Changes in the transcriptome can serve as a metric of physiological plasticity in metazoans in response to changes in their abiotic environment [1, 4, 6, 7, 38]. The goal of this study was to assess patterns of differential gene expression in the polar pteropod, Limacina helicina antarctica, in response to pH conditions that reflect both present-day, seasonal variability in pH, and pH conditions predicted for future ocean acidification. In general, we found that the L. h. antarctica transcriptome changed in response to pH treatments that represented pH values for both seasonal, and ocean acidification conditions. In addition, the L. h. antarctica transcriptome changed as a function of time of exposure to the various pH treatments used in the experiment. Lastly, the expression patterns observed involved genes found in key processes that would be physiologically significant to a calcifying marine mollusk under these abiotic conditions in that these conditions may challenge biogenic calcification and energy allocation in a juvenile marine calcifier. Specifically, these four groups of genes are those involved in shell formation, the cellular stress response, metabolism, and neural function.
Differential gene expression analysis
Acute transcriptomic responses
We found that L. h. antarctica had a robust transcriptional response to acute pH changes that represent present-day seasonal pH values (Tables 1 and 2). Specifically, in comparisons of expression for pH 8.13 to 8.01 at day 1, strong up-regulation of transcripts involved with large-scale cellular function indicated a regulatory response to more alkaline conditions that typifies the transition from winter into summer . Changes in transcript abundance of genes associated with DNA binding and epigenetic modifications such as DNA methylation (e.g. methyltransferases) indicated that the response may have involved epigenomic modifications that could act as mechanisms to change the transcriptome (Additional file 2).
Along with these genomic/epigenomic changes, we also observed increases in transcripts for calcium binding proteins (e.g. calmodulin-like proteins), ion binding, and transmembrane transporters, all genes known to be important in biogenic calcification (Additional file 2). In addition, functional enrichment analysis revealed enrichment of transcripts involved with cytoskeleton function and calcium ion binding (Additional file 4). The up-regulation of these gene groups in the high-pH treatment suggests pteropods increased shell formation when held in conditions highly favorable for calcification.
Together, these results provide evidence that exposure to high pH conditions, which are presumably not physiologically stressful, led to broad scale transcriptomic changes that are focused on genome reorganization, and the maintenance and synthesis of cytoskeletal and calcified structures. Following 7 days of exposure these trends further intensified with 1554 up-regulated transcripts that expanded the d1 regulated set to include anatomical structure development, and enrichment of cytoskeletal protein binding (Additional file 2). The continued increase in expression of genes associated with both the cytoskeleton and calcification indicated that pteropods up-regulate gene pathways associated with enhancing biogenic calcification when exposed to high-pH (8.13) conditions, the pH value observed during the peak of summer.
An examination of the suppressed transcripts for the acute seasonal pH treatment revealed minor down-regulation in three gene types: nucleotide binding, helicase activity, and cytoskeleton structure (Additional file 2). While these results are similar to the transcripts identified in the up-regulated category, they represent a small number of isoforms that may have additional functions within these polar gastropods. Indeed, after 7 days of exposure, these contrasting transcripts were no longer present, and the residual suppressed transcripts were primarily involved in protein degradation (Additional file 2).
In general, differential gene expression analysis in juvenile L. h. antarctica revealed that acute exposure to pH 8.13 produced dramatic transcriptional changes that were focused on cytoskeletal development and genome organization. While a robust transcriptional response is consistent with previous studies of the arctic form of L. h. helicina and the Mediterranean pteropod Heliconoides inflatus [22, 37], our results are the first to characterize the effects of pH on the Antarctic form of L. helicina. In addition, these data suggest that the juvenile pteropods were responding positively to seawater conditions that are typical of summer, a time when food supply is high and the growth potential is presumably also optimal for this polar species.
