E-05 I

GO:0031274 Positive regulation of pseudopodium assembly P 3.70E-05 1.70E-04 Z

GO:0071822 Protein complex subunit organization P 8.90E-05 O

GO:0051603 Proteolysis involved in cellular protein catabolic process P 2.50E-05 E

GO:0000079 Regulation of cyclin-dependent protein kinase activity P 1.80E-05 D



components. Mitochondrial ribosomal proteins display

high transcription rates in the cold (Appendix S2 cluster

1, cat. J, Supporting information). This may relate to

high mitochondrial densities found in cold-adapted fish

at habitat temperatures or other cold-acclimated poikilo-

therm species (Clarke & Johnston 1999; O’Brien 2011).

These transcripts are less expressed at intermediate and

warm temperatures, whereas 40S, 60S ribosomal pro-

teins and eukaryotic translation initiation factors are

progressively induced (Table 1: GO:0005852, GO:00022

625, GO:0003735, GO:0006414, GO:0042254; Appendix

S2 cluster 1, cat. J, Supporting information). The protein

synthesis machinery is one of the largest multi-enzyme

complexes in cells involving 50–80 structural protein

subunits (for review, see Korobeinikova et al. 2012). The

aforementioned higher thermal sensitivity of multi-sub-

unit enzyme assemblies seems to hold in this case as

well. A functional impairment of the protein biosynthe-

sis was shown earlier for this species by lower transla-

tional capacities in vitro at 5 °C (Storch et al. 2005).

Although we did not measure protein biosynthesis, it is

likely that excess de novo protein synthesis compen-

sates for protein damage at intermediate and warm

temperatures.

The induction of genes encoding heat-shock proteins

(HSP) such as HSP71 and HSP105 (Appendix S2 cluster

2, cat. O, Supporting information) at 7 and 9 °C indi-

cates a chronic demand for chaperones at warm tem-

peratures. As HSP function depends on ATP binding

(cf. database by Kumar et al. 2012), their chronic induc-

tion will increase metabolic energy demands. Combined

with high translational rates of ribosomal components,

our findings imply higher metabolic costs of protein

synthesis and maintenance in the warmth and to some

extent also in the cold, which may contribute to reduce

animal growth at the respective temperatures (Fig. 1A).

Protein degradation

Multiple components of the proteasome encoded by

genes for regulatory proteins and ubiquitin-conjugating

enzymes are up-regulated in the cold (Table 1,

GO:0005839, GO:0004298; Appendix S2 cluster 2, cat. O,

Supporting information) but are repressed in the

warmth (Table 2, same terms and GO:0071822). Our

data resemble findings in Austrofundulus limnaeus (Po-

drabsky & Somero 2004) where higher transcript levels

for the 26S ubiquitin subunit were accompanied by con-

stant levels of de novo-synthesized proteins after

chronic cold exposure. High levels of ubiquitin-conju-

gated proteins were also found in other Antarctic fish

species and were discussed to represent an energetic

constraint in cold-adapted species (Todgham et al. 2007;

Shin et al. 2012). High rates of ATP-dependent

ubiquitination and degradation due to ‘nonproductive’

protein folding may be connected to high metabolic

costs. Nevertheless, sufficient amounts of energy seem

to be available for ATP-dependent protein degradation

at low temperatures. At warm temperatures, the pre-

ferred protein degradation pathway shifts completely

towards employment of the lysosomal pathway. This

shift is possibly compensating for a loss of function of

the proteasome, which is assembled from at least 50

single subunits (Lander et al. 2012). Furthermore, con-

straints in oxygen supply at higher temperatures may

reduce the energy available for protein degradation.

The lysosomal pathway has been described by Lum

et al. (2005) as a mechanism of autophagy during star-

vation and metabolic stress causing intracellular dam-

age. However, we did not identify higher expressions

of the respective gene network including the associated

ATG genes (Mizushima 2007), which are represented

on the array by 33 probes for 11 transcripts. Neverthe-

less, repression of proteasomal genes occurs from 3 °Conwards, whereas lysosomal transcripts are up-regu-

lated (see Appendix S2 cluster 2, cat. T&O, Supporting

information). This may be beneficial under energetically

constrained conditions as the lysosomal pathway does

not consume ATP as needed for the ubiquitination and

proteasome pathway. Lysosomal activation is paralleled

by an up-regulation of cell cycle control genes, such as

cyclin-G1-inhibiting cellular growth (also see Table 1,

GO:0000079, GO:0007095).

