Abstract
Background
The green crab Carcinus maenas is known for its high acclimation
potential to varying environmental abiotic conditions. A high ability
for ion and acid-base regulation is mainly based on an efficient
regulation apparatus located in gill epithelia. However, at present it
is neither known which ion transport proteins play a key role in the
acid-base compensation response nor how gill epithelia respond to
elevated seawater pCO[2 ]as predicted for the future. In order to
promote our understanding of the responses of green crab acid-base
regulatory epithelia to high pCO[2], Baltic Sea green crabs were
exposed to a pCO[2 ]of 400 Pa. Gills were screened for differentially
expressed gene transcripts using a 4,462-feature microarray and
quantitative real-time PCR.
Results
Crabs responded mainly through fine scale adjustment of gene expression
to elevated pCO[2]. However, 2% of all investigated transcripts were
significantly regulated 1.3 to 2.2-fold upon one-week exposure to CO[2
]stress. Most of the genes known to code for proteins involved in osmo-
and acid-base regulation, as well as cellular stress response, were
were not impacted by elevated pCO[2]. However, after one week of
exposure, significant changes were detected in a calcium-activated
chloride channel, a hyperpolarization activated nucleotide-gated
potassium channel, a tetraspanin, and an integrin. Furthermore, a
putative syntaxin-binding protein, a protein of the transmembrane 9
superfamily, and a Cl^-/HCO[3]^- exchanger of the SLC 4 family were
differentially regulated. These genes were also affected in a
previously published hypoosmotic acclimation response study.
Conclusions
The moderate, but specific response of C. maenas gill gene expression
indicates that (1) seawater acidification does not act as a strong
stressor on the cellular level in gill epithelia; (2) the response to
hypercapnia is to some degree comparable to a hypoosmotic acclimation
response; (3) the specialization of each of the posterior gill arches
might go beyond what has been demonstrated up to date; and (4) a
re-configuration of gill epithelia might occur in response to
hypercapnia.
Background
With increasing atmospheric pCO[2], a decrease in global surface ocean
pH of between 0.4 to 0.8 units is predicted due to oceanic CO[2 ]uptake
[[34]1,[35]2]. Although large changes in atmospheric CO[2 ]have been
recorded throughout earth history, the current anthropogenic increase
in pCO[2 ]is much more rapid and severe than the cyclic changes of
pCO[2 ]during the last 20 million years [[36]3]. The resulting changes
in carbonate chemistry speciation, termed 'ocean acidification', may
become a general stress factor modulating future marine communities by
differentially influencing the fitness of marine species [[37]4-[38]7].
Many studies suggest complications for marine ectothermic metazoans in
response to future ocean acidification: reduced growth and
calcification rates, reduced rates of development, altered energy
budgets, disturbed acid-base status, disturbed chemosensory function
and even increased rates of mortality have been measured
[[39]4,[40]7-[41]14]. On the other hand, increases in growth and
calcification rate were shown primarily in organisms that accumulate
significant concentrations of bicarbonate in their body fluids, e.g.
decapod crustaceans [[42]12], cephalopods [[43]15] and fish [[44]16].
Coping with ocean acidification
Biological impacts of acidification are strongly related to life
history, genetic pre-disposition and physiological acclimation
potential of the species in question. Elevated environmental pCO[2
]results in an increased extracellular CO[2 ]partial pressure, as
positive diffusion gradients of CO[2 ]have to be maintained in order to
excrete metabolic CO[2 ][[45]5]. This can then lead to an acidification
of extracellular fluids [[46]17-[47]19]. However, several active, high
metabolic species with pH sensitive respiratory pigments regulate
extracellular pH (pH[e]): active modulation of the extracellular
carbonate system leads to bicarbonate accumulation and pH compensation
while maintaining pCO[2 ]values sufficiently high for diffusive CO[2
]flux out of the animal [fish: 20; crustaceans: 21; cephalopods: 22,
23]. In teleost fish, cephalopods, and decapod crustaceans, the
majority of the acid-base relevant ion regulatory apparatus is located
in gill epithelia. It is thought that net proton extrusion is primarily
achieved via active (V-type H^+-ATPase) and secondarily active ion
transport molecules (e.g. sodium proton exchangers, NHE; sodium
bicarbonate cotransporters, NBC), with a strong supporting role of
carbonic anhydrases (CAs) and Na^+/K^+-ATPase [[48]24].
Carcinus maenas
In the case of the hyperosmoregulator Carcinus maenas (green or shore
crab), the three posterior gills number 7-9 have been found to be
involved in osmo- and acid-base regulation, while the anterior six gill
pairs serve the demands of gas exchange [[49]25-[50]27]. Studies of
Truchot [[51]25] demonstrated that C. maenas actively and rapidly
accumulates HCO[3]^- in its hemolymph during acute exposure to elevated
seawater pCO[2 ](690 Pa). However, active adjustment of the
extracellular carbonate system might not only be a short-term response
in this species, but a persistent physiological response in order to
maintain high extracellular pH (pHe). Despite efficient pHe regulatory
control, other physiological aspects might be negatively impacted by
hypercapnia and might show effects on the animal's performance only in
the long run. Up to date, only few studies investigated long-term
effects of acid-base disturbance in crustaceans on the physiological
level. Potential reponses may include an altered calcification rate, as
demonstrated for three crustacean species (the crab Callinectes
sapidus, the shrimp Penaeus plebejus and lobster Homarus americanus) by
Ries et al. [[52]12], and other invertebrates, such as the cuttlefish
Sepia officinalis [[53]15]. Other adaptations to long-term hypercapnia
may be similar to what has been observed as responses upon hypoxia,
both factors being closely linked to each other. These might include
energy conserving strategies (e.g. down-regulation of protein synthesis
and modification of certain regulatory enzymes) as can be observed in
response to hypoxia in mussels [[54]28], fish [[55]29] or reptiles
[[56]30] (reviewed by [[57]31]).
On the molecular level, it is not known at present which ion
transporters play a key role in the short- and the long-term response
to hypercapnia in decapod crustaceans. Although models for the
organization of the gill epithelia of euryhaline crabs have been
postulated [[58]32,[59]33], the transporter inventory in decapod
crustacean gill epithelia and their functional interactions are not
fully understood at present. As C. maenas is exposed to regular
short-term fluctuations in pH in its highly variable habitat in the
western Baltic Sea [[60]19], not only changes in gene expression levels
may play a role in its acid-base compensation response. A compensatory
stress response can as well take place on the post-transcriptional
level e.g. through covalent modification of certain regulatory enzymes
and their activity through phosphorylation-dephosphorylation reactions.
This has been shown e.g. for the enzyme phosphofructokinase, the key
enzyme in controlling anaerobic carbon flow in glycolysis in mussels in
response to hypoxia [[61]34], and fish [[62]35]. A post-translational
alteration has also been hypothetized for the enzyme carbonic anhydrase
in the blue crab Callinectes sapidus, a close relative of C. maenas
[[63]36].
Aim of the study
In order to promote our understanding of ion and acid-base regulatory
processes, green crabs from the Baltic Sea were exposed to control (39
Pa) and elevated (400 Pa) pCO[2 ](100 Pa = 987 μatm). The experimental
conditions in the laboratory were chosen to mimick the parameters in
the crabs' natural environment, the brackish water system of the
western Baltic Sea. The chosen experimental level of pCO[2 ]enrichment
(400 Pa) represents a stress scenario that could occur in the natural
habitat within the next 100 years: pH in Kiel Fjord can reach values as
low as 7.5 (corresponding pCO[2 ]= 230 Pa) in surface waters during
summer and autumn. Future changes in pCO[2 ]will be more severe in this
habitat than in the average surface ocean [[64]19]. As a strong
extracellular acid-base reaction has been observed in C. maenas exposed
to hypercapnia [[65]25], we hypothesize that the ion regulatory
transcriptome of C. maenas will respond to hypercapnia with a short-
and long-term adjustment of expression of important ion- and acid-base
transporter candidates (e.g. Na^+/K^+-ATPase, Na^+/H^+ exchanger (NHE),
V - type H^+-ATPases, Cl^-/HCO[3]^- exchangers and carbonic anhydrases;
[[66]33]). As little is known about candidate genes for acid-base
regulation in crustaceans, we chose to utilize a microarray approach to
screen for previously unrecognized candidates. Therefore, gene
expression profiles of gills of short-term (3 and 7 days) exposed crabs
were investigated using a 4,462-feature microarray assay recently
developed by Towle et al. [[67]37]. Expression levels of distinct
candidate genes were further investigated using quantitative real-time
PCR analysis following long-term exposure to hypercapnia (11 weeks).
Expression profiles obtained in our study were compared to those
obtained in a recent hyposmotic study, in which gene expression changes
after transfer of C. maenas from a salinity of 32 to 15 was
investigated using the same microarray [[68]37]. A second focus of
interest was placed on genes known to be in involved in the cellular
stress-response (e.g. heat-shock proteins, superoxide dismutase,
glutathione reductase [[69]38]).
This is the first study that elucidates the impact elevated seawater
pCO[2 ]has on the gill transcriptome of a decapod crustacean.
Methods
Animals, exposure, and tissue sampling
Short-term CO[2 ]experiments
Green crabs (Carcinus maenas) were caught in traps in April 2009 in
Kiel Fjord (Baltic Sea) at the IFM-GEOMAR pier (westshore building) at
3-4 m depth (54°19.8'N; 10°9.0'E). In order to acclimate the animals to
laboratory conditions, they were kept in a flow-through tank (H30 × W30
× L50 cm) in a climate chamber at IFM-GEOMAR with ambient aerated
Baltic Sea brackish water at 13°C for 3 to 7 days. Experimental animals
were fed ad libitum with mussels (Mytilus edulis) during this time.
