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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: heidrun.windisch@awi.de
© 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).
© 2014 John Wiley & Sons Ltd
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*
Cold Intermediate Warm
9Acclimation temperature (°C)
–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
Cold Intermediate Warm
***
9Acclimation temperature (°C)
–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%).
© 2014 John Wiley & Sons Ltd
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.
© 2014 John Wiley & Sons Ltd
3472 H. S . WINDISCH ET AL.
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.
© 2014 John Wiley & Sons Ltd
GENE EXPRESSION PATTERNS IN COLD- ADAPTED FISH 3473
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
© 2014 John Wiley & Sons Ltd
3474 H. S . WINDISCH ET AL.
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
© 2014 John Wiley & Sons Ltd
GENE EXPRESSION PATTERNS IN COLD- ADAPTED FISH 3475
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
© 2014 John Wiley & Sons Ltd
3476 H. S . WINDISCH ET AL.
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),
© 2014 John Wiley & Sons Ltd
GENE EXPRESSION PATTERNS IN COLD- ADAPTED FISH 3477
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
© 2014 John Wiley & Sons Ltd
3478 H. S . WINDISCH ET AL.
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
Cold Intermediate Warm
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.
© 2014 John Wiley & Sons Ltd
GENE EXPRESSION PATTERNS IN COLD- ADAPTED FISH 3479
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.
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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.
© 2014 John Wiley & Sons Ltd
3482 H. S . WINDISCH ET AL.
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