Oxidative stress

Excess oxygen availability and high mitochondrial den-

sities may lead to a higher rate of ROS production in

the cold that requires reduction equivalents from the

glutathione redox system. Transcripts of the glutathione

S-transferase (GST) are highly expressed in the cold and

1.7-fold repressed in the warmth (Table 2: GO:0004364;

Appendix S2 cluster 2, cat. O, Supporting information).

High GST levels may constitute a cold adaptation fea-

ture. Similar observations were made in Antarctic inver-

tebrates (for review, see Abele & Puntarulo 2004) as

well as in a recent transcriptomic study in the notothei-

oid fish Pagothenia borchgrevinki (Bilyk & Cheng 2013).

ROS formation was discussed to increase after warm

acclimation due to the induction of uncoupling proteins

(Mark et al. 2006). Under oxygen-limited conditions in

the warmth, uncoupling proteins may compensate for a

disturbed electron transfer and too high membrane

potentials caused by an insufficient entry of oxygen into

the respiratory chain. In fact, mitochondrial uncoupling

proteins were identified in this study as part of the

long-term stress response to warm temperatures

(Appendix S2 cluster 3, cat. C, Supporting information),



possibly indicating an impairment of energy formation

at elevated temperatures.

Signalling

Cold-specific signal transduction mechanisms comprise

GTP binding and MAPK and JNK signalling (Tables 1

and 2, GO:0030742, GO:0031435, GO:0046330; repression

in the warmth: GO:0005525, GO:0003924; Appendix S2

cluster 2, cat. T, Supporting information). These genes

are involved in signalling cascades initiating cell prolif-

eration and differentiation (Ip & Davis 1998; Goldsmith

& Dhanasekaran 2007). Chemokines, ras suppressor

protein-1 and GTP-binding protein-8 are candidate

genes in this group.

At intermediate temperatures and above, coagulation

factors and acute-phase proteins of the complement sys-

tem (Appendix S2 cluster 2, cat. O, Supporting informa-

tion) indicate a reaction similar to chronic responses to

warming in temperate or acute warming in Antarctic

fish (Podrabsky & Somero 2004; Buckley & Somero

2009; Thorne et al. 2010). These marker proteins label

damaged cells and organize inflammation and healing

processes. One potential mediator of wound healing is

fibronectin (Appendix S2 cluster 2, cat. O, Supporting

information), an integrin promoting cell adhesion and

phagocytosis of opsonized cells (Grinnell 1984). Here, it

may contribute as a chemo-attractant maintaining the

integrity of connective tissue after cellular damage. Fur-

thermore, structural remodelling of the cytoskeleton

(mediated by the four-and-a-half lim domains protein

1), angiogenesis (angiopoietin-related protein 3) and the

development of neurons (neogenin) are stimulated. In

addition, higher haematocrit levels and the expression

of haptoglobin at warm temperatures boost an

improved oxygen supply (Appendix S2 cluster 3, cat.

Q, Supporting information). Thus, mechanisms involv-

ing extracellular matrix organization, vascularization

and tissue remodelling counter the effects of the CSR

under higher temperatures. Although these mechanisms

seem suitable to sustain prolonged exposure to the

warmth or to hypoxemic events, permanent survival

under these conditions is impaired by limited energetic

reserves, remnant acclimation capacities as well as the

cold-adapted architecture of proteins.

Cytoskeleton

Along with the different exposures, large rearrangements

in the cellular substructure became visible in the COG/

KOG analyses (Fig. 3 and Appendix S2 cluster 2, cat. Z &

U, Supporting information) as well as by GO enrich-

ments. The solubility and viscosity of the cytosol is lar-

gely affected by temperature, and their maintenance

requires cytoskeleton remodelling as already mentioned

above. The subcellular structure in the cold is dense, indi-

cated by high levels of dynein, myosin and tubulin,

which are largely reduced at other temperatures. Also,

genes associated with cellular communication (i.e. syncy-

tium formation, pseudopodium assembly, filopodium

assembly, adherence junction organization) are more

strongly expressed in the cold than in the warmer treat-

ments as indicated by several GO terms (Table 1,

GO:0006949, GO:0031274, GO:0046847, GO:0034332;

Table 2, GO:0034332, GO:0046847, GO:0031274).

