Maintenance of coelomic fluid ph in sea urchins exposed to elevated CO 2 : the role of body cavity epithelia and stereom dissolution

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1 Marine Biology Supplementary Online Resource For: Maintenance of coelomic fluid ph in sea urchins exposed to elevated CO 2 : the role of body cavity epithelia and stereom dissolution Wiebke C. Holtmann 1, Meike Stumpp 1,2,3, Magdalena A. Gutowska 1,2, Stephanie Syré 2, Nina Himmerkus 1, Frank Melzner 2, Markus Bleich 1 1 Physiologisches Institut, CAU Kiel, D Kiel, Germany 2 GEOMAR Helmholtz-Zentrum für Ozeanforschung Kiel, D Kiel, Germany 3 Department of Biological and Environmental Sciences, The Sven Lovén Centre for Marine Science, University of Gothenburg, Kristineberg, Fiskebäckskil, Sweden Correspondence: Markus Bleich, Phone: +49 (0) , Fax: +49 (0) , m.bleich@physiologie.uni-kiel.de

2 Online Resource 1 In some ossicle pairs growth measurements were not possible. Thus, for classification of the measurements, three categories were introduced. The first category growth described successful calcein labelling and two lines (one per ossicle) were distinguishable. In no growth calcein labelling was successful but only one line was visible. Thus, growth was set to zero and was included in the analysis. The third category no labelling included ossicles without a calcein signal at the putative growing side. These ossicles were excluded from the analysis. Category assignment of ossicle growth measurements. Percentage of ossicles falling under the three categories growth, no growth and no labelling in the three regions aboral I, II and oral. Growth decreased from aboral to oral and with increasing CO 2. The percentage of "no growth" increased with increasing CO 2. Calcein labelling was not dependent on these conditions. aboral I growth (%) no growth (%) no labelling (%) aboral II growth (%) no growth (%) no labelling (%) oral growth (%) no growth (%) no labelling (%)

3 Online Resource 2 Membrane staining and FITC diffusion The properties of the peritoneal epithelium were investigated by staining the membranes with the nontoxic FM 1-43 membrane probe (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl)pyridinium dibromide, obtained from Life Technologies GmbH, Germany). A piece of the peritoneal epithelium was dissected, stained with 20 x 10-6 M FM 1-43 and transferred to a slide. A z-stack (1 µm stacks) was performed with a LSM 510 (Zeiss, Germany), excitation 488 nm, HFT 488, LP 505. a) Confocal micrograph of FM 1-43 stained peritoneal epithelium (1 µm stacks). (a-1) gives an example of the luminal side (PCF facing) where cilia are visible and (a-2) shows the connective tissue of the basolateral side (stereom facing). b) FITC fluorescence as a result of transepithelial diffusion as function of incubation time. Peritoneal epithelium integrity and size selectivity is visible from the low permeation of molecules >0.3 kda. Relative fluorescence units (RFU) are normalised to the starting molarities of luminally applied FITC. Significant differences between RFU of the starting point 0 min and the respective sampling point are marked with an asterisk (N = 4-6). Means are given ± SEM.

4 Online Resource 3 Fracture measurements of spines Distribution of the breaking force in all measured ossicles (a, open squares) and spines (b, open circles; only lengths of mm were used for the analysis). The means ± SEM of true replicates are shown next to the respective distributions (see also Table 2). N for distribution data: ossicles = 22, ossicles = 23, ossicles = 18; spines = 24, spines = 23, spines = 20; N for means = 6.

5 Online Resource 4 Calculation of dissolution Assumptions: Sea urchin diameter: Volume of perivisceral coelomic fluid: Height of granular structures: 3.2 cm 2.6 ml 100 nm First, the volume of dissolved material was calculated under the assumption that 100 nm of ossicle thickness over the whole surface is dissolved and that 50% of ossicle volume consists of CaCO 3. The density of calcite is 2.71 g (cm 3 ) -1 and was used to calculate the amount of dissolved CaCO 3 and the respective concentration increase of CO 2-3. ( ) This would result in a concentration increase by 1.68 mm of dissolved CO 2-3 if the whole surface would be eroded. Taking into account that the SEM analysis revealed that 50% of the est, 50% of the and 12.5% of the CO 2 treated ossicles exhibited pitted surfaces, the following increases in CO 2-3 concentration were estimated. High CO 2 treatment, released CO 3 2- : Intermediate CO 2 treatment, released CO 3 2- : Control CO 2 treatment, released CO 3 2- : 0.84 mm 0.84 mm 0.21 mm In SEM pictures it was visible that not all ossicle regions were characterised by the granular structures. But as we analysed only a small area and as we don t know the dissolution pattern in the inner stereom parts we don t give any further estimations. At the given ph s for the three CO 2 treatments it can be further assumed that most of the released CO 2-3 is available as HCO - 3.

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