In contrast, exposure to pH conditions of ocean acidification (pH 7.71) resulted in suppression of calcification, lipid transport, and metabolic function; suggesting that these conditions are physiologically stressful (Additional file 3). During the first 24 h of exposure, the dominant signature was a large-scale down-regulation of transcripts with enrichment in 20 gene ontologies (Additional file 5). Among these gene ontologies, three are noteworthy with regard to this suppression of gene expression in Limacina: (1) calcium ion binding, (2) chitin binding, and (3) the proteinaceous extra-cellular matrix. Within these down-regulated ontological groups are transcripts that code for tyrosinase, multiple chitin-binding proteins, calmodulin, carbonic anhydrase, Von Willebrand factor type A, Sushi, and bone morphogenic protein 1-like (Additional file 3). These genes are all known to have fundamental roles in the formation of the calcium carbonate shell of mollusks [39,40,41,42,43]. The suppression of these transcripts after 24 h of exposure to low pH conditions indicates that pH-stressed pteropods rapidly suppress biogenic calcification.
In addition to changes in genes related to calcification, we also found significant impacts on genes associated with lipid transport, suggesting a shift in energetic needs in response to conditions of ocean acidification (Additional file 3) . The down-regulation of lipid transport genes points to a global disruption of energy allocation that corroborates other observations in studies on marine invertebrates including oysters, Antarctic krill and corals [45,46,47]. Changes in energy allocation for L. h. antarctica have been previously reported, wherein energy reserves in eggs were decreased when adults were exposed to low-pH conditions . Similar effects of pH have also been reported in green sea urchins  and copepods, where low pH resulted in significant impacts on energy allocation and egg production .
That exposure to low-pH conditions impacted the energy budget of L. h. antarctica was further underscored by the down-regulation of metabolically important transcripts such as carbonic anhydrase, glucose dehydrogenase and malate dehydrogenase (Additional file 3). These genes are central to ATP production in metabolic pathways, and the integrity of energy metabolism has been identified as a key indicator of tolerance to abiotic stress . Specifically, down-regulation of glucose and malate dehydrogenase indicates a reduced ability to produce NADPH, a critical cofactor involved in oxidation-reduction, glutathione generation, and lipid synthesis. Down-regulation of these genes in response to conditions of ocean acidification has been documented in corals, oysters, and the great spider crab [45, 51, 52].
By day 7, differential expression between the mid-pH (8.01) and the low-pH (7.71) was reduced to a total of 40 differentially expressed transcripts (Table 2). The up-regulated transcripts were concentrated around transcription-related processes while down-regulated transcripts were involved in developmental processes. With so few differentially expressed transcripts, no gene ontological enrichment was present. The shift from high numbers of differentially expressed genes to this muted response suggests the transcriptomic profiles for the mid- and low-pH treatments were converging, an observation further supported by gene expression analysis of genes involved in physiological and cellular processes.
Differential expression following short-term acclimation exposure
As was observed with the acute exposures of juvenile Limacina, longer-term acclimation (14 to 21 days) to low pH conditions resulted in significant changes in gene expression (Table 2). In general, gene expression varied from the short-term response in that we observed a higher percentage of down-regulated transcripts at both d14 and d21. Specifically, at d14 the largest signal was among down-regulated transcripts with enrichment in 26 gene ontologies, an indication of large-scale reduction in transcription (Additional file 6). This was further supported by down-regulation of metabolic and ion binding transcripts, and indicated significant suppression of genes involved in biogenic calcification similar to the differential gene expression patterns observed in the acute response (Additional file 3). This pattern continued into d21 with down-regulation of cytoskeleton and calcium ion binding transcripts. Enrichment at d21 identified 9 enriched gene ontologies that reflect patterns identified during the acute exposure to low pH conditions (Additional file 6). With 872 down-regulated transcripts that continued to span calcium ion binding, metabolic function and cytoskeleton development, this pattern of differential expression is evidence that present-day pteropods genotypes do not modulate expression of candidate calcification genes that may be critical to continued calcification during the under-saturated austral winter conditions forecast for the Southern Ocean.