In contrast, anchor- and structure-maintaining pro-

teins such as spectrin (associated protein), dystrobrevin

(alpha) and desmoplakin are induced at 5 °C and

above. These proteins stabilize the inner side of the

plasma membrane, link the cytoskeleton to the extracel-

lular matrix and connect neighbouring cells more clo-

sely. From these observations, it seems that cells are

losing shape and volume in the warmth, in line with

the aforementioned pattern of resorbing body-own

substrate reserves.

Metabolism

Energy production (Appendix S2 cluster 3, cat. C, Sup-

porting information) in the cold was linked to an

increased provision of substrates to the citric acid cycle

(cytoplasmic aconitase hydratase) as well as an increase

of reduction equivalents (NADP-dependent isocitrate

dehydrogenase; Table 1, GO:0004450, GO:0019643, GO:0

006102). Together with higher expression levels of

respiratory chain components (subunits of complexes I,

III, IV and ATP synthase), the array of induced tran-

scripts indicates an enhanced functional capacity of

mitochondria in the cold.

Higher enzyme capacities of complex IV at constant

transcript levels were found in this species after

6 weeks of acclimation to 5 °C (Windisch et al. 2011).

This indicates improved aerobic capacity – probably by

post-translational modification – suitable to promote

energy allocation into growth at intermediate treat-

ments. Higher transcript levels of complexes I and ~IVseen here may then be required to counter higher rates

of protein turnover at warm temperatures.

Cold-adapted fish accumulate lipids and use a lipid-

based metabolism. This facilitates intracellular oxygen

transport and exploits the excess availability of ambient

oxygen (P€ortner et al. 2005). Acclimation to higher tem-

peratures, however, results in a metabolic shift towards

the formation of glycogen stores and carbohydrate-based

metabolism (Brodte et al. 2006; Windisch et al. 2011).

These alterations were thought to promote ‘warm hardi-

ness’, as carbohydrates can be metabolized during oxy-

gen deficiency under hypoxaemia as induced in the



warmth. High expression levels of genes involved in

lipid metabolism (I) prevail in the cold (Tables 1 and 2,

GO:0043552), whereas increased transcript levels related

to carbohydrate metabolism (G) are found in the warmth

(Fig. 3; Appendix S2 cluster 3, Supporting information).

The induction of glycogen branching enzyme is in line

with glycogen accumulation at 5 °C (Windisch et al.

2011) and prevails at temperatures above confirming

physiological patterns at the transcriptomic level.

A new aspect is the intense induction of genes associ-

ated with amino acid transport and metabolism

(category E) in the cold. Transcripts of the glycine

cleavage system (GCS) are highly expressed only at

these temperatures (Table 1, GO:0005960, GO:0051603,

GO:0019464; while being repressed at the other treat-

ments: Table 2, GO:0005960, GO:00015171, GO:0019464,

GO:0015807, GO:0051603). Free amino acids may not

only be used by protein synthesis or as solutes, but also

seem to play an important role as an energy-rich sub-

strate (especially glycine). Kikuchi et al. (2008) reviewed

the composition and reaction mechanism of the mito-

chondrial GCS protein complex. This pathway is widely

distributed from bacteria to plants as well as animals

and is responsible for serine and glycine catabolism

with turnover rates being highest in kidney, brain and

liver of vertebrates. However, the general functional

importance of the GCS complex in cold-adapted polar

organisms remains to be confirmed in further compara-

tive studies.

Transcripts with unknown function

The bulk of differentially expressed genes was observed

among poorly characterized genes. When analysing the

distribution of associated functional terms across the

total response (Fig. 3), it became evident that these tran-

scripts contribute to the acclimation process and can be

classified as thermally responsive genes. It remains to

be investigated whether these responsive transcripts

point to species-specific mechanisms or have general

importance. It is likely that within this set of genes,

new candidate genes will be identified that represent

functions crucial in thermal adaptation.

Synopsis

To our knowledge, this is the first study monitoring

long-term temperature-conditioned transcriptomes of an

Antarctic fish and, at the same time, the first to address

the molecular underpinning of temperature-dependent

physiological performance as a result of long-term accli-

mation. A comprehensive picture of the temperature

response became visible by linking liver expression data

to physiological performance parameters.