Female crabs (n = 24, 6 animals per CO[2 ]treatment and day) with a
mean carapace width of 4.6 ± 0.2 cm were then chosen for the short-term
experiment and two each were transferred to 20 L tanks of a
flow-through seawater CO[2 ]manipulation system (flow rate = 100 mL
min^-1) that was supplied with ambient Baltic Sea brackish water of a
salinity of 14 - 15 (Practical salinity scale, as it will be used
throughout the script). Temperature was held constant in the storage
tank by a thermostat set to 13°C. A light-dark cycle of 12:12 h was
established. The system design was identical to that presented in
Thomsen et al. [[70]19].
After 24 hours of acclimation in the experimental system resembling the
natural parameters encountered in Kiel Bight (S = 14.9, T = 12.9 ±
0.1°C), crabs were exposed to control (53 Pa, pH = 8.11 ± 0.04) and
elevated (440 Pa, pH = 7.24 ± 0.03) pCO[2 ]for 3 or 7 days. CO[2 ]was
provided by a central automatic CO[2]-mixing-facility (Linde Gas, HTK
Hamburg, Germany). Animals were fed ad libitum with crushed mussels
(Mytilus edulis).
After 3 days, one of the two animals in each tank was removed for
analysis, anaesthetized on ice and then killed by destroying its
ventral ganglion and removal of the carapace. Left gills #7 and #9 were
carefully removed with forceps and immediately stored in RNAlater^®
(Ambion, #AM7021) until microarray analysis at the Mount Desert Island
Laboratory, Salisbury Cove, Maine/USA, or quantitative real-time PCR
(qRT-PCR at the IFM-GEOMAR, Kiel. The procedure was repeated for the
second animal of each tank after 7 days of incubation.
For qRT-PCR on gills of short-term exposed animals, a repeated
incubation of Carcinus maenas specimens was performed in May 2010.
Again, Baltic Sea green crabs were caught in traps in Kiel Fjord at the
IFM-GEOMAR pier. Females had a mean carapace width of 4.8 ± 0.6 cm.
System parameters in the acclimation phase were S = 13.4 and T = 10.8 ±
0.1°C. The animals (n = 12, 6 animals per CO[2 ]treatment and day) were
then exposed to seawater of pH 7.99 ± 0.02 (68 Pa CO[2]) and pH 7.28 ±
0.05 (361 Pa CO[2]) as described above. Gills were isolated only after
7 days in this experiment.
Abiotic seawater parameters
To evaluate the carbonate system parameters, water samples (500 mL)
were taken of 4 randomly chosen tanks each for day 3 and day 7 of the
short-term hypercapnia experiment in 2009. For the second short-term
exposure in 2010, water samples of 4 control and 4 experimental aquaria
were taken only on day 7. Total dissolved inorganic carbon (C[T ]=
[CO[2]*] + [HCO[3]^-] + [CO[3]^2-]) was measured according to Dickson
and Millero [[71]39]. In case of the second 1 week exposure in 2010,
C[T ]was measured using an AIRICA CT analyzer (Marianda GmbH, Kiel,
Germany). In all experiments, salinity, pH and temperature were
assessed daily using a pH- and salinometer (WTW 340i pH-analyzer/WTW
SenTix 81-measuring chain, WTW cond 315i/WTW TETRACON 325-measuring
chain).
Seawater total alkalinity (A[T]) and pCO[2 ]was calculated from pH and
C[T ]using CO2SYS software [[72]40] and the appropriate parameter and
constants (dissociation constants K1 and K2 according to Roy et al.
[[73]41], KHSO[4 ]dissociation constant after Dickson [[74]42], NBS
scale [mol/kg H[2]O]; table [75]1).
Table 1.
Carbonate system parameters of all hypercapnia experiments with
Carcinus maenas
1 week experiment 2009 1 week experiment 2010 11 week experiment 2009
Parameter control experiment control experiment control experiment
__________________________________________________________________
C[T ][μmol/kg] (measured) 2025 ± 29 2242 ± 37 1879 ± 7 2047 ± 10 1999 ±
18 2209 ± 24
__________________________________________________________________
A[T ][μmol/kg] (calculated) 2098 ± 29 2062 ± 26 1904 ± 5 1887 ± 9 2046
± 19 2061 ± 20
__________________________________________________________________
pH (measured) 8.12 ± 0.02 7.24 ± 0.02 8.00 ± 0.02 7.28 ± 0.01 8.06 ±
0.01 7.36 ± 0.01
__________________________________________________________________
S (measured) 15.35 ± 0.06 15.30 ± 0.00 13.40 ± 0.02 13.4 ± 0.02 14.80 ±
0.13 14.80 ± 0.13
__________________________________________________________________
T (measured) [°C] 12.90 ± 0.16 12.98 ± 0.05 10.87 ± 0.02 10.77 ± 0.13
12.90 ± 0.07 12.90 ± 0.07
__________________________________________________________________
pCO[2 ](calculated) [Pa] 53 ± 2 440 ± 25 68 ± 3 361 ± 9 40 ± 5 230 ± 21
[76]Open in a new tab
Measured parameters of the carbonate system were assessed daily during
the acclimation phase of 1 or 11 weeks, while A[T ]and pCO[2 ]were
calculated applying the measured parameters and CO2SYS software (see
Material and Methods for details). Under experimental conditions
(elevated pCO[2]), a drop in pH of 0.7 - 0.9 units could be observed.
The carbonate system remained stable during the acclimation phase in
both, short- (1 week) and long-term (11 weeks) experiments. C[T ]=
total dissolved inorganic carbon, A[T ]= total alkalinity, S =
salinity, T = temperature, pCO[2 ]= partial pressure of CO[2]. Values
are given as mean with standard deviation.
Long-term CO[2 ]experiments
Green crabs (Carcinus maenas) for the long-term experiment were part of
a study by Appelhans et al. (under review) that was conducted in
parallel. Experimental animals for this long-term study were caught in
traps in April 2009 in Kiel Fjord (Baltic Sea) at the IFM-GEOMAR pier
(westshore building) in 3 to 4 m water depth (54°19.8'N; 10°9.0'E).
Animals with a carapace width of 4.5 ± 0.5 cm were chosen
irrespectively of their sex (n = 9 animals per CO[2 ]treatment). Crabs
were exposed to control (40 Pa, pH = 8.06 ± 0.01) and elevated pCO[2
](230 Pa, pH = 7.36 ± 0.01). Animals were fed with 2 mussels (size =
2.50 ± 0.35 cm) three times a week. To evaluate the carbonate system
parameters, water samples of three aquaria per treatment level were
taken at the beginning, the intermediate phase and at the end of the
experiment, respectively. Analysis of C[T ]was coulometric (SOMMA
System autoanalyser, Marianda GmbH, Kiel, Germany) and A[T ]analyzed
via potentiometric titration (VINDTA autoanalyser, Marianda; table
[77]1). An overview over the different experiments and sampling points
is depicted in Figure [78]1.
Figure 1.
[79]Figure 1
[80]Open in a new tab
Overview over the experiments performed on Carcinus maenas specimen of
the Baltic Sea. A short-term incubation experiment was conducted in
April 2009 and microarray analysis was performed on isolated RNA of
gills of these animals exposed to control (nominal 39 Pa) and elevated
(nominal 400 Pa) pCO[2 ]for 3 and 7 days. The experiment was repeated
in 2010 and this time, RNA was isolated for quantitiative real-time
polymerase chain reaction (qRT-PCR) of gill 9 after 7 days. In
parallel, a long-term experiment was performed on Baltic Sea C. maenas
in 2009 by Appelhans et al. (under review), and RNA of gill 7 was
isolated and analysed by qRT-PCR.
Microarray experiment
RNA extraction
RNA from the first short term experiment (2009) was extracted from
whole left gills #7 and #9 (control pCO[2], n = 6; elevated pCO[2], n =
6) using the RNeasy Midi-Kit from Qiagen (#75144). Gills were
homogenized with an OMNI international TH/G7-195STW for 50 s in 4 mL
buffer RLT from the Qiagen kit. Extraction then followed the
manufacturer's description. RNA quality was monitored by
electrophoresis via the Agilent 2100 Bioanalyzer system. Each RNA
sample was then reversely transcribed into cDNA and in parallel
fluorescence-labeled using the SuperScript™ Plus Direct cDNA Labeling
System from Invitrogen (#L1015-06), following the provided protocol.
Using both anchored oligo(dT)20 and random hexamer primers included in
the kit, control cDNA was labeled with AlexaFluor555 (green
fluorescent, equivalent to Cy™3; excitation: 555 nm, emission: 565 nm)
while cDNA of the CO[2]-treatment samples was labeled with
AlexaFluor647 (red fluorescent, equivalent to Cy™5; excitation: 650 nm,
emission: 670 nm).
Microarray assay for Carcinus maenas
The C. maenas microarray was developed by D. W. Towle et al. and
prepared as described in Towle et al. [[81]37]. The annotation of the
respective contigs included in the microarray, based on a Carcinus
maneas EST library (deposited at [82]http://www.ncbi.nlm.nih.gov), was
performed by Towle et al. [[83]37] using BLASTX analysis on a local
TimeLogic DeCypher server (Active Motif, Inc.). The most informative
BLASTX hit for each contig sequence was selected manually from the ten
highest scoring hits identified by the programm. In order to improve
the information on each included gene, a re-annotation of the contigs
has been performed in 2009 by the authors of this study and compared to
the existing annotation (Additional File [84]1 Table S1). A sequence
blast with the online portal Blast2GO [[85]43] was performed to improve
the quality of the annotations. Additionally, a mapping and substance
Gene Ontology (GO) annotation analysis was performed to amend
information of biological functions [[86]44], including: (1) Enzyme
Commission number (EC), (2) InterPro scan, (3) annotation augmentation
through the Second Layer concept (ANNEX) and (4) summarizing annotation
results by creating a subset of the gene ontology vocabulary
encompassing key ontological terms (GOslim). Additionally, direct NCBI
blasts were performed for selected genes of special interest.
