Sibling species differently distributed around a CO2 vent show transplantation proteomic remodelling, while displaying metabolomic signatures associated with their origin

Abstract

The cellular homeostatic response (CHR) and cellular stress response (CSR) work together to maintain homeostasis. Studying phylogenetically closely-related species inhabiting different environments can help investigate the interplay between the CHR and CSR. We conducted reciprocal in situ transplant experiments in a natural CO₂ vent (Ischia, Italy), using the sibling annelid species Platynereis cf.. dumerilii and Platynereis cf.. massiliensis which have been shown to have different preferential distributions around the CO₂ vent. Following transplantations, we characterised the response of each individual's proteome, metabolome, and lipidome, to short or long-term exposure to different pCO₂ regimes (i.e., high and low), and confirmed its genetic identity. Here we show that different components of the CHR and CSR are utilised at different rates when Platynereis spp. are exposed to different pCO₂ regimes, with cellular responses shown to be conserved across species. Metabolome and lipidome responses were dependent on regime of origin, and changed relatively slowly, whereas proteome responses were dependent on transplant type and changed more rapidly. Our results provide new insights to improve our understanding of the interplay between different cellular physiological responses involved in defining the functional phenotype of marine species, and their ability to acclimatise to future projected high pCO₂ conditions.

Keywords: Platynereis; Cellular stress response (CSR); Lipidomics; Metabolomics; Ocean acidification; Proteomics.

Fig. 1

Metabolome (polar extracts) characterization of marine annelids Platynereis spp. reciprocally transplanted between high or low pCO₂ conditions (four treatments, CC = collected in low pCO₂ and transplanted to low pCO₂, CA = collected in low pCO₂ and transplanted to high pCO₂, AA = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). (a) Principal Components Analysis 2D score plot of the four groups with 95% confidence intervals, (b) Sample cluster analysis using Euclidean distance and Ward’s clustering algorithm, (c) Principal Components Analysis 2D score plot of the metabolome of annelids according to regime of origin (control, including group CC and CA; and acidified, including group AA and AC) with 95% confidence intervals, (d) Principal Components Analysis 2D score plot of the metabolome of annelids according to transplant type, namely between the same environment (SE, which includes CC and AA annelids), or different environment (DE, which includes CA and AC annelids) with 95% confidence intervals. (e) Volcano plot representing differentially abundant metabolites (n = 129, coloured as red dots) according to worm regime of origin (control vs. acidified), based on t-tests with 250 randomizations, FDR 0.05 and s 0.1. (f) Volcano plot highlighting that there are no differences in the metabolome of annelids according to transplant type, based on t-tests with 250 randomizations, FDR 0.05 and s 0.1.

Fig. 2

Metabolome (polar extracts) profiling of marine annelids Platynereis spp. according to regime of origin, namely control (including CC annelids = collected in low pCO₂ and transplanted to low pCO₂, and CA = collected in low pCO₂ and transplanted to high pCO₂) and acidified (including AA annelids = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). (a) Heat map representation of the 129 significantly different m/z peaks, based on a clustered data matrix (distance: Spearman correlation; linkage: complete) in which cells denote the normalized metabolite abundances (glog transformed and Z-scored), ranging from down-accumulated (blue) to up-accumulated (orange). (b) Top enriched metabolite classes based on mummichog and GSEA algorithms. (c) Top enriched pathways based on mummichog and GSEA algorithms. Graphs are coloured according to significance in mummichog (blue) and significance in GSEA (pink). The size and colour of the circles correspond to their transformed combined p-values. More details on enrichment results can be consulted on Table S4 and S5.

Fig. 3

Lipidome characterization of marine annelids Platynereis spp. reciprocally transplanted between high or low CO₂ conditions (four treatments, CC = collected in low pCO₂ and transplanted to low pCO₂, CA = collected in low pCO₂ and transplanted to high pCO₂, AA = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). (a) Principal Components Analysis 2D score plot of the four groups with 95% confidence intervals, (b) Sample cluster analysis using Euclidean distance and Ward’s clustering algorithm, (c) Principal Components Analysis 2D score plot of the lipidome of annelids according to regime of origin (control, including group CC and CA; and acidified, including group AA and AC) with 95% confidence intervals, (d) Principal Components Analysis 2D score plot of the lipidome of annelids according to transplant type, namely between the same environment (SE, which includes CC and AA annelids), or different environment (DE, which includes CA and AC annelids) with 95% confidence intervals. (e) Volcano plot representing differentially abundant lipids (coloured as red dots) according to annelid regime of origin (control vs. acidified), based on t-tests with 250 randomizations, FDR 0.05 and s 0.1. (f) Volcano plot highlighting that there are no differences in the lipidome of annelids according to transplant type, based on t-tests with 250 randomizations, FDR 0.05 and s 0.1.

Fig. 4

Lipidome profiling of marine annelids Platynereis spp. according to regime of origin, namely control (including CC annelids = collected in low pCO₂ and transplanted to low pCO₂, and CA = collected in low pCO₂ and transplanted to high pCO₂) and acidified (including AA annelids = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). (a) Heat map representation of the clustered data matrix (distance: Spearman correlation; linkage: complete) in which cells denote the normalized lipid abundances (glog transformed and Z-scored), ranging from down-accumulated (blue) to up-accumulated (orange). (b) Main lipid classes among the significant m/z peaks and corresponding ions. (c) Biological functions (retrieved from the Encyclopedia of Lipidomics and HMDB Metabocards) associated with the lipid classes of the identified ions (each ion may be associated to several biological functions). PC – glycerophosphocholines, PE – glycerophosphoethanolamines, LC-SFA – long chain saturated fatty acids.

Fig. 5

Proteome characterization of marine annelids Platynereis spp. reciprocally transplanted between high or low CO₂ conditions (four treatments, CC = collected in low pCO₂ and transplanted to low pCO₂, CA = collected in low pCO₂ and transplanted to high pCO₂, AA = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). Species were identified (using COI) as Platynereis cf.. dumerilii, Platynereis cf.. massiliensis or unknown. (a) Principal Components Analysis 3D score plot, (b) Principal Components Analysis 2D score plot with 95% confidence intervals (c) Sample cluster analysis using Euclidean distance and Ward’s clustering algorithm.

Fig. 6

Differentially abundant proteins (DAPs) and pathways regulated by marine annelids Platynereis spp. reciprocally transplanted between the same (SE) or different environments (DE), namely high or low pCO₂ conditions. The group SE includes CC annelids (collected in low pCO₂ and transplanted to low pCO₂) and AA annelids (collected in high pCO₂ and transplanted to high pCO₂). The group DE includes CA annelids (collected in low pCO₂ and transplanted to high pCO₂) and AC annelids (collected in high pCO₂ and transplanted to low pCO₂). (a) Volcano plot representing the proteome difference between annelids transplanted to the SE and DE, based on t-tests with 250 randomizations, FDR 0.05 and s 0.1. (b) Network analysis carried out using Cytoscape v3.8.0 and plugins ClueGO v2.5.7 and CluePedia v1.5.7. The analysis was based on differentially abundant proteins (DAPs) between SE and DE (species: Caenorhabditis elegans, ontologies: GO_ImmuneSystemProcess-GOA_17.11.2016_10h53, KEGG_17.11.2016, GO_BiologicalProcess-GOA_17.11.2016_10h53, enrichment/depletion: two-sided hypergeometric with Bonferroni step down correction and p-value of 0.05, GO level 3 to 8, minimum number of genes 1, minimum percentage of genes 4%, Kappa score 0.4).

Fig. 7

Proteomic profiling of marine annelids Platynereis spp. reciprocally transplanted between the same environment (SE) or different environment (DE), namely high or low CO₂ conditions (four treatments, CC = collected in low pCO₂ and transplanted to low pCO₂, CA = collected in low pCO₂ and transplanted to high pCO₂, AA = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). Heat map representation of the clustered data matrix (distance: Spearman correlation; linkage: complete) in which cells denote the normalized protein abundances (Log₂ transformed and Z-scored), ranging from down-accumulated (blue) to up-accumulated (orange). Differentially abundant proteins are highlighted with an arrow (based on a volcano plot - t-tests with 250 randomizations, FDR 0.05 and s 0.1). Three clusters were defined, and protein profiles were plotted for each cluster.

Fig. 8

Multi-omics characterization of marine annelids Platynereis spp. reciprocally transplanted between high or low CO₂ conditions (four treatments, CC = collected in low pCO₂ and transplanted to low pCO₂, CA = collected in low pCO₂ and transplanted to high pCO₂, AA = collected in high pCO₂ and transplanted to high pCO₂, and AC = collected in high pCO₂ and transplanted to low pCO₂). Species were identified (using COI) as Platynereis cf.. dumerilii, Platynereis cf.. massiliensis or unknown. Principal Components Analysis score plot of the (a) proteome, (b) metabolome, namely lipid extracts (c) metabolome, namely polar extracts.