The changing expression levels in various functions

and across temperature-dependent GP indicate a signifi-

cant capacity of P. brachycephalum to acclimate success-

fully to temperatures up to 6 °C. Although the natural

thermal niche of P. brachycephalum ranges between �1

and 1 °C, a growth maximum at 3 °C indicates that the

species exists permanently below its optimum physiolo-

gical performance temperature. At the transcriptomic

level, this was paralleled by the lowest regulative effort

seen as the smallest number of thermally responsive

transcripts. Nonetheless, P. brachycephalum has occupied

a niche below its thermal optimum, but may benefit

from the availability of food, space or less competition.

More detailed analyses of molecular phenotypes (and

genotypes) in natural populations would be helpful to

understand the correlation between gene expression

and adaptive genetic divergence (Pavey et al. 2010).

Differentially expressed functions above and below

the thermal optimum revealed cellular mechanisms that

require adjustment for maintenance at cold and warm

temperatures (summarized in Fig. 4). Accordingly, the

regulatory effort in gene expression increases towards

the edges of the thermal tolerance window. Some


Lipids

Lysosomal

Ubiquitindependent

CarbohydatesAmino acids

Anchor proteins

Filament density

Inflammation

Cytoskeleton organisation

Angiogenesis

Post-translational modification

Translation

–1 0 3 5 7 9

Stor

age

fuel

sPr

otei

nde

grad

atio

nC

yto-

skel

eton

Sign

allin

gPr

otei

ntu

rnov

er

Acclimation temperature (°C)

Fig. 4 Summary scheme of temperature-specific gene regula-

tion. Specific patterns identified in this study are summarized

in a qualitative and semi-quantitative overview. The graphs

reflect transcript diversity as well as relative expression levels

of altered pathways over the assessed temperature range.



changes in mRNA levels may indicate new steady

states in the balancing of diverse functions; others may

reflect that homeostasis becomes more costly at extre-

mely cold and warm temperatures.

In the cold, transcripts and correlated functions indi-

cate elevated protein turnover rates and ATP-dependent

proteasomal degradation. This may lead to unfavour-

able shifts in energy budget at the expense of growth.

Nevertheless, GP remains positive and HSI levels

highly reflecting a surplus of energy allocated into

growth. With temperatures increasing to above the opti-

mum, energy metabolism is remodelled from an amino

acid and lipid-based to a carbohydrate-based one. The

induction of ribosomal proteins indicates a compensa-

tory response to counter functional losses due to an

increasing instability of the protein synthesis machinery

at higher temperatures.

A strong decrease of total body and liver weight is

noted for temperatures between 6 and 7 °C. The activa-

tion of an inflammatory response as well as lysosomal

degradation processes and cell cycle arrest points to criti-

cal cellular damages and energetic deficiencies at temper-

atures from 7 °C upwards. Together, these data indicate

that events at the gene expression level mirror the upper

pejus temperature determined earlier in this species in

physiological studies at whole organism level, including

capacity limitations of the cardiovascular system leading

to insufficient blood flow and – at the intracellular level –

a deviation from alphastat–pH regulation above 6 °C(Mark et al. 2002). Various molecular mechanisms, com-

prising the augmentation of the haematocrit, angiogene-

sis, cell structure and storage compound remodelling,

were identified here to cope with the increasingly

adverse conditions of the whole organism (Fig. 4).

However, it is likely that fundamental constraints

ultimately limit the acclimation capacity of cold-

adapted ectotherms, because functions at various levels

of systemic organization seem to be affected at similar

threshold temperatures. As proteins in this species dis-

play signatures of cold adaptation through a composi-

tional bias of amino acids increasing their flexibility

(Windisch et al. 2012), we hypothesize that complex

protein assemblies limit the scope of a thermal acclima-

tion at the molecular level due to higher instabilities of

the quaternary structure at warmer temperatures. Such

disturbances can involve the capacity of transport

mechanisms, for example for nutrition, as well as

increase energy demand overproportionally with rising

temperatures, thereby disturbing the balances in whole

animal energy budget. Broadscale studies correlating

molecular size and number of subunits of large enzyme

complexes to their thermal sensitivity would be

required to address a potential limitation at this level.

Acknowledgements

The study was partly funded by Deutsche Forschungsgemeins-

chaft (LU1463/1-2). It is a contribution to the PACES research

program (work package 1.6) of the Alfred Wegener Institute

funded by the Helmholtz Association. The authors would like

to thank the crew of RV Polarstern and Nils Koschnick for

excellent technical support during expeditions. Nicole Hilde-

brand is acknowledged for taking care of the animals during

the incubations. The authors are grateful to the anonymous ref-

erees for their comments and suggestions which helped to sig-

nificantly improve the article.

