DataSheet_1_Transcriptomic Responses of Adult Versus Juvenile Atlantids to Ocean Acidification.pdf

Shelled holoplanktonic gastropods are among the most vulnerable calcifiers to ocean acidification. They inhabit the pelagic environment and build thin and transparent shells of aragonite, a metastable form of calcium carbonate. While shelled pteropods have received considerable attention and are widely regarded as bioindicators of ocean acidification, atlantids have been much less studied. In the open ocean, atlantids are uniquely positioned to address the effects of ocean acidification at distinct trophic levels. From juvenile to adult, they undergo dramatic metamorphosis. As adults they are predatory, feeding primarily on shelled pteropods, copepods and other zooplankton, while as juveniles they feed on algae. Here we investigated the transcriptome and the impact of a three-day CO2 exposure on the gene expression of adults of the atlantid Atlanta ariejansseni and compared these to results previously obtained from juveniles. Individuals were sampled in the Southern Subtropical Convergence Zone (Atlantic Ocean) and exposed to ocean chemistry simulating past (~mid-1960s), present (ambient) and future (2050) conditions. In adults we found that the changes in seawater chemistry had significantly affected the expression of genes involved in biomineralization and the immune response, although there were no significant differences in shell growth between the three conditions. In contrast, juveniles experienced substantial changes in shell growth and a broader transcriptomic response. In adults, 1170 genes had the same direction of expression in the past and future treatments when compared to the ambient. Overall, this type of response was more common in adults (8.6% of all the genes) than in juveniles (3.9%), whereas a linear response with decreasing pH was more common in juveniles (7.7%) than in adults (4.5%). Taken together, these results suggest that juveniles are more sensitive to increased acidification than adults. However, experimental limitations including short incubation times, one carboy used for each treatment and two replicates for transcriptome analysis, require us to be cautious about these conclusions. We show that distinct transcriptome profiles characterize the two life stages, with less than 50% of shared transcripts. This study provides an initial framework to understand how ocean acidification may affect the molecular and calcification responses of adult and juvenile atlantids.</p

DataSheet_2_Transcriptomic Responses of Adult Versus Juvenile Atlantids to Ocean Acidification.xlsx

Shelled holoplanktonic gastropods are among the most vulnerable calcifiers to ocean acidification. They inhabit the pelagic environment and build thin and transparent shells of aragonite, a metastable form of calcium carbonate. While shelled pteropods have received considerable attention and are widely regarded as bioindicators of ocean acidification, atlantids have been much less studied. In the open ocean, atlantids are uniquely positioned to address the effects of ocean acidification at distinct trophic levels. From juvenile to adult, they undergo dramatic metamorphosis. As adults they are predatory, feeding primarily on shelled pteropods, copepods and other zooplankton, while as juveniles they feed on algae. Here we investigated the transcriptome and the impact of a three-day CO2 exposure on the gene expression of adults of the atlantid Atlanta ariejansseni and compared these to results previously obtained from juveniles. Individuals were sampled in the Southern Subtropical Convergence Zone (Atlantic Ocean) and exposed to ocean chemistry simulating past (~mid-1960s), present (ambient) and future (2050) conditions. In adults we found that the changes in seawater chemistry had significantly affected the expression of genes involved in biomineralization and the immune response, although there were no significant differences in shell growth between the three conditions. In contrast, juveniles experienced substantial changes in shell growth and a broader transcriptomic response. In adults, 1170 genes had the same direction of expression in the past and future treatments when compared to the ambient. Overall, this type of response was more common in adults (8.6% of all the genes) than in juveniles (3.9%), whereas a linear response with decreasing pH was more common in juveniles (7.7%) than in adults (4.5%). Taken together, these results suggest that juveniles are more sensitive to increased acidification than adults. However, experimental limitations including short incubation times, one carboy used for each treatment and two replicates for transcriptome analysis, require us to be cautious about these conclusions. We show that distinct transcriptome profiles characterize the two life stages, with less than 50% of shared transcripts. This study provides an initial framework to understand how ocean acidification may affect the molecular and calcification responses of adult and juvenile atlantids.</p

MOESM2 of Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance

Additional file 2: Transcripts of L. bulimoides with homology to biomineralisation proteins

MOESM1 of Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance

Additional file 1: Supporting information containing Appendices S1-5 and Tables S1-2

MOESM3 of Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance

Additional file 3: 82-mer probe sequences for L. bulimoides

Molecular composition of the skeletal organic matrix from A. millepora.

<p>(A) Analysis of electrophoresis on gel after AgNO3 staining.(B) PVDF membrane revealed by autoradiography with 45Ca, calmodulin (CaM)was used as positive control. (C) Infrared absorption spectra of ASM and AIM fractions with assignment of the main peaks. MM – Molecular marker, ASM – Acid soluble matrix, AIM – Acid insoluble matrix.</p

Skeleton morphology and microstructure.

<p>(A) Skeletal fragments after treatment in NaOCl (5%, vol/vol) for 72 h prior to longitudinal and transversal cuts. Scanning electron microscopy images from the skeleton morphology: (B) Axial corallite, (C) Radial corallites, (D) Closer view into a radial corallite showing different septa. Polished and EDTA-etched sections from a transversal cut (E–G) and longitudinal cut (H–I). EMZ – early mineralization zone.</p

Characterization of the 36 skeletal organic matrix proteins (SOMPs).

<p>Computed parameters: molecular weight (Mw), isoelectric point (pI), most abundant amino acid (Major aa%), post-translational modifications (PTM) and other sequence features: signal peptide (SP), transmembrane domain (TM), glycosylphosphatidylinositol (GPI anchor), complexity regions (LCR), regions of biased composition, motifs and repeats.</p><p>*Properties calculated based on the primary sequence of the mature protein, <i>i.e.</i> without peptide signal.</p

SEM images of CaCO₃ crystals grown <i>in vitro</i> with the addition of different concentrations of ASM: (A) 0 µg.ml⁻¹, (B) 0.1 µg.ml⁻¹, (C) 1 µg.ml⁻¹, (E) 5 µg.ml⁻¹, (E) 10 µg.ml⁻¹ and (F) 20 µg.ml⁻¹.

<p>(G) Corresponding FTIR(ATR) absorbance spectra on the whole precipitated for the following ASM concentrations: 0, 0.1, 1, 10 and 20 µg.ml⁻¹.</p

Quantification of neutral, aminated and acidic monosaccharides in the ASM (blue) and in the AIM (red) of <i>A. millepora</i>.

<p>Samples were hydrolyzed with 2°C (4 h). (A) Total Wt. % in the skeletal organic matrix (SOM, either ASM or AIM) are indicated in the graph (bars). (B) Concentrations (ng/µg) and relative molar percentages are shown in the table for both matrices.</p