Green crabs, especially those of the Baltic Sea, are known to be effective ion and acid-base regulators [25, 49, 50]. When exposed to hypercapnia, Carcinus maenas is characterized by a rapid pHe compensatory response that involves the accumulation of bicarbonate in its hemolymph (). This high HCO3
- level is sustained even over a period of 11 weeks (Appelhans et al., under review). The enrichment of HCO3
- 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 [13, 23, 51–53]. We hypothesized that an increased demand for ion transport and modifications in epithelial CO2 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 pCO2 (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 pCO2) 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 . A negative effect of hypercapnia on transcripts related to energy metabolism, as has been shown in sea urchin larvae , was not observed in our study. Transcripts known to be involved in acid-base responses were not regulated strongly in response to elevated pCO2. 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 . Usually, cellular responses through HSP occur rapidly and transiently [55, 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; ) corroborates the interpretation that the applied changes in seawater pCO2 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 [37, 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 . 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 . 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 ). They are generally described as molecular 'facilitators' or 'organizers' at the plasma membrane [58, 59]. Furthermore, integrin-tetraspanin complexes play an important role in cell-cell-adhesion at cellular junctions [56, 59, 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 . 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 [62, 63]. It thus can be hypothesized that exposure to elevated seawater pCO2 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 [17, 26, 27]. However, a more individualized specialization of each of the posterior gills has been shown for other species with respect to salinity changes [64, 65]. For C. maenas, Siebers et al.  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.  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  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  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 pCO2. Nevertheless, acute hyposmotic acclimation also does not lead to a pronounced CSR . 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, ). 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, pCO2 and temperature observed in Kiel Fjord , 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 [23, 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  and Schimmöller et al.  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 .
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 [71, 72], but a high variability within this class of channels with respect to physiological roles and mechanisms of regulation was observed [73, 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 , reviewed ). Chloride channels have been hypothesized to be situated in the basolateral membrane of epithelial cells in gills of osmoregulating crabs [32, 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 HCO3
- during the extracellular pH regulatory reaction. Extracellular HCO3
- accumulation is probably enabled by Cl-/HCO3
- exchangers . While respective Cl-/HCO3
- 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 . In acid secretion, a respective exchanger is postulated to transport HCO3
- 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-/HCO3
- exchanger can be discussed to favor the electroneutral exchange of Cl- and HCO3
- against an unfavorable Cl- gradient . A Cl--channel like the identified CaCC could be an important additional player and facilitator in this Cl-/HCO3
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 . 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 . 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 [33, 51, 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 . 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.  documented that in teleost fish, NKA mRNA concentration decreased initially (day 4), only to increase 2-fold after 6 weeks of exposure to a pCO2 of 1 kPa. In contrast, O'Donnell et al.  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 pCO2 of 0.4 kPa, no change in expression was detected in juveniles under comparable conditions . We did not find changes in NKA expression in C. maenas gill tissue in the present study. Only one Cl-/HCO3
--anion exchanger from the SLC family 4 (member 1; [GenBank: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:DN202373.1]), and a vacuolar H+-ATPase [GenBank: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 . The enzyme catalyzes the highly energy-demanding transition of H2O and CO2 to HCO3
- and H+ and vice versa. It is of great importance in osmoregulation (especially the cytoplasmic pool ), acid-base regulation and CO2 excretion in the gills of crustaceans [71, 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: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 pCO2. 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.