References

Abele D, Puntarulo S (2004) Formation of reactive species and

induction of antioxidant defence systems in polar and tem-

perate marine invertebrates and fish. Comparative Biochemistry

and Physiology Part A: Molecular & Integrative Physiology, 138,

405–415.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)

Basic local alignment search tool. Journal of Molecular Biology,

215, 403–410.Anderson ME (1990) Zoarcidae. In: Fishes of the Southern Ocean

(eds Gon O & Heemstra P), pp. 256–276. J.L.B. Smith Insti-

tute for Ichthyology, Grahamstown, South Africa.

Bilyk K, Cheng C-H (2013) Model of gene expression in

extreme cold – reference transcriptome for the high-Antarctic

cryopelagic notothenioid fish Pagothenia borchgrevinki. BMC

Genomics, 14, 634.

Bilyk K, Evans C, DeVries A (2012) Heat hardening in Antarc-

tic notothenioid fishes. Polar Biology, 35, 1447–1451.

Brodte E, Knust R, P€ortner HO (2006) Temperature-dependent

energy allocation to growth in Antarctic and boreal eelpout

(Zoarcidae). Polar Biology, 30, 95–107.Buckley BA, Somero GN (2009) cDNA microarray analysis

reveals the capacity of the cold-adapted Antarctic fish Tre-

matomus bernacchii to alter gene expression in response to

heat stress. Polar Biology, 32, 403–415.Busacker GP, Adelman IR, Goolish EM (1990) Physiological

and biochemical indices of growth. In: Methods For Fish Biol-

ogy (eds Schreck C & Moyle PB), American Fisheries Society,

Baltimore, Maryland.

Clark M, Fraser K, Burns G, Peck L (2008) The HSP70 heat

shock response in the Antarctic fish Harpagifer antarcticus.

Polar Biology, 31, 171–180.Clarke A, Johnston NM (1999) Scaling of metabolic rate with

body mass and temperature in teleost fish. Journal of Animal

Ecology, 68, 893–905.

Conesa A, Goetz S, Garcia-Gomez JM, Terol J, Talon M, Robles

M (2005) Blast2GO: a universal tool for annotation, visualiza-

tion and analysis in functional genomics research. Bioinfor-

matics, 21, 3674–3676.

van Dijk PL, Tesch C, Hardewig II, P€ortner HO (1999) Physio-

logical disturbances at critically high temperatures: a com-

parison between stenothermal antarctic and eurythermal

temperate eelpouts (Zoarcidae). Journal of Experimental Biol-

ogy, 202(Pt 24), 3611–3621.Douglas SE (2006) Microarray studies of gene expression in

fish. OMICS: A Journal of Integrative Biology, 10, 474–489.



Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in

cold adaptation. Nature Reviews Microbiology, 1, 200–208.Fraser KPP, Clarke A, Peck LS (2002) Low-temperature protein

metabolism: seasonal changes in protein synthesis and RNA

dynamics in the Antarctic limpet Nacella concinna Strebel

1908. Journal of Experimental Biology, 205, 3077–3086.Gille ST (2002) Warming of the Southern Ocean since the

1950s. Science, 295, 1275–1277.Goldsmith ZG, Dhanasekaran DN (2007) G Protein regulation

of MAPK networks. Oncogene, 26, 3122–3142.Gracey AY, Fraser EJ, Li W et al. (2004) Coping with cold: an

integrative, multitissue analysis of the transcriptome of a

poikilothermic vertebrate. Proceedings of the National Academy

of Sciences, USA, 101, 16970–16975.Grinnell F (1984) Fibronectin and wound healing. Journal of Cel-

lular Biochemistry, 26, 107–116.Hochachka PW, Somero GN (2002) Temperature. In: Biochemical

Adaptation-Mechanisms and Process in Physiological Evolution,

pp. 290–438. Oxford University Press, Oxford.

Hofmann G, Buckley B, Airaksinen S, Keen J, Somero G (2000)

Heat-shock protein expression is absent in the Antarctic fish

Trematomus bernacchii (family Nototheniidae). Journal of

Experimental Biology, 203, 2331–2339.

Hudson H, Brauer P, Scofield M, Petzel D (2008) Effects of

warm acclimation on serum osmolality, cortisol and hemato-

crit levels in the Antarctic fish, Trematomus bernacchii. Polar

Biology, 31, 991.