Microarray analysis of the short-term samples (3 and 7 days)
Hybridization of the microarray slides was performed using the MAUI
Hybridization system (MAUI Mixer SC Hybridization Chamber mixers,
Biomicro Cat. no. 02A00830) using the Pronto!Universal Hybridization
Kit. Slides were scanned in an Axon GenePix 4000B dual wavelength
scanner using the GenePix 6.0 software.
Initial within-slide normalization (lowess-normalization) was performed
by the Acuity 4.0 software (Input parameter: Smoothing = 0.19 (chosen
by graphical/visual evaluation), Iterations = 3 (default), Delta = 0.01
(default)). For further processing and statistical analysis, data was
then exported to Excel. Filtering of the data set started with
excluding low-quality features according to the "flagging" performed in
Acuity 4.0. Genes were defined "absent" and excluded from further
analysis, when the fluorescence intensity of one or both channels was
less than 30% higher than the background fluorescence intensity. Also,
transcripts with a variance of more than 20% in the 4 technical
replicates included in each slide were not included in the data set.
Gene expression was calculated as the log[2 ]of the ratio of the
fluorescence intensity of the CO[2]-treatment cDNA to the fluorescence
intensity of the control cDNA (log[2]-ratio = F635/F532) and as the
median and median deviation (MD) of each 6 replicate microarray slides
per block (gill7day3, gill7day7, gill9day3, gill9day7).
Quantitative polymerase chain reaction (qRT-PCR)
RNA extraction
RNA from whole left gills #9 (only day 7, control CO[2], n = 6;
elevated CO[2], n = 6) from the second 1 week experiment (2010) and
from whole left gills #7 (control CO[2], n = 9; elevated CO[2], n = 9)
of the 11 week experiment was extracted using the RNeasy Midi-Kit from
Qiagen (#75144). Gills were first homogenized with an OMNI
international TH/G7-195STW for 50 s in 4 mL buffer RLT from the kit.
Extraction then followed the manufacturer's description.
Primers
A number of transcripts shown to undergo changes in the 1 week
microarray experiment and/or known to be involved in acid-base
regulation were selected for analysis by qRT-PCR, according to their
relevant function and statistical significance. Primers were designed
according to the database previously generated by D.W.Towle [[87]37],
using PrimerExpress software (Version 2.0, Applied Biosystems). Primer
quality and specificity was tested by blasting with the EST library for
Carcinus maenas from NCBI. Forward and reverse primers were generated
with the following parameters: GC content 30-80%, primer length 9-40 bp
(opt. 20 bp), Amplicon melting temperature 0-85°C, Amplicon length
70-150 bp (Additional File [88]1 Table [89]2). Performance and
efficiency of each primer pair was tested in triplets in a cDNA
dilution series (1:40, 1:80, 1:160, 1:320, 1:640). Performance was
evaluated as suitable if the R^2 of the linear regression of the
dilution series was > 0.98 and no background noise (unspecific binding)
was detected. A melting curve analysis was performed for each reaction
to ensure a single PCR amplicon. Efficiency was calculated as follows:
Table 2.
Enrichment analysis in the short-term hypercapnia microarray study on
Carcinus maenas
Test-set GO name FDR Over-/Under- representation
down day 3 GO:0050794 regulation of cellular process 0.00 under
__________________________________________________________________
down day 3 GO:0007165 signal transduction 0.00 under
__________________________________________________________________
down day 3 GO:0023060 signal transmission 0.00 under
__________________________________________________________________
down day 3 GO:0023046 signaling process 0.00 under
__________________________________________________________________
down day 3 GO:0003824 catalytic activity 0.00 under
__________________________________________________________________
down day 3 GO:0050789 regulation of biological process 0.04 under
__________________________________________________________________
up day 3 no enrichment - - -
__________________________________________________________________
down day 7 no enrichment - - -
__________________________________________________________________
up day 7 no enrichment - - -
__________________________________________________________________
up gill 7 GO:0007010 cytoskeleton organization 0.05 under
__________________________________________________________________
down gill 7 GO:0005623 cell 0.04 over
__________________________________________________________________
up gill 9 GO:0005198 structural molecule activity 0.03 over
__________________________________________________________________
down gill 9 no enrichment - - -
[90]Open in a new tab
Results of the enrichment analysis (Fisher's Exact Test with multiple
testing correction; FDR < 0.05) as implemented in Blast2GO [[91]43].
Tested were subsets of all significantly regulated transcripts as
identified by sign test (up- and down-regulated separately for each
gill and day or each combination of both). Reference-set in each
analysis was the complete microarray sequence Blast2GO database
generated from the contig list. GO = Gene Ontology term, name = GO name
of the functional group, FDR = false discovery rate for correction of
multiple testing, Over-/Underrepresentation = a GO term is considered
over-/under-represented if it appears significantly more often/less
often in the reference-set than it does in the test-set.
E = 10 ^(-1/S), with S being the slope of the linear regression of the
dilution series. Only primer pairs with an efficiency between 1.9 - 2.1
were chosen for analysis.
qRT-PCR
Following DNase digestion of 2 μg total RNA with the DNAfree™ Kit from
Ambion (#AM1906), 0.4 μg DNAfree RNA was then transcribed into cDNA
using the High Capacity cDNA RT Kit from Applied Biosystems (Part no.
4368814). Real-time PCR was performed on 80-fold diluted cDNA in
96-well plates using the FastSYBR^®Green Master Mix (Part no. 4385612)
and the StepOnePlus™ Real-Time PCR System from Applied Biosystems
(PCR-conditions: 20 s at 95°C, 40 cycles of 5 s at 95°C and 30 s at
62°C). On each plate, one control cDNA and one experimental cDNA were
analyzed with the same primer pairs, as well as the respective DNAfree
RNA for each of the two samples as control for DNA contamination, and
arginine kinase as housekeeping gene to eliminate within-plate
variation. Arginine kinase was chosen as housekeeping gene according to
[[92]36] and was tested to be appropriate (stability value = 0.007)
with NormFinder ([[93]45], available at
[94]http://www.mdl.dk/publicationsnormfinder.htm). Only those samples
were included in the analysis that showed no or low background in the
DNAfree RNA control compared to the cDNA samples (difference in cycles
> 9 ≈ 0.3%). Gene expression was calculated based on the
C[T]-threshold. First, absolute quantities Q[X ]of all genes were
calculated as
[MATH: QX(gene<
/mi>)=(E)CT :MATH]
, followed by the calculation of normalized quantities
[MATH: QN(gene<
/mi>)=Q<
mrow>X(gene<
/mi>)<
mrow>QX(HKge<
/mi>ne)
:MATH]
, with Q[X (HK gene) ]being the absolute quantity of the housekeeping
gene arginine kinase. For matters of consistency with the microarray
data, gene expression resulting from qRT-PCR analysis was also
calculated as the log[2]-ratio of normalized gene quantity in elevated
pCO[2 ]treated animals to normalized gene quantity in control pCO[2
]animals.
Statistics
Because of the high biological variance within replicate samples,
non-parametric statistical tests were chosen. To identify significant
changes in gene expression, a two-sided sign test with α = 0.05 was
performed on the fluorescence intensities of the filtered and
lowess-normalized data set for each of the four experimental blocks
(gill7day3, gill7day7, gill9day3, gill9day7). For each individual
transcript in each block, the fluorescence intensities for the
F635-dye, representing the animals being exposed to elevated pCO[2],
were tested against the control animals (F532-dye). The test only
allowed for including those transcripts that were represented by 5 or 6
replicates.
Sign test was also applied on the experimental vs. control normalized
gene quantities of the qRT-PCR.
In order to detect differences between gills and days of the short-term
exposed animals, Wilcoxon's matched pairs test as implemented in
STATISTICA 8 was applied with α = 0.05. Prior to testing, microarray
values were transformed into a matrix with 1 = significantly
up-regulated, -1 = significantly down-regulated, 0 = not significantly
regulated transcript. Data of each sampling day and gill were then
tested against each other.
Additionally, intensity values for both fluorescence dyes of the
processed microarray data (lowess-normalized and filtered as described
above) was read into the R software environment (version 2.13.1,
[95]http://www.R-project.org), using the R/maanova package (version
2.22.0, [[96]46]). As the package does not allow for missing data, only
transcripts represented by all 6 replicates could be tested by this
method. A linear mixed model
[MATH: yijk=μ+CO2i+Gj+Dk+eijk :MATH]
was fitted to the data, with yijk as the normalized-transformed gene
expression, μ as the group mean, CO2i as the effect of the CO[2 ]level,
Gj as the effect of ith gill, Dk as the effect of jth day, and eijk as
the sample effect (random error). The significance of effects of the
fixed factors "CO[2 ]treatment level", "gill" and "day" on the
differential expression of genes was tested by appropriate contrasts in
F-tests between groups in a multifactorial ANOVA design. The F-tests
were calculated with the James-Stein shrinkage estimate (Fs),
incorporating shrinkage estimates of variance components [[97]47]. P
values were calculated by performing 1,000 permutations of samples to
break their association to expression values, then corrected for
multiple comparisons by false discovery rate transformation (FDR),
using the qvalue package (jsFDR, version 1.26.0 [[98]48]) with a 5% FDR
cutoff.
