Digestion in sea urchin larvae impaired under ocean acidification

Transcription

1 SUPPLEMENTARY INFORMATION DOI: /NCLIMATE2028 Digestion in sea urchin larvae impaired under ocean acidification Supplementary information for: Digestion in sea urchin larvae impaired under ocean acidification Meike Stumpp #, Marian Hu #, Isabel Casties, Reinhard Saborowski, Markus Bleich, Frank Melzner, Sam Dupont * # These authors contributed equally to the present work *To whom correspondence should be addressed: Sam Dupont sam.dupont@bioenv.gu.se phone: +46(0) This PDF file includes: Figs S1 to S5 Tables S1 to S2 References NATURE CLIMATE CHANGE 1

2 Supplemental figures Growth and development In order to correct physiological parameters on the developmental stage, growth, and development of larvae were monitored (Fig. S1 A+B). Body length (BL) growth followed a significant logarithmic relationship with time (dpf) for the 3 tested ph and the two experiments. In both experiments, ph had a significant effect on growth rates (exp1, F=229.9, p<0.0001; exp 2, F=3308.7, p<0.0001). Fig. S1 Growth curves of sea urchin larvae reared under different ph conditions from the two experiments used in the present study. (A) Growth curves from the experiment used for gastric ph measurements (experiment 1). (B) Growth curves from the experimental trial used for enzyme and feeding experiments (experiment 2). Growth curves were fitted by logarithmic curves (y=a*ln(x) + b). Gastric ph measurements Using non-selective electrodes we could demonstrate that no potential exists between the stomach lumen and the bath solution (Fig. S2 A). In contrast H + -selective electrodes recorded a negative potential of approximately 100 mv between gastric lumen a bath solution (Fig 2S B). Acute changes (up to 20 min) of the bath solution ph could not affect the gastric ph (Fig. S2 C). We additionally tested the effects of starvation on gastric ph homeostasis demonstrating decreased gastric ph in starved animals (Fig. S3). As the maintenance of high proton gradients between stomach lumen and the environment can be considered energetically expensive, decreased gastric ph can be considered an energy saving mode in response to starvation.

3 Fig. S2 Original time traces of voltage measurements (time line from left to right) using non-selective (A) or H + selective (B, C) microelectrodes. Nonselective voltage electrode measurements demonstrating no trans-epithelial potential difference between the bath solution and stomach lumen of Strongylocentrotus droebachiensis larvae (A). H + -selective voltage electrode measurements demonstrating a -100 mv potential difference between the bath solution and stomach lumen (A). Acute changes in the perfusion bath ph in time scales up to 20 minutes did not affect gastric ph homeostasis in Strongylocentrotus droebachiensis (C). Fig. S3 Gastric ph of fed and starved Strongylocentrotus droebachiensis echinopluteus 7-12 days post fertilization. Values are given as mean ± SD letters denote significant differences (n = 6-11).

4 Immunocytochemistry Fig. S4 Immunohistochemical detection of Na + /K + -ATPase (NKA) rich cells in the stomach and intestinal epithelium in Strongylocentrotus droebachiensis echinopluteus. NKA is distributed in stomach ionocytes in a salt and pepper pattern (A) (left: fluorescence image; right fluorescence + brightfield). NKA is localized in luminal membranes of the stomach and intestinal epithelium (B). Negative controls were performed by omitting the primary antibody (C). Western blot analysis of pluteus larvae exctracts, demonstrating the detection of a 115 KDa protein by the monoclonal NKA specific antibody (D). In vivo digestion In order to test the digestive potential of live larvae, we used the natural emission wavelength of chlorophyll a at 680 nm fluorescence to detect the breakdown of ingested Rhodomonas sp. algae 1. Algal cells are collected via ciliated epidermal cells located on the arms of the larvae and transported to the mouth. Cells are collected and concentrated within the oesophagus (Fig. S4 upper panel, cells are indicated by arrows). No degradation of the signal was recorded when cells were stored within the oesophagus, indicating the stability of the fluorescence signal. However, when sufficient cells were collected, the larvae swallows and transfers algal cells into the stomach where the fluorescence intensity decreases in a linear fashion, reflecting the digestive potential (Fig. S4). Algal cells within the stomach (indicated by dashed circle) stay in place during recordings. No changes in fluorescence intensity due to movements out of the focus plane were observed. Since the maximum fluorescence in each swallow event and the digestion rate differed in response to the no of swallowed algae, the slope of each swallow event was normalized (mgv norm ) onto the maximum fluorescence signal observed within each swallow event.