Ip YT, Davis RJ (1998) Signal transduction by the c-Jun N-ter-

minal kinase (JNK) - from inflammation to development.

Current Opinion in Cell Biology, 10, 205–219.

Kikuchi G, Motokawa Y, Yoshida T, Hiraga K (2008) Glycine

cleavage system: reaction mechanism, physiological signifi-

cance, and hyperglycinemia. Proceedings of the Japan Academy.

Series B, Physical and Biological Sciences, 84, 246–263.

Korobeinikova AV, Garber MB, Gongadze GM (2012) Ribo-

somal proteins: structure, function, and evolution. Biochemis-

try (Moscow), 77, 562–574.K€ultz D (2005) Molecular and evolutionary basis of the cellular

stress response. Annual Review of Physiology, 67, 225–257.Kumar RR, Nagarajan NS, Arunraj SPSP et al. (2012) HSPIR: a

manually annotated heat shock protein information resource.

Bioinformatics, 28, 2853–2855.

Lander GC, Estrin E, Matyskiela ME, Bashore C, Nogales E,

Martin A (2012) Complete subunit architecture of the protea-

some regulatory particle. Nature, 482, 186–191.Logan CA, Somero GN (2011) Effects of thermal acclimation on

transcriptional responses to acute heat stress in the euryther-

mal fish Gillichthys mirabilis (Cooper). American Journal of

Physiology: Regulatory, Integrative and Comparative Physiology,

300, R1373–R1383.

Lowe CJ, Davison W (2005) Plasma osmolarity, glucose concen-

tration and erythrocyte responses of two Antarctic notothe-

niid fishes to acute and chronic thermal change. Journal of

Fish Biology, 67, 752–766.

Lum JJ, DeBerardinis RJ, Thompson CB (2005) Autophagy in

metazoans: cell survival in the land of plenty. Nature Reviews

Molecular Cell Biology, 6, 439–448.Mark FC, Bock C, P€ortner HO (2002) Oxygen-limited thermal

tolerance in Antarctic fish investigated by MRI and 31P-

MRS. American Journal of Physiology: Regulatory, Integrative

and Comparative Physiology, 283, R1254–R1262.

Mark FC, Lucassen M, P€ortner HO (2006) Thermal sensitivity

of uncoupling protein expression in polar and temperate

fish. Comparative Biochemistry and Physiology Part D: Genomics

and Proteomics, 1, 365–374.Mizushima N (2007) Autophagy: process and function. Genes &

Development, 21, 2861–2873.O’Brien KM (2011) Mitochondrial biogenesis in cold-bodied

fishes. Journal of Experimental Biology, 214, 275–285.Pavey SA, Collin H, Nosil P, Rogers SM (2010) The role of

gene expression in ecological speciation. Annals of the New

York Academy of Sciences, 1206, 110–129.

Peck LS, Morley SA, Richard J, Clark MS (2014) Acclimation

and thermal tolerance in Antarctic marine ectotherms. Journal

of Experimental Biology, 217, 16–22.Podrabsky JE, Somero GN (2004) Changes in gene expression

associated with acclimation to constant temperatures and

fluctuating daily temperatures in an annual killifish Austro-

fundulus limnaeus. Journal of Experimental Biology, 207, 2237–2254.

P€ortner HO (2002) Climate variations and the physiological

basis of temperature dependent biogeography: systemic to

molecular hierarchy of thermal tolerance in animals. Compar-

ative Biochemistry and Physiology Part A: Molecular & Integra-

tive Physiology, 132, 739–761.P€ortner HO (2010) Oxygen- and capacity-limitation of thermal

tolerance: a matrix for integrating climate-related stressor

effects in marine ecosystems. Journal of Experimental Biology,

213, 881–893.

P€ortner HO, Knust R (2007) Climate change affects marine

fishes through the oxygen limitation of thermal tolerance.

Science, 315, 95–97.P€ortner HO, Lucassen M, Storch D (2005) Metabolic biochemis-

try: its role in thermal tolerance and in the capacities of

physiological and ecological function. In: The Physiology of

Polar Fishes(eds Farrell AP & Steffensen JF), pp. 79–154. Else-vier Academic Press, California, San Diego.

R-Development-Core-Team. (2011) R: A Language and Environ-

ment for Statistical Computing. R Foundation for Statistical

Computing, ISBN 3-900051-07-0, http://www.R-project.org/.