To identify overall affected pathways, enrichment analysis (FDR < 0.05)
was performed using Fisher's exact test as implemented in Blast2GO
[[99]43]. All significantly regulated genes identified by sign test,
divided into subsets for each gill and day, were used as test-sets and
tested against a complete microarray sequence Blast2GO database
generated from the contig list. It has to be considered that Carcinus
maenas is a non-model organism and that therefore the annotation of
gene functions/Gene ontology (GO) terms is not comprehensive.
Results
General findings of the microarray analysis
Using variance-based analysis utilizing the R/maanova package in R, we
only identified the factor "CO[2]" to have a significant effect on gene
expression, while factors "Gill" and "Day" did not significantly
influence expression patterns. 956 out of 3,720 tested transcripts were
found to be differentially expressed between the two CO[2 ]levels. This
accounts for 26% and is comparable to the result of the sign test (24%,
see below). 378 (40%) of the transcripts identified to be
differentially expressed for the factor CO[2 ]in the variance-base
approach were also identified as significantly regulated by sign test,
including 11 (65%) of the transcripts of special interest as presented
in table [100]3. The sign test identified as many as 1,056 (24%) out of
the 4,462 transcripts on the microarray to be significantly up- or
down-regulated in at least one gill or at one sampling time point
during the short-term response to elevated seawater pCO[2 ](p ≤ 0.05;
Figure [101]2; Additional File [102]1 Table S3). Significant
up-regulation was observed in 541, down-regulation in 502 genes (13
genes showed a mixed regulation pattern). However, magnitudes of change
were generally low: expression of only 84 genes of the significantly
regulated transcripts (sign test, accounting for 2% of the whole
microarray and 8% of the significantly altered transcripts) were
modulated by a log[2]-ratio > |0.200| (sign test; equivalent to a >
1.3-fold change). F-test identified 16 additional transcripts with a
log[2]-ratio > |0.200|.
Table 3.
Regulation of transcripts of special interest in the short-term
hypercapnia study on Carcinus maenas
CO[2 ]experiments 3 + 7 days CO[2 ]experiments 11 weeks Salinity
experiments 7 days
[MA; med. log2-ratio ± MD] [qPCR; med. log2-ratio ± MD] [MA; mean
log2-ratio ± SD]
rank ACC. no. Name gill 7 - day 3 gill 9 -day 3 gill 7 - day 7 gill 9
-day 7 gill 7 - 10 weeks gill 8 - day 7
1 [103]DV944570.1 senescence-associated protein -0.12 ± 0.50 -0.03 ±
0.03 -1.16* ± 0.64 -0.07 ± 0.25 0.69 ± 0.57 -0.06 ± 0.03
__________________________________________________________________
2 [104]DW250369.1 pg1 protein 1.01 ± 0.61 0.60 ± 1.14 -0.34 ± 0.49
-0.29 ± 0.56 - 1.644 ± 0.414
__________________________________________________________________
3 [105]DN161290.1 unknown (putative Syntaxin binding protein 2) 0.32 ±
0.32 0.42 ± 0.10 0.88* ± 0.10 0.67 ± 0.31 0.49 ± 0.66 2.86 ± 0.02
__________________________________________________________________
4 [106]DN796131.1 23S ribosomal RNA gene (a) -1.20 ± 0.92 -0.25 ± 0.18
-1.14 ± 1.16 -0.87* ± 0.46 - 0.36 ± 0.34
__________________________________________________________________
5 [107]DW584691.1 calcium-activated chloride channel 0.25 ± 0.40 0.81*
± 0.34 -0.09 ± 0.30 0.16 ± 0.31 1.12 ± 0.88 -
__________________________________________________________________
6 [108]DN635040.1 23S ribosomal RNA gene (b) -0.16 ± 1.42 0.09 ± 0.64
-0.80* ± 0.22 -0.27 ± 0.49 - 1.29 ± 0.16
__________________________________________________________________
7 [109]DV944193.1 rRNA intron-encoded homing endonuclease 0.54 ± 1.00
0.80 ± 0.81 0.06 ± 0.22 0.59 ± 0.41 - 1.073 ± 0.38
__________________________________________________________________
8 [110]DW249535.1 unknown -0.12 ± 1.43 0.04 ± 0.55 -0.71* ± 0.62 -0.41
± 0.65 - 0.99 ± 0.29
__________________________________________________________________
9 [111]DY656451.1 unknown -0.15 ± 0.23 0.05 ± 0.26 0.57* ± 0.09 0.04 ±
0.27 - -0.15 ± 0.30
__________________________________________________________________
10 [112]DW251049.1 unknown -0.35 ± 0.24 -0.53* ± 0.28 -0.05 ± 0.22
-0.10 ± 0.19 - -0.86 ± 0.23
__________________________________________________________________
11 [113]DN551127.1 unknown 0.54 ± 0.25 0.26 ± 0.14 0.48* ± 0.31 0.37 ±
0.48 - 2.34 ± 0.07
__________________________________________________________________
12 [114]DW250828.1 thymosin-repeated protein 1 -0.39 ± 0.21 -0.46* ±
0.14 0.13 ± 0.21 0.19 ± 0.21 - -0.05 ± 0.06
__________________________________________________________________
13 [115]DN202592.1 multispanning membrane protein (transmembrane 9
superfamily protein member 4) 0.35 ± 0.23 0.21 ± 0.14 0.46* ± 0.11 0.36
± 0.34 - 2.15 ± 0.08
__________________________________________________________________
14 [116]DW250260.1 hyperpolarization activated cyclic nucleotide-gated
potassium channel-2 -0.26 ± 0.30 -0.14 ± 0.08 -0.46* ± 0.24 -0.14* ±
0.08 -0.13 ± 0.26 -0.11 ± 0.09
__________________________________________________________________
18 [117]DV944030.1 tetraspanin 3 (Transmembrane super family 4 member
8) -0.22 ± 0.11 -0.41* ± 0.05 0.01 ± 0.27 0.09 ± 0.03 - -0.13 ± 0.06
__________________________________________________________________
25 [118]DY307809.1 potential integrin alpha-7 -0.12 ± 0.36 0.26 ± 0.21
-0.35* ± 0.28 -0.03 ± 0.26 - -
__________________________________________________________________
29 [119]DV642643.1 kazal-type proteinase inhibitor 0.25* ± 0.17 0.21* ±
0.12 0.33* ± 0.20 0.24* ± 0.15 0.12 ± 0.84 1.04 ± 0.11
__________________________________________________________________
31 [120]DN635395.1 unknown -0.17* ± 0.04 -0.14* ± 0.04 -0.32* ± 0.06
-0.13* ± 0.06 0.25 ± 0.68 -0.11 ± 0.11
__________________________________________________________________
63 [121]DV943872.1 predicted ATPase -0.34 ± 0.33 -0.24 ± 0.10 -0.58 ±
0.39 -0.23* ± 0.09 - -0.65 ± 0.23
__________________________________________________________________
88 [122]CX994129.1 anion/bicarbonate transporter family member (SLC
family 4, member 1) -0.31 ± 0.11 -0.20* ± 0.09 -0.22 ± 0.07 -0.22 ±
0.11 0.18 ± 0.54 0.41 ± 0.11
__________________________________________________________________
- [123]DN739347.1 glycosyl-phosphatidylinositol-linked carbonic
anhydrase VII -0.09 ± 0.05 -0.06 ± 0.06 -0.13* ± 0.07 -0.08* ± 0.03
0.40 ± 0.35 -0.92 ± 0.07
__________________________________________________________________
- [124]DN551450.1 glutathione peroxidase 0.02 ± 0.06 0 ± 0.05 0.03* ±
0.02 -0.03 ± 0.05 -0.90 ± 0.24 -0.81 ± 0.14
__________________________________________________________________
- [125]DY656042.1 vacuolar-type H+-ATPase subunit A -0.05 ± 0.37 0.11 ±
0.09 -0.11 ± 0.09 0.07 ± 0.06 0.45 ± 0.65 -0.17 ± 0.27
__________________________________________________________________
- [126]DY657461.1 v-type proton ATPase subunit E 0.02 ± 0.03 -0.02 ±
0.04 0 ± 0.03 -0.02 ± 0.03 0.30 ± 0.39 -0.12 ± 0.17
__________________________________________________________________
- [127]DW251095.1 cytoplasmic carbonic anhydrase 0.07 ± 0.01 -0.03 ±
0.06 0 ± 0.07 0.01 ± 0.01 -0.12 ± 1.20 1.78 ± 0.16
__________________________________________________________________
- [128]DN202373.1 anion/bicarbonate transporter family member (SLC
family 4, member 11) -0.02 ± 0.03 0.02 ± 0.06 -0.02 ± 0.10 0.01 ± 0.02
0.02 ± 0.64 -1.29 ± 0.47
__________________________________________________________________
- [129]DN796352.1 K+/Cl- symporter (solute carrier family 12, member 6)
-0.04 ± 0.04 0.22 ± 0.17 0.01 ± 0.04 -0.07 ± 0.07 0.91 ± 0.30 0.25 ±
0.23
__________________________________________________________________
- [130]DV111894.1 Na+/K+/2Cl- cotransporter 0.05 ± 0.05 0.01 ± 0.05 0 ±
0.06 0 ± 0.10 0.62 ± 0.57 -0.20 ± 0.09
__________________________________________________________________
- [131]DW250552.1 Na+/K+ ATPase alpha subunit 0.04 ± 0.05 0.02 ± 0.05
0.05 ± 0.10 0.03 ± 0.16 0 ± 1.11 1.38 ± 0.11
__________________________________________________________________
- [132]DY308103.1 Na+/H+ exchanger (NHE 3) - - - - 0.27 ± 0.03 -0.13 ±
0.14
[133]Open in a new tab
Included are the 14 most strongly regulated transcripts (as identified
by sign test and F-test), as well as several transcripts which have a
potential function during structural modifications or in ion- and
acid/base regulation. Transcripts are ordered according to the rank in
absolute regulation. Accession numbers (ACC. no.) refer to the ESTs
generated for the C. maenas microarray by Towle et al. [[134]37] in the
database GenBank (NCBI). Rank 1 = highest regulation; bold and
underlined = significant regulation relative to the control identified
by sign test; bold = identified to be differentially expressed by both
tests; italic = identified to be differentially expressed only by
F-test; "-" = a rank was not assigned in case the regulation was less
than log[2]-ratio = |0.20|.