Analysis of differential gene expression in physiologically relevant processes
In order to better characterize the effect of pH on L. h. antarctica, we focused on 4 major processes that are key to pteropod physiology: shell formation, metabolism, the cellular stress response, and neural function. Overall, the differential gene expression analysis found that all 4 of these groups were predominately down-regulated when pteropods were exposed to low pH conditions. Below we briefly discuss each set of transcripts in these 4 categories.
Shell formation response
Expression patterns for genes involved in shell formation at the acute time points (d1 and d7) revealed a pattern of increased expression under the high-pH condition of 8.13, and very close clustering of expression profiles for both the mid-pH and low-pH conditions (Fig. 2a,b). At the acclimatory time points (d14 and d21), expression patterns for the three treatments partitioned into 3 categories that were associated with treatment conditions: high expression (pH 8.13), mid expression (pH 8.01), and low expression (pH 7.71) (Fig. 2b). These results support our position that long-term exposure to under-saturated conditions, mimicking those that L. h. antarctica are predicated to experience in winter by the year 2050, could limit the ability for pteropods to maintain calcified structures, and highlights the necessity for further functional validation of these effects.
Previous gene expression analysis for calcifying marine invertebrates has focused on a subset of shell formation genes that are impacted by changes in pH including calmodulin-like protein, chitin synthases, C-type lectines, perlucin, and collagen associated transcripts [2, 22, 37, 53]. Within this subset of shell formation related transcripts, there was a clear down-regulation associated with exposure to the low pH treatment (Additional file 6). Among these, calmodulin-like transcripts have been identified as important for calcification in the pearl oyster , and have been shown to be down-regulated in response to low pH in both oysters and corals [41, 53]. Chitin synthase is also critical in coordinating shell formation for mollusks , and has also been reported to be down-regulated in Limacina spp. . Finally, collagen-related transcripts (including C-type lectine, perlucin, and collagen-associated transcripts) have been shown to be important in shell formation in the disk abalone (Haliotis discus discus), and have been reported to be highly abundant in the mantle tissue of the Mediterranean mussel (Myilus galloprovincialis) [55, 56].
The heatmap displaying the broad patterns of gene expression in these shell formation genes (Fig. 2a, b) highlighted a subset of genes that were activated in response to low pH. Among these up-regulated transcripts were tyrosinase tyr-3, chromatin assembly factor 1 subunit B-like, ribosome biogenesis homolog, ribosome-releasing factor mitochondrial-like, and a RNA-directed DNA polymerase from mobile element jockey-like gene (Additional file 6). Among these, tyrosinase tyr-3 has previously been shown to be up-regulated in the biomineralization process in the blue mussel (Mytlius edulis), and is associated with formation of new periostracum [57, 58].
Expression patterns assessed in the metabolic response category revealed both temporal and pH-related patterns (Fig. 3a,b). Among these metabolic transcripts the highest levels of expression observed were in the T0 samples, and in the high pH treatments (Fig. 3a,b). The maintenance of T0 levels of expression for the first week of exposure, and continued grouping of the high pH treatments, suggests that food limitation was not a major obstacle for pteropods held in the pH 8.13 treatment. The mid and low-pH treatments however showed significant depression of metabolism-associated transcripts that grouped the low and mid pH treatments with time of exposure. This suppression of metabolic genes in response to low pH has been documented in corals, urchins, and the Mediterranean pteropod Heliconoides inflautus [2, 37, 59, 60]. The enrichment of genes in both the mid-pH and high-pH treatments along with a dramatic reduction in metabolic transcript expression suggests energy demands under short-term exposure to low pH conditions were not met by an increase in metabolism. This result may reflect the limited feeding opportunities experienced in our flow-through Z-system; however, in situ, little is known about food availability throughout the light-limited austral winter.
Cellular stress response
Differential expression patterns for genes involved in the CSR provided additional evidence of a compromised transcriptional response in L. h. antarctica in response to low pH. Up-regulated CSR transcripts associated with exposure to the low pH treatment were: HSP 70, heat-shock factor 2 binding protein-like gene, hydroxysteroid dehydrogenase 2-like gene, and a short-chain dehydrogenase/reductase family member 11-like gene (Additional file 6). In contrast, the transcripts down-regulated in the low pH conditions included those coding for multiple components of the cytochrome p450 pathway, the universal stress A-like protein, HSP 90-beta, and a glutathione mitochondrial-like protein.