Vienna, Austria.

Rustici G, Kolesnikov N, Brandizi M et al. (2013) ArrayExpress

update–trends in database growth and links to data analysis

tools. Nucleic Acids Research, 41, D987–D990.

Saeed AI, Bhagabati NK, Braisted JC et al. (2006) TM4 micro-

array software suite. Methods in Enzymology, 411, 134–193.Shin SC, Kim SJ, Lee JK et al. (2012) Transcriptomics and com-

parative analysis of three Antarctic Notothenioid Fishes.

PLoS One, 7, e43762.

Smith TF (2008) Diversity of WD-repeat proteins. SubCellular

Biochemistry, 48, 20–30.

Smyth GK, Speed T (2003) Normalization of cDNA microarray

data. Methods, 31, 265–273.

Somero GN, Dahlhoff E, Lin JJ (1996) Stenotherms and eury-

therms: mechanisms establishing thermal optima and toler-

ance ranges. In: Animals and Temperature: Phenotypic and

Evolutionary Adaptations (eds Johnston IA & Bennett AF), pp.

538–578. Cambridge University Press, Cambridge.

Storch D, Lannig G, P€ortner HO (2005) Temperature-dependent

protein synthesis capacities in Antarctic and temperate

(North Sea) fish (Zoarcidae). Journal of Experimental Biology,

208, 2409–2420.



Tetens V, Wells RMG, Devries AL (1984) Antarctic fish blood:

respiratory properties and the effects of thermal acclimation.

Journal of Experimental Biology, 109, 265–279.

Thorne MAS, Burns G, Fraser KPP, Hillyard G, Clark MS

(2010) Transcription profiling of acute temperature stress in

the Antarctic plunderfish Harpagifer antarcticus. Marine Ge-

nomics, 3, 35–44.

Todgham A, Hoaglund E, Hofmann G (2007) Is cold the new

hot? Elevated ubiquitin-conjugated protein levels in tissues

of Antarctic fish as evidence for cold-denaturation of pro-

teins in vivo. Journal of Comparative Physiology. B, Biochemical,

Systemic, and Environmental Physiology, 177, 857–866.Turner J, Colwell SR, Marshall GJ et al. (2005) Antarctic climate

change during the last 50 years. International Journal of Clima-

tology, 25, 279–294.

Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of

microarrays applied to the ionizing radiation response. Pro-

ceedings of the National Academy of Sciences, USA, 98, 5116–5121.Windisch HS, Kath€over R, P€ortner H-O, Frickenhaus S, Lucas-

sen M (2011) Thermal acclimation in Antarctic fish: transcrip-

tomic profiling of metabolic pathways. American Journal of

Physiology: Regulatory, Integrative and Comparative Physiology,

301, R1453–R1466.

Windisch H, Lucassen M, Frickenhaus S (2012) Evolutionary

force in confamiliar marine vertebrates of different tempera-

ture realms: adaptive trends in zoarcid fish transcriptomes.

BMC Genomics, 13, 549.

H.W. and M.L. developed the concept and design of the

experiment in cooperation with R.K. (growth study), S.F.

and U.J. (array design and experimental design). H.W.

performed the experiments and analysed the data with

the help of S.F. and U.J. M.L. contributed to data interpre-

tation. H.W. wrote the manuscript, which was revised by

H.O.P., M.L., S.F. and U.J.

Data accessibility

The arrays were designed by utilization of sequences of

a transcriptomic cDNA library of P. brachycephalum

(SRA049761). Microarray data are available at ArrayEx-

press (Rustici et al. 2013), under IDs E-MTAB-2252

(design: A-MEXP-2248 for the 8 9 60 k format) and E-

MTAB-2256 (design: A-MEXP-2249 for the 2 9 105 k

format). Sequence annotations of the transcripts repre-

sented on the 60 K format as well as whole animal and

sampling data are available at Dryad doi:10.5061/dy-

rad.40rk0.

Supporting information

Additional supporting information may be found in the online ver-

sion of this article.

Appendix S1. Detailed information on statistics of physiologi-

cal data.

Appendix S2. Detailed expression profiles of responsive

probes in COG categories of known functions.

Appendix S3. Analyses of sex-specific expression patterns

within the 9°C incubation group.



Documents

Stress response or beneficial temperature acclimation: transcriptomic signatures in Antarctic fish ( Pachycara brachycephalum )