Figure 2.
[135]Figure 2
[136]Open in a new tab
Differentiation of the sum of all significantly regulated transcripts
for each gill and day of Carcinus maenas upon short-term exposure to
hypercapnia. For each of the experimental blocks of the short-term
hypercapnia experiment (gill7day3, gill7day7, gill9day3 and gill9day7),
up- and down- regulated transcripts were counted and represented as
bars. Additionally, a matrix was generated for all significantly
regulated transcripts (sign test) for each block with 1 = significantly
up-regulated, 0 = not significantly regulated, -1 = significantly
down-regulated. Statistical comparison of the four experimental blocks
was done applying Wilcoxon's matched pairs test on the matrices. No
significant difference was found in gill 7 comparing the two sampling
points, while gill 9 on day 3 differed significantly compared to gill 9
on day 7 (p < 0.05). Additionally, gill 9 in general differed
significantly in comparison to gill 7 on both days (p < 0.05).
Different letters a, b, c denote these significant difference.
Enrichment analysis demonstrated that "structural molecule activity"
(Gene ontology term (GO):0005198) was over-represented in all
significantly up-regulated transcripts of gill 9 (table [137]3).
Additionally, "cell" (GO:0005623) was over-represented in all
significantly down-regulated transcripts of gill 7. No other
over-representation of any GO-term was identified. However, 7 GO-terms
were under-represented for different subsets of all significant
regulated transcripts in the same analysis.
Regulation of specific groups
Stress response
Out of 137 identified transcripts associated with the cellular stress
response (according to [[138]38]), only 30 genes were significantly
regulated (sign test with p < 0.05), albeit less than 1.1-fold (20 up-,
9 down-regulated transcripts; Additional File [139]1 Table S4).
Variance-based linear modeling identified 23 genes to be differentially
expressed, including 5 that were identified also by sign test.
Expression of only 5 out of 26 heat-shock proteins (HSPs) was
significantly altered (2 up-, 3 down-regulated transcripts) in sign
test, while 5 different HSPs were identified by variance-based
analysis. Additionally, stress-associated GO-terms were not found to be
significantly enriched in the gills of short-term exposed animals
(Fisher's exact test). The expression level of glutathione peroxidase
[GenBank: [140]DN551450.1] was also investigated in the long-term
exposed animals (qRT-PCR analysis) and did not show a significant
change.
Structural modification
In total, 77 transcripts were associated with GO: 0005198 (structural
molecule activity). Significant up-regulation of 13 genes (sign test,
10 of them also identified by F-test) from this group resulted in the
over-representation of this GO-term in gill 9 (Additional File [141]1
Table S5). These up-regulated genes are eight ribosomal proteins, a
cuticular protein, two alpha-tubulins, a keratin-associated protein and
a vitelline membrane outer layer protein. The identification of a
1.4-fold up-regulated transcript, encoding for a multispanning
endomembrane protein of the transmembrane 9 superfamily (protein member
4 = TM9SF4, [GenBank:[142]DN202592.1]), also suggests structural
modification of gill membranes. It was up-regulated in both gills on
both sampling days (sign test with p < 0.05). This transcript was also
identified to be up-regulated in the low salinity study [[143]37].
Additionally, two other transmembrane proteins were identified to be
strongly down-regulated (sign test with p < 0.05): a tetraspanin-like 8
protein ([GenBank:[144]DV944030.1], included in the GO-class "cell")
was found to be strongly down-regulated during short-term hypercapnia
(1.3-fold) on day 3 and slightly during low salinity exposure. A
putative Integrin-alpha-7 [GenBank:[145]DY307809.1] was identified to
be strongly down-regulated (1.3-fold, sign test with p < 0.05) on day 7
(table [146]3, Additional File [147]1 Table S5).
Specialization of the gills
Although no effect of the factor "gill" on individual gene expression
was identified by variance-based linear modeling, Wilcoxon's matched
pairs test identified a significant difference in the overall gene
expression patterns in the different experimental blocks (gill7day3,
gill7day7, gill9day3, gill9day7; p < 0.05; Figure [148]2, Figure
[149]3A), based on total numbers of up-, down-, or not regulated
transcripts. Fewer genes were significantly regulated in gill 9 after
both 3 and 7 days of exposure than in gill 7 (sign test). In total, 756
genes were significantly regulated in gill 7, whereas only 510 in gill
9. Upon hypercapnia stress, gill 7 in general shows stronger responses
to hypercapnia than gill 9. For example, the strongest overall
significant change in gene expression was observed in gill 7 of a
senescence-associated protein (sign test and F-test with p < 0.05,
[GenBank:[150]DV944570.1]), a gene that was not altered in gill 9,
regarding both, day 3 and 7. On the other hand, gene expression of a
calcium-activated chloride channel [GenBank:[151]DW584691.1] was only
significantly and strongly affected in gill 9 on both days of the
short-term exposure (sign test and F-test with p < 0.05). Additionally,
according to the enrichment analysis, structural changes in gill
epithelia are linked mainly to gill 9.
Figure 3.
[152]Figure 3
[153]Open in a new tab
Comparison of effects in transcript regulation in gills of Carcinus
maenas upon short-term exposure to hypercapnia and low salinity. (A)
Regression analysis of gill 9 vs. gill 7 (both days) in the short-term
hypercapnia experiment and (B) of gill 7/day 7 (short-term hypercapnia
experiment, this study) vs. gill 8/day 7 (salinity dilution experiment
by Towle et al. [[154]37]). The long-dashed black line represents a
theoretical regression ratio of 1:1 (equal influence of both factors),
while the black line shows the actual regression. Transcripts outside
the short-dashed horizontal and vertical lines are more than 2-fold
regulated. Please note the different scaling in (A) and (B). Numbers
are according to the gene's rank in table 3, indicating transcripts
discussed in further detail in the manuscript. (1)
senescence-associated protein [GenBank:[155]DV944570.1], (3) putative
Syntaxin binding protein 2 [GenBank:[156]DN161290.1], (4) 23S ribosomal
RNA gene (a) [GenBank:[157]DN796131.1], (5) calcium-activated chloride
channel [GenBank:[158]DW584691.1], (6) 23S ribosomal RNA gene (b)
[GenBank:[159]DN635040.1], (13) multispanning membrane protein
(transmembrane 9 superfamily protein member 4
[GenBank:[160]DN202592.1], (25) potential integrin alpha-7
[GenBank:[161]DY307809.1].
Comparison with gene expression patterns in response to low salinity
When compared to gene expression changes observed after 7 days of
acclimation to a 2-fold reduction in salinity (from S = 32 to S = 15;
[[162]37]), short- and long-term acidification (10-fold increase in
pCO[2]) resulted in smaller changes in expression levels in fewer
transcripts: only 8 transcripts were regulated > 1.5-fold (log[2]-ratio
> |0.60|) in the present study, while 533 transcripts were regulated >
1.5-fold in the low salinity acclimation study; maximum expression
changes of up to 14-fold (log[2]-ratio = 3.8) were observed in the low
salinity acclimation study (Figure [163]3B, [164]4). Nevertheless, a
considerable amount of transcripts were regulated during acclimation to
both abiotic stressors (table [165]2).
Figure 4.
[166]Figure 4
[167]Open in a new tab
Comparison of regulation frequencies in Carcinus maenas gills in
salinity and CO[2 ]experiments. (A) Frequency distribution of all gene
expression levels in gill 7 and 9 on day 7 as response to short-term
hypercapnia (this study). (B) Frequency distribution of all gene
expression levels in gill 8 on day 7 as response to low salinity (study
of Towle et al. [[168]37]). Please note the different scaling of the
y-axis in (A) and (B). Exposure to low salinity (B) clearly resulted in
more genes to be changed on a higher level compared to hypercapnia (A),
where levels of change were mainly between a log[2]-ratio of -0.125 -
0.125. (C) Numbers of genes found to be regulated in expression
simultaneously in response to a salinity dilution, as well as
short-term hypercapnia (only transcripts with a log[2]-ratio > |0.200|
were considered).
Comparison of qRT-PCR and microarray analysis
In the long-term experiment, no significant changes in expression were
found, likely due to the high biological variance within samples
(coefficients of variation of the ratio 400 Pa/39 Pa CO[2 ]between 15
to 79%, Figure [169]5). In the short-term experiment, 7 out of the 8
tested genes responded in the same direction as in the microarray
analysis (Additional File [170]2 Figure S1). In one case
(glycosyl-phosphatidylinositol-linked carbonic anhydrase VII
[GenBank:[171]DN739347.1]), the transcript was slightly, but
significantly down-regulated in the microarray analysis, whereas it was
up-regulated in the qRT-PCR analysis. Variation in both, qRT-PCR and in
the microarray experiment was high.
Figure 5.
[172]Figure 5
[173]Open in a new tab
Change of expression levels of distinct transcripts in gill 7 as
response of Carcinus maenas to long-term exposure (11 weeks) to
hypercapnia analysed by quantitative real-time polymerase chain
reaction. Bars represent the median log[2]-ratio and median deviation
(error bars) of the respective gene. Numbers and abbreviations refer to
primers and genes as used in the qRT-PCR (see Additional File [174]1
Table S2 for primer sequences and full names). No statistical
significance was detected (sign-test with p < 0.05), but several
transcripts exhibit a tendency to be up- or down-regulated, e.g.
chloride associated transporters (no. 16, 18, 21).
Most strongly affected transcripts
The most down-regulated gene coded for a senescence-associated protein
[GenBank:[175]DV944570.1] in gill 7 on day 7 with a median log[2]-ratio
of -1.16 ± 0.64 (2.2-fold change, sign test and F-test with p < 0.05,
table [176]3). Its expression was not altered in the low-salinity
experiment. An unknown transcript [GenBank:[177]DN161290.1], suggested
to encode for Syntaxin binding protein 2 by Towle et al. [[178]37]),
was the most strongly up-regulated transcript in gill 7 on day 7 with a
median log[2]-ratio of +0.88 ± 0.36 (1.9-fold change, sign test and
F-test with p < 0.05). This transcript was also the most strongly
up-regulated gene in gill 8 in response to low salinity transfer
[[179]37]. Pg1 protein [GenBank:[180]DW250369.1], only identified by
F-test, was another highly up-regulated transcript on day 3, while it
showed strong down-regulation on day 7. In gill 9, the most strongly
down-regulated gene was identified on day 7 as a 23S ribosomal RNA gene
[GenBank:[181]DN796131.1] with a median log[2]-ratio of -0.87 ± 0.46
(1.9-fold change, sign test and F-test with p < 0.05). This gene was
strongly up-regulated during the low-salinity experiment. Additionally,
a different 23S ribosomal gene was found to be equally strongly
affected on day 7 in gill 7 ([GenBank:[182]DN635040.1], log[2]-ratio =
0.80 ± 0.22, 1.7-fold change, sign test with p < 0.05, not included in
F-test). The most strongly up-regulated transcript in gill 9 at day 3
was a calcium-activated chloride channel [GenBank:[183]DW584691.1] with
a median log[2]-ratio of +0.81 ± 0.34 (1.7-fold change, sign test and
F-test with p < 0.05). It was also the most highly up-regulated
transcript in the long-term exposed animals (log[2]-ratio = 1.12 ±
0.88, 2.2-fold change), although not tested significant due to high
variance between replicates. Additionally, rRNA intron-encoded homing
endonuclease [GenBank:[184]DV944193.1] was identified by F-test as
being differentially expressed. Besides 4 unknown, but strongly changed
transcripts (rank 8-11, table [185]2), a thymosin isoform was
identified to be strongly down-regulated in gill 9 on day 3
(log[2]-ratio = -0.46 ± 0.14, 1.4-fold change, sign test with p <
0.05). In addition, a hyperpolarization-activated cyclic
nucleotide-gated potassium channel (HCN2, [GenBank: [186]DW250260.1]),
was significantly down-regulated in both gills on day 7 (gill 7 with a
log[2]-ratio = -0.46 ± 0.24/1.4-fold, and gill 9 with -0.14 ±
0.08/1.1-fold, sign test and F-test with p < 0.05) with a tendency for
down-regulation in both gills on day 3. In the low-salinity study, it
was only slightly down-regulated [[187]37].
Acid/base and ion regulation
None of the acid-base regulatory candidate genes were regulated more
than 1.3-fold in our microarray experiment at days 3 and 7. Of 9
candidate genes tested on the microarray, only 2 were significantly
regulated with respect to controls. Among them were a Cl^-/HCO[3]^-
exchanger ([GenBank:[188]CX994129.1], -1.1-fold, sign test and F-test
with p < 0.05) and the glycosyl-phosphatidylinositol-linked carbonic
anhydrase VII ([GenBank:[189]DN739347.1], -1.1-fold, sign test and
F-test with p < 0.05). In contrast, during acclimation to reduced
salinity, Towle et al [[190]37] found 5 of this 9 acid-base regulatory
candidate genes to be altered by more than 1.3-fold, including the
cytoplasmic carbonic anhydrase ([GenBank:[191]DW251095.1], + 3.4-fold),
the Na^+/K^+-ATPase alpha subunit ([GenBank:[192]DW250552.1], +
2.6-fold), a member of the anion/bicarbonate transporter family
(abts-3) ([GenBank:[193]DN202373.1], similarity to a Na^+/HCO[3]^-
transporter (SLC4A11), - 2.4-fold), the
glycosyl-phosphatidylinositol-linked carbonic anhydrase VII
([GenBank:[194]DN739347.1], - 1.9-fold) and the Cl^-/HCO[3]^- exchanger
(SLC4A1; [GenBank:[195]CX994129.1], + 1.3-fold).
In the long- term acclimation study (11 weeks), no significant changes
in expression could be found, but variances were extremely high
(coefficients of variation of the ratio 400 Pa/39 Pa CO[2 ]ranged from
15 to 79%). Only in the case of the sodium-hydrogen exchanger NHE3,
variance was smaller (1.3%).
Discussion
Green crabs, especially those of the Baltic Sea, are known to be
effective ion and acid-base regulators [[196]25,[197]49,[198]50]. When
exposed to hypercapnia, Carcinus maenas is characterized by a rapid
pH[e ]compensatory response that involves the accumulation of
bicarbonate in its hemolymph ([[199]17]). This high HCO[3]^- level is
sustained even over a period of 11 weeks (Appelhans et al., under
review). The enrichment of HCO[3]^- in the crabs' hemolymph may be
mediated by altered ion regulation processes in the gills and thus can
be expected to also leave a footprint in the gills' transcriptome.
However, long-term hypercapnia incubations may additionally result in
other physiological responses, pointing to trade-offs in energy
allocation in favor of extra-/intracellular ion homeostasis vs. protein
metabolism and growth, as has been concluded for molluscs, teleost fish
and echinoderms [[200]13,[201]23,[202]51-[203]53]. We hypothesized that
an increased demand for ion transport and modifications in epithelial
CO[2 ]permeability would influence expression patterns of the
respective gene transcripts in gills in both, short- and long-term
hypercapnia scenarios.
Variance-based analysis of the microarray identified a significant
effect of pCO[2 ](53 vs. 440 Pa) on 26% of the transcripts. However,
expression profiles in this study revealed that short-term hypercapnia
(10-fold increase in seawater pCO[2]) does not act as a strong stressor
on gill tissues. Expression changes were moderate and it has to be
considered that some of the observed changes are random noise, given
also that only few significant effects persisted over both time points
(3 and 7 days) and considering the partially differing results from
both statistical tests applied (40% of trancripts identified to be
differentially expressed by both, sign test and variance-based
analysis). Only relatively few transcripts (2% of all tested
transcripts) were differentially regulated more than 1.3-fold, with
maximum observed changes of 2.2-fold. Even these changes are much lower
than those elicited by a 2-fold decrease in salinity (transfer from S =
32 to S = 15), where maximum observed changes in expression were
14-fold [[204]37]. A negative effect of hypercapnia on transcripts
related to energy metabolism, as has been shown in sea urchin larvae
[[205]53], was not observed in our study. Transcripts known to be
involved in acid-base responses were not regulated strongly in response
to elevated pCO[2]. Instead, new acid-base regulatory candidate genes
were identified to be differentially expressed, including a calcium
activated chloride channel, a multispanning membrane protein
(transmembrane 9 superfamily protein member 4), a hyperpolarization
activated cyclic nucleotide-gated potassium channel-2, tetraspanin 3
(transmembrane super family 4 member 8) and a potential integrin
alpha-7.
Short-term hypercapnia does not lead to a pronounced stress response
Only few stress-associated genes were differentially regulated in in
gills in response to short-term hypercapnia. Therefore, short-term
hypercapnia does not evoke a pronounced cellular stress-response (CSR)
in the gills of adult Carcinus maenas from the Baltic Sea. A lack of
pronounced changes in the expression of heat-shock proteins supports
this interpretation, as does the finding that in general only small
expression level changes occured. Heat-shock proteins (HSPs) are highly
conserved among species and involved in acute stress responses towards
various stressors [[206]54]. Usually, cellular responses through HSP
occur rapidly and transiently [[207]55,[208]56]. Thus, it cannot be
excluded that a CSR occurred and ended before our first sampling after
3 days. However, the observation that a hypoosmotic acclimation with
its much stronger changes in gene expression patterns also did not
elicit a pronounced CSR (even within the first hours of exposure;
[[209]37]) corroborates the interpretation that the applied changes in
seawater pCO[2 ]do not trigger a prolonged or pronounced CSR in gills
of C. maenas. As focus has been laid upon the gills, it should be
considered that hypercapnia may indeed provoke a CSR in other organs.
Although magnitudes of change in gene expression were comparatively low
(e.g. compared to [[210]37,[211]53]), we found indications for a
re-arrangement of the green crabs' gill epithelia and support for
specialization of each single posterior gill.
Re-arrangement of the gill epithelium and epithelial cell membranes
Enrichment analysis supports that a reorganization of gill epithelia
occurs during the initial stages (< 7 days) of hypercapnia acclimation.
The GO-term "structural modification" is the only biological process
that was enriched within up-regulated genes in response to hypercapnia.