5 Fig. S5 In vivo digestion traces (uncorrected raw data), measured as change of mean grayscale values (mgsv) per time in a selected region of interest (ROI, white circles) using confocal microscopy and the natural emission wavelength of chlorophyll a at 680 nm. Sequential images of a larva during the digestion process indicating the degradation of ingested algae. Note the maintained fluorescence intensity in collected algae within the oesophagus (arrows). Mgsv in the ROI in the stomach increase directly after swallowing (grey dashed line) and decrease linearly (red line) during digestion of ingested algae. The slope of the linear decrease in mgsv was used as a measure of the digestion efficiency. ph perturbation experiments ph perturbation experiments for studies on the gastric ph in sea urchin larvae (Strongylocentrotus droebachiensis) were carried out in 2011 at GEOMAR and CAU Kiel (Germany; exp 1; duration: 10 days). For enzyme assays and feeding experiments another ph pertubation experiment was conducted in spring 2012 at the Sven Lovén Centre for Marine Sciences (Kristineberg, Sweden; exp 2; duration: 24 days). Adult S. droebachiensis were collected in Winter 2011/2012 in the Oslo Fjord (Dröbak, Norway; same population used in a previous study 2 ) by SCUBA-divers. For each e periment eggs of t o females ere collected in separate ea ers ashed and fertili ed y adding dry sperm of t o males ( ; to a final concentration of sperm m -1, allowing a fertilization success >95 %). Zygotes were allowed to divide once before they were pooled, concentrated to 25 ml, and separated at a density of 10 embryos per ml into 2L (exp 1) or 5L (exp 2) Erlenmeyer culture flasks (three

6 replicates per ph treatment) which were pre-equilibrated with the respective ph/pco 2 levels. After 5 days, larvae used for all experiments (except for starved larvae used for gastric ph measurements) were fed daily with the cryptophyte algae Rhodomonas sp., which were raised in B1 medium 3 at 20 C under a 12:12 h light:dark cycle. Starved larvae were never fed prior to ph measurements and were not exposed to elevated seawater pco 2. Algal strains were provided by the Marine Algal Culture Centre at Gothenburg University (GUMACC). The carbon content of the algae was estimated based on biovolume measurements as equivalent spherical diameter (ESD) with an electronic particle analyzer (Elzone 5380, Micrometrics, Aachen, Germany) and equations provided by Mullin et al. 4. To prevent changes in food concentration, algae concentration and size were checked daily using a Coulter counter (Elzone 5380, Micrometrics, Aachen, Germany) and then adjusted in the experimental bottles to the ma imum concentration of 5 μg C L -1 ( 6,000 cells ml -1 for diameters ranging et een 6 and 8 μm). At the chosen algae concentration ( 5 μg C L -1 ), time of exposure (24 h, algae added daily), seawater ph treatment levels and temperature had no impact on algal growth and survival 5. The ph in the culture flasks was controlled by a central automatic gas mixing device (Linde Gas, HTK Hamburg, Germany; exp 1) or by a computer-controlled system (AquaMedic, Bissendorf, Germany; exp 2) which regulates the ph (NBS scale) by addition of pure gaseous CO 2 directly into the seawater (+/. ph units). Three scenarios ere tested: (i) ph corresponding to the average ph experienced by the sea urchin larvae today; (ii) ph , corresponding to the average ph expected by 2100 under ocean acidification scenarios and the extreme of the ph environmental variability naturally experienced today; (iii) , corresponding to the extreme of the ph variability expected by 2100 under ocean acidification scenarios. Water ph NBS, salinity, and temperature were monitored daily during the incubation period. Seawater samples for total dissolved inorganic carbon (C T ), seawater ph T (ph total) and total alkalinity (A T ) were collected twice a week. C T was determined with an AIRICA analyzer (Marianda). A T was determined with a titration system (titroline alpha plus, SI Analytics). Seawater carbonate system speciation was calculated from ph T and C T or ph T and A T with the open-source program CO2SYS 6 using the dissociation constants by Mehrbach et al. 7 as refitted by Dickson and Millero 8. Water parameters measured during the experimental periods are summarized in table S1. All experiments were in accordance with the German law for animal welfare and were approved by the animal welfare officers of Christian Albrechts University, Kiel and the University of Gothenburg.

7 Table S1 Seawater physicochemical conditions Incubation group Temp ( C) ph T CO 2 (μatm) C T A T Experiment 1 Control 9.42 ± ± ± ± ± 0.01 CO 2 ~ 1000 ppm 9.43 ± ± ± ± ± 0.04 CO 2 ~ 2400 ppm 9.48 ± ± ± ± ± 0.02 Experiment 2 Control 9.01 ± ± ± ± ± 0.01 CO 2 ~ 1200 ppm 9.05 ± ± ± ± ± 0.03 CO 2 ~ 3000 ppm 9.07 ± ± ± ± ± 0.01 Seawater physicochemical conditions during the two ph experiments (10 d: ph measurements and 24 d: enzyme and feeding). ph T (ph total), A T, total alkalinity; C T, total dissolved inorganic carbon; pco 2, partