Gill transcriptome response to changes in environmental calcium in the green spotted puffer fish

Abstract

Background: Calcium ion is tightly regulated in body fluids and for euryhaline fish, which are exposed to rapid changes in environmental [Ca2+], homeostasis is especially challenging. The gill is the main organ of active calcium uptake and therefore plays a crucial role in the maintenance of calcium ion homeostasis. To study the molecular basis of the short-term responses to changing calcium availability, the whole gill transcriptome obtained by Super Serial Analysis of Gene Expression (SuperSAGE) of the euryhaline teleost green spotted puffer fish, Tetraodon nigroviridis, exposed to water with altered [Ca2+] was analysed.

Results: Transfer of T. nigroviridis from 10 ppt water salinity containing 2.9 mM Ca2+ to high (10 mM Ca2+ ) and low (0.01 mM Ca2+) calcium water of similar salinity for 2-12 h resulted in 1,339 differentially expressed SuperSAGE tags (26-bp transcript identifiers) in gills. Of these 869 tags (65%) were mapped to T. nigroviridis cDNAs or genomic DNA and 497 (57%) were assigned to known proteins. Thirteen percent of the genes matched multiple tags indicating alternative RNA transcripts. The main enriched gene ontology groups belong to Ca2+ signaling/homeostasis but also muscle contraction, cytoskeleton, energy production/homeostasis and tissue remodeling. K-means clustering identified co-expressed transcripts with distinct patterns in response to water [Ca2+] and exposure time.

Conclusions: The generated transcript expression patterns provide a framework of novel water calcium-responsive genes in the gill during the initial response after transfer to different [Ca2+]. This molecular response entails initial perception of alterations, activation of signaling networks and effectors and suggests active remodeling of cytoskeletal proteins during the initial acclimation process. Genes related to energy production and energy homeostasis are also up-regulated, probably reflecting the increased energetic needs of the acclimation response. This study is the first genome-wide transcriptome analysis of fish gills and is an important resource for future research on the short-term mechanisms involved in the gill acclimation responses to environmental Ca2+ changes and osmoregulation.

Figure 1

Blood plasma total calcium and ecac gill mRNA expression upon exposure to different water [Ca²⁺]. Each bar is the mean ± S.E.M of (A) the plasma total calcium levels (mM) and (B) the relative expression of the epithelial calcium channel mRNA in the gills (ecac expression quantified by qPCR and normalized to the reference gene rps18) of T. nigroviridis individuals (n = 5-6/group) after 2 or 12 h transfer to 10 ppt artificial water containing 2.9 mM Ca²⁺ (control water, black bars), 10 mM Ca²⁺ (HighCa water, light grey bars) or 0.01 mM Ca²⁺ (LowCa water, dark grey bars). Different letters indicate statistically significant differences (p < 0.05) between groups, evaluated by two-way ANOVA.

Figure 2

Results from K-means clustering of differentially expressed SuperSAGE tags according to their expression patterns. Pattern of expression of all tags for each cluster (mean ± S.E.M of expression, normalized by making the sum of tag counts for all the analyzed libraries equal to 100%), (A) for the LowCa analysis (effects of transfer to 0.01 mM Ca²⁺ at 2 or 12 h), (B) for the 12 h analysis, describing tag expression at 12 h exposure to LowCa (0.01 mM Ca²⁺), control (2.9 mM Ca²⁺) or HighCa (10 mM Ca²⁺) water. The number of tags grouped in each cluster is shown in each graph. C- Bubble graph showing the relation between clusters of the two analyses. Bubble area is proportional to the number of tags in common by each pair of cluster number (LowCa and 12 h analysis). Tags falling in clusters numbered "zero" were those not analyzed (due to failure of inclusion criteria, see methods) for a given analysis (LowCa or 12 h). Solid line - control; dashed line - LowCa.

Figure 3

Relationship between relative change of gene expression measured by qPCR and SuperSAGE. Relative change of gene expression (fold change) between Ca²⁺-challenged (Low or HighCa water) and control gills for the same response period (2 or 12 h) measured by qPCR (target gene/rps18) and SuperSAGE (sum of tag counts annotating to the same gene). r is the Pearson correlation coefficient.

Figure 4

Main biological processes affected by transfer to water at different Ca²⁺ concentrations in Tetraodon gills. Each triangle represent the differential expression of genes contained in the most represented biological processes (represented in the lower panel and grouped into broad categories in the upper panel) between LowCa (0.01 mM Ca²⁺) or HighCa (10 mM Ca²⁺) water and control water (2.9 mM Ca²⁺, blue triangle). Triangle width is inversely proportional to the adjusted p values obtained for biological process GO categories in our GO enrichment/clustering analysis (wider for most enriched processes); shown categories were selected among the most enriched GOs (adjusted p values > 10^-4).