During salinity acclimation, posterior gills of C. maenas also undergo
structural modification through extension of the apical plasma membrane
infolding system of the thick prismatic salt-transporting epithelium
and through an increase of the subcuticular space [[212]50]. Although
no genes encoding for cellular junction proteins such as claudins,
occludins, cadherins or selectins were tested on the microarray used in
the present study, our analysis identified several other genes
potentially involved in structural rearrangements. A member of the
tetraspanin family was found to be strongly down-regulated during
short-term hypercapnia and slightly during low salinity acclimation
[[213]37]. Tetraspanins are a group of four-transmembrane-domain
proteins that are expressed in epithelia and known to be involved in
diverse cellular processes (e.g. morphogenetic re-organization of
monolayers of epithelial cells [[214]57]). They are generally described
as molecular 'facilitators' or 'organizers' at the plasma membrane
[[215]58,[216]59]. Furthermore, integrin-tetraspanin complexes play an
important role in cell-cell-adhesion at cellular junctions
[[217]56,[218]59,[219]60]. A putative Integrin-alpha-7 was identified
to be significantly and strongly down-regulated in the short-term
acidification experiment. Due to their essential role in cell adhesion
and cell-cell communication, biochemical functions of integrins are
likely to be highly conserved in metazoans [[220]61]. The common and
distinct regulation of a tetraspanin and an integrin indicate that the
possible complex of both could play a role during re-arrangement of the
gill. Another strongly up-regulated transcript encodes for a
multispanning endomembrane protein of the transmembrane 9 superfamily
(protein member 4 = TM9SF4), also known as p76 in humans. TM9SF4 plays
an important role in cellular adhesion, membrane reconfiguration and
vesicle mediated transport [[221]62,[222]63]. It thus can be
hypothesized that exposure to elevated seawater pCO[2 ]has an influence
on the membrane composition of gill epithelial cells, and on the cell
composition of the epithelium of the gills of C. maenas. This warrants
detailed studies on structural changes of the gills in response to
hypercapnia (eg. light or electron microscopy and (immune-)
histochemical investigations), including studies on the involvement of
the three membrane proteins mentioned above.
Specialization of the gills
As described above, only the three posterior gills 7 to 9 were found to
be involved in ion- and acid-base regulation in C. maenas. The anterior
six gill pairs are primarily important for gas exchange
[[223]17,[224]26,[225]27]. However, a more individualized
specialization of each of the posterior gills has been shown for other
species with respect to salinity changes [[226]64,[227]65]. For C.
maenas, Siebers et al. [[228]26] detected a salinity-dependent
activation of Na^+/K^+-ATPase with increasing activity at decreasing
salinities mainly in the posterior gills, but only subtle differences
between the individual gills 7-9. Henry et al. [[229]66] made a
corresponding observation with respect to carbonic anhydrase. However,
based on the distribution of V-type H^+-ATPase in the posterior gills
of several (intertidal) crab species, Tsai and Lin [[230]65] showed
that the functional differentiation in crab gills generally is not only
between anterior and posterior, but also within individual gill
lamellae. While the variance-based analysis of the data set suggested
no significant effect of the factor "gill" on gene expression levels,
enrichment analysis indicated different and specific responses to
hypercapnia acclimation between posterior gills 7 and 9. Gill 7
responded stronger to hypercapnia than gill 9 with respect to both, the
number of genes affected and the overall magnitude of change in gene
expression. Most transcripts were either significantly regulated for
one or the other gill, only 11% were regulated in parallel. The result
of the enrichment analysis further suggested that structural changes
are mainly associated with gill 9. However, the conflicting results of
the two statistical methods used demand more detailed investigations on
the cell ultrastructural level to substantiate potential differences in
function between gills.
Comparison with gene expression levels in response towards low salinity
When responses to hypercapnia (this study) and hypoosmotic acclimation
[[231]37] are compared, it becomes obvious that hyposmotic acclimation
to a 2-fold reduction in salinity results in far larger expression
changes than acclimation to a 10-fold increase in seawater pCO[2].
Nevertheless, acute hyposmotic acclimation also does not lead to a
pronounced CSR [[232]37]. C. maenas from the western Baltic Sea show an
increased ability for hyperosmoregulation when exposed to low
salinities (salinity < 20) than animals from the more saline North Sea
(salinity > 30, [[233]47]). Consequently, they also must possess a very
high ion regulatory capacity and as salinity was low in our experiment
(S = 15), the gill ion regulatory machinery was working at a
comparatively high load. Microarray analysis only allows detection of
relative changes in gene expression. If a certain transcript is already
highly expressed, even a small relative change on the level of
transcript expression could result in a highly effective regulatory
capacity on the protein level. Even small changes may allow for
successful acclimation. Furthermore, the strong fluctuations (both in
rate and magnitude) in salinity, pCO[2 ]and temperature observed in
Kiel Fjord [[234]19], might have led to an adaptation towards excess
ion regulatory capacity that can be recruited upon demand. This
recruitment might take place on the post-transcriptional level
[[235]23,[236]67] and would therefore remain undetected in our
microarray analysis.
Old and new candidate genes for hypercapnia acclimation in crustaceans
In addition to the above discussed potential role of the multispanning
membrane protein TM9SF4 in structural re-arrangement of gill epithelia,
its hypercapnia induced up-regulation might be relevant for cellular
acid-base regulation. TM9SF4 is known to participate in vesicular
transport [[237]68] and Schimmöller et al. [[238]69] suggested its
association with endosomes (acidic compartments/vesicles in mammalian
cells). In posterior C. maenas gills, intracellular vesicles were
postulated to be involved in cellular acid-base regulation via
V-H^+-ATPases [[239]70].
A calcium-activated chloride channel (CaCC) was strongly up-regulated
in gill 9 on day 3 and it was also up-regulated after 11 weeks of
hypercapnia. Beside others, CaCCs have been shown to play a key role in
epithelial secretion [[240]71,[241]72], but a high variability within
this class of channels with respect to physiological roles and
mechanisms of regulation was observed [[242]73,[243]74]. A member of a
CaCC subfamily has also been shown to act in cell-cell adhesion through
interaction with an integrin (e.g. in lung cancer [[244]67], reviewed
[[245]75]). Chloride channels have been hypothesized to be situated in
the basolateral membrane of epithelial cells in gills of osmoregulating
crabs [[246]32,[247]33]. The identified CaCC as described above might
be an important candidate gene in Cl^- regulation. In order to achieve
electroneutrality, Cl^- typically is the counter-ion of HCO[3]^- during
the extracellular pH regulatory reaction. Extracellular HCO[3]^-
accumulation is probably enabled by Cl^-/HCO[3]^- exchangers [[248]20].
While respective Cl^-/HCO[3]^- exchangers (AE) have been postulated to
be situated in the apical membrane in crustaceans, molecular
identification and/or biochemical characterization is still lacking. On
the other hand, a basolateral-situated AE can be discussed to play a
role in pH and volume regulation [[249]33]. In acid secretion, a
respective exchanger is postulated to transport HCO[3]^- ions from the
cell into the hemolymph in exchange for Cl^- ions and therefore is
argued to sit in the basolateral membrane. As has been postulated in
fish, other transporters in close proximity to a Cl^-/HCO[3]^-
exchanger can be discussed to favor the electroneutral exchange of Cl^-
and HCO[3]^- against an unfavorable Cl^- gradient [[250]76]. A
Cl^--channel like the identified CaCC could be an important additional
player and facilitator in this Cl^-/HCO[3]^- exchange.
Additionally, a hyperpolarization-activated cyclic nucleotide-gated
potassium channel (HCN) was significantly down-regulated in the
short-term hypercapnia study. So far, HCNs (1+4) have been shown to be
situated in the plasma membrane of vallate papillea taste cells in rat
tongue. Those transporters/receptors are associated with the
basolateral membrane and play a role in response to sour stimuli
(extracellular protons) by mediating an inwardly directed current
[[251]77]. On the other hand, lowered intracellular pH has been shown
to lead to a decreased opening speed of the channel in thalamocortical
neurons of the rat ventrobasal thalamic complex [[252]78]. Thus, an
altered extracellular acid-base status might interact with the function
of this protein.
Future studies should characterize these candidate genes with respect
to localization and function. In the case of the CaCC and HCN,
electrophysiological experiments could reveal in which way these
transporters mediate ion fluxes across the gill epithelium.