The differential expression of key chaperones such as HSP 70 and HSP 90, genes critical for maintaining protein structure/function during oxidative stress, indicates that pH exposure in polar waters does not illicit a classic cellular stress response. Rather, exposure to low-pH conditions led to slight differential expression of these constitutively expressed genes. Within Antarctic species, high-levels of constitutive expression of CSR genes has been observed in Antarctic ectotherms, a response that is hypothesized to be due to increased levels of oxidative damage  and protein damage  caused by life at subzero temperatures. In this study, the differential expression of genes within the CSR indicated a mixed physiological response of L. h. antarctica to low pH conditions.
In our project on Limacina, we specifically explored differential expression patterns in genes involved in neural function because previous work with H. inflatus correlated pH exposure to elevated expression of neural genes . In our experiments however, exposure to low pH resulted in the opposite trend - namely, a decrease in expression levels of neural transcripts with the highest percentage found in the low pH treatment on d21 (Fig. 4b). Acute exposure times clearly still grouped the high pH treatment with the T0 samples while both the mid and the low pH treatment exhibit a decrease in expression levels (Fig. 4a). Within the up-regulated neural gene set on d21, we found 5 neuronal acetylcholine receptors, and 4 NXPE type-2 transcripts (Additional file 9). These up-regulated transcripts correspond to similar up-regulation of acetylcholine and neural transcripts observed in H. inflatus. The down-regulated transcripts included an acetylcholine receptor subunit delta-like, 4 transcripts for small conductance calcium-activated potassium channels and 5 skeletal receptor tyrosine kinase-like transcripts (Additional file 9). The proportion of down-regulated neural transcripts represent a more-pronounced suppression of neural transcripts than was found in the H. inflatus transcriptome, potentially due to the shorter exposure time (3 days) and narrower gap in treatment conditions (pH 7.9 vs. pH 8.01). The up-regulation of neuronal receptors coupled with the down-regulation of potassium channels and skeletal tyrosine kinases suggest that exposure to the low-pH treatments will negatively affect locomotion. Here, prolonged exposures to low pH conditions might impact the ability for L. h. antarctica to maintain normal neuronal function that could, for example, impede feeding mechanisms and vertical migration under seawater conditions expected in the winter by the year 2050.
Other studies of comparative transcriptomic and ocean acidification
Other investigators have assessed the impact of pCO2 levels on the transcriptome of marine invertebrates. These organisms include corals , oysters , sea urchins [7, 60, 63], mussels [56, 64], and pteropods [22, 23, 37]. In general, our results are in alignment with these other studies, with the general result that changes in pCO2 exposure does alter the transcriptome. In addition, our results on the Antarctic form of Limacina helicina are largely consistent with what has been reported in pteropods as a taxonomic group. Specifically, working with the Arctic form of Limacina helicina, Koh and colleagues found significant down-regulation of biomineralization genes . These results on polar Limacina spp. contrasts with what has been reported in a distantly related Mediterranean pteropod H. inflatus where low pH exposure elicited an up-regulation of biomineralization-related genes . Within the Limacina complex this observed divergence in response suggests pteropods from the Southern Ocean may be more sensitive then the temperate pteropod H. infaltus to under-saturation conditions of ocean acidification, and did not maintain expression of calcification-related genes when exposed to low pH conditions. Lastly, although the effects of pH on calcification in Limacina spp. have been well documented [13, 16, 20, 21, 65,66,67], there remains an ongoing debate regarding the ability for Limacina spp. to utilize internal shell repair mechanisms to minimize the deleterious impacts of ocean acidification [65, 68, 69]. However, this study along with the transcriptome study presented by Koh et al.  suggests that all forms of Limacina helicina are challenged to maintain calcification in low pH conditions.