Several transporters and channels shown to be involved in ion or
acid-base regulation during hypercapnia acclimation in other studies on
diverse marine organisms [[253]33,[254]51,[255]79] were not affected in
C. maenas gill tissue. The sodium pump, Na^+/K^+-ATPase (NKA), is
crucial for the maintenance of ion gradients that drive acid-base
regulation [[256]80]. This primary active transporter is a key player
in establishing the characteristic ion gradient that is used by many
secondary active transporters. NKA transcript and protein level, as
well as activity increased in response to decreased salinities in
diverse crabs, including C. maenas [C. maenas: 37, 69; others: 64, 65,
81]. During hypercapnia acclimation, Deigweiher et al. [[257]52]
documented that in teleost fish, NKA mRNA concentration decreased
initially (day 4), only to increase 2-fold after 6 weeks of exposure to
a pCO[2 ]of 1 kPa. In contrast, O'Donnell et al. [[258]53] found that
in sea urchin larvae, NKA mRNA was down-regulated in response to
hypercapnia. Although NKA expression decreased significantly in
cephalopod embryos and hatchlings exposed to a pCO[2 ]of 0.4 kPa, no
change in expression was detected in juveniles under comparable
conditions [[259]23]. We did not find changes in NKA expression in C.
maenas gill tissue in the present study. Only one Cl^-/HCO[3]^--anion
exchanger from the SLC family 4 (member 1; [GenBank:[260]CX994129.1])
was significantly down-regulated in the short-term experiment
(1.1-fold), while a different anion-bicarbonate exchanger, similar to
the SLC family 4, member 11 [GenBank:[261]DN202373.1]), and a vacuolar
H^+-ATPase [GenBank:[262]DY656042.1] were not affected. Another
important transporter for acid-base regulation, a Na^+/H^+ exchanger
(NHE3) was not significantly regulated in C. maenas gill tissue in the
long- term experiment. C. maenas also possesses two branchial isoforms
of carbonic anhydrase (CA) in its posterior gills, a
membrane-associated and a cytoplasmic form [[263]82]. The enzyme
catalyzes the highly energy-demanding transition of H[2]O and CO[2 ]to
HCO[3]^- and H^+ and vice versa. It is of great importance in
osmoregulation (especially the cytoplasmic pool [[264]66]), acid-base
regulation and CO[2 ]excretion in the gills of crustaceans
[[265]71,[266]83]. However in this study, no response of CAs was
observed in short-term hypercapnia experiments, except for a slight
significant down-regulation of glycosyl-phosphatidylinositol-linked
carbonic anhydrase VII [GenBank:[267]DN739347.1] in both, gill 7 and 9,
on day 7. In agreement with the results of the short-term experiment,
we also found no significant expression changes following long-term
exposure to hypercapnia.
We suggest that the ion regulatory apparatus of Baltic C. maenas
already works at a high load due to the demands of a hyposmotic habitat
with large fluctuations in pCO[2]. Therefore, only moderate mRNA
expression level changes might be necessary to compensate hypercapnia
in C. maenas. Compensation of the ion regulatory apparatus might
additionally take place on the post-transcriptional level or is
facilitated by transcripts not included on this microarray. It has to
be considered though, that to some extend, effects might be hidden
under the experimental error. Nevertheless, those few genes for which
we have identified relatively strong changes in expression levels are
particularly interesting. They are likely key players of hypercapnia
acclimation of crustacean gill tissues.
Conclusions
The response of Carcinus maenas to elevated seawater pCO[2 ]based on
the gill transcriptome does not suggest that seawater acidification
acts as a strong stressor for the western Baltic population of this
species. Following short-term hypercapnia, the low but specific
responses of the gills indicate that (1) the response to hypercapnia is
partially similar to the response to hypoosmotic conditions; (2) a
multispanning membrane protein (TM9SF4), a calcium-activated chloride
channel (CaCC) and a hyperpolarization-activated cyclic
nucleotide-gated potassium channel (HCN) are strongly regulated in
response to hypercapnia and may be involved in acid-base regulation;
(3) posterior gills might respond differently to hypercapnia; and (4) a
re-configuration of the epithelial gill membrane, including the
involvement of a tetraspanin-integrin complex, might occur.
Due to the low salinity environment in the Baltic Sea in this
hypercapnia study, effects on distinct gene expression levels to one of
the respective abiotic factors might be influenced by the other. It is
therefore essential to study both abiotic factors separately and in
more detail. Here, potential pathological effects of the long-term
consequences of elevated blood bicarbonate concentrations on various
tissues and organs should also deserve particular attention. We also
expect that stronger effects of hypercapnia are to be found on the
proteome and metabolome level.
Authors' contributions
SF carried out the short-term incubations, microarray experiments and
real-time PCR, analyzed the results and drafted the manuscript. Most of
the facts displayed here are content of SF's Diploma thesis at the
IFM-GEOMAR, Kiel, Germany 2010. RK contributed to the analysis,
illustration and interpretation of the data and the preparation of the
manuscript. YA carried out the long term incubation experiments and
participated in the sampling procedure. DWT passed away during the
preparation of the manuscript; before his untimely death, he
significantly contributed to the microarray and study design and
coordination, and provided the microarray facilities. FM and MZ
conceived the study, participated in its design, coordination and
analysis, supplied infrastructure and material and helped to draft the
manuscript. All authors read and approved the final manuscript.
Supplementary Material
Additional File 1
Tables S1-S6.
Table S1. Annotation details on transcripts for the Carcinus maenas the
microarray assay applied in this study. Annotation results on
transcripts for the Carcinus maenas microarray assay applied in this
study from a newly performed bioinformatic analysis conducted with the
internet portal Blast2GO [[268]43] in 2009 by the authors of this
study, compared to a first annotation provided by Towle et al.
[[269]37]. Included are the sequence length of the aligned sequence (=
seq. length), the number of hits for the transcript (= #hits), the mean
similarity of the alignment (= mean sim), the number of Gene Ontology
(GO) terms assigned to that blast hit (= #GOs), a description of the
assigned GOs (= GOs), Enzyme Codes (= EC), results of the InterProScan,
the exact hit description of the best hit, as well as the accession
number (= hit ACC, E-value, similarity, score, alignment length and
number of positive matches bases of the best hit.
Table S2. Details on primers for quantitative real-time polymerase
chain reaction (qRT-PCR) to assess responses of Carcinus maenas to
hypercapnia. Primer sequences (5' → 3') and descriptions of the
targeted genes used in the real-time polymerase chain reaction
(qRT-PCR) in the short- and long-term hypercapnia experiments on
Carcinus maenas. Numbers ('no.') are according to the numbers used in
Figure 4 and Additional File [270]2 Figure S1. Accession numbers (ACC.
no.) refer to the ESTs generated for the C. maenas microarray by Towle
et al. [[271]37] and the database GenBank (NCBI). R^2 and efficiency
were tested in a qRT-PCR dilution series (for details, see Material &
Methods).
Table S3. Transcripts significantly regulated in response to
hypercapnia in Carcinus maenas. Transcripts significantly regulated in
response to hypercapnia in Carcinus maenas as identified by
variance-based linear modelling (F-test) and sign test. Out of 1634
genes, 678 were identified by sign test, 578 genes were identified by
F-test, and 378 were identified to be affected by both statistical
tests. Transcripts are sorted alphabetically after the accession number
(ACC. no., database GenBank (NCBI)). Bold and underlined =
significantly regulated as identified by sign test, p < 0.05; bold =
significantly regulated as identified by both statistical tests (sign
test and F test, p < 0.05); italic = differentially expressed as
identified by F-Test. Accession numbers refer to the ESTs generated for
the C. maenas microarray by Towle et al. [[272]37]. Values are given as
median and median deviation (MD).
Table S4. Significantly regulated transcripts associated with a
cellular stress-response in Carcinus maenas when exposed to
hypercapnia. Significantly regulated transcripts in the short-term
hypercapnia study on Carcinus maenas associated with a cellular
stress-response acoording to [[273]38]. Bold and underlined (values) =
significantly regulated (sign test, p < 0.05), bold (ACC. no.) =
transcripts identified to be significantly regulated by both tests
(sign test and F-test, p < 0.05). Accession numbers (ACC. no.) refer to
the GenBank database (NCBI).
Table S5. Significantly up-regulated transcripts in the short-term
hypercapnia study associated with structural molecule activity in gills
of Carcinus maenas. Transcripts identified to be responsible for the
over-representation of the GO-term "structural modification"
(GO:0005198) in Carcinus maenas gills exposed to hypercapnia
(enrichment analysis with Fisher's exact test). Bold and underlined
(values) = significantly regulated as identified by sign test (p <
0.05); bold (ACC. no.) = identified to be significantly regulatd by
both tests (sign test and F-test, p < 0.05). Accession numbers (ACCs)
refer to the database GenBank (NCBI).
Table S6. Details on transcripts of special interest identified by
microarray analysis on gills of Carcinus maenas after exposure to
short-term hypercapnia. Identification and details on transcripts of
special interest identified by microarray analysis, after an additional
NCBI blastx. * = NCBI blastn; ** = see Towle et al. [[274]37] for
details. Accession numbers (ACC. no.) refer to the database GenBank
(NCBI).
[275]Click here for file^ (1,018.1KB, XLSX)
Additional file 2
figure S1. Comparison of distinct transcripts of the microarray
analysis vs. qRT-PCR for the response of Carcinus maenas to short-term
hypercapnia. Comparison of the regulation of distinct transcripts of
gill 9 for the Carcinus maenas response to short-term hypercapnia (1
week, April 2009) in the microarray analysis with results of the
qRT-PCR experiment performed on the respective genes from the
short-term incubation conducted in April 2010. In 7 of 8 cases, both
techniques show the same tendency in regulation. Values represent
median log[2]-ratios with median deviation (error bars). Transcript
numbers according to Additional File [276]1 Table S2. Senesc. ass. prot
= senescence-associated protein, put. syntaxin = putative Syntaxin
binding protein 2, gCA = glycosyl-phosphatidylinositol-linked carbonic
anhydrase VII, prot. inh. = hemozyte kazal-type proteinase inhibitor,
K-channel = hyperpolarization activated cyclic nucleotide-gated
potassium channel 2, NKA = Na^+/K^+-ATPase alpha subunit, Cl-channel
(Ca-act.) = calcium acitvated chloride channel.
[277]Click here for file^ (25.3KB, PDF)
Contributor Information
Sandra Fehsenfeld, Email: umfehsen@cc.umanitoba.ca.
Rainer Kiko, Email: rkiko@ifm-geomar.de.
Yasmin Appelhans, Email: yappelhans@ifm-geomar.de.
David W Towle, Email: .
Martin Zimmer, Email: martin.zimmer@sbg.ac.at.
Frank Melzner, Email: fmelzner@ifm-geomar.de.
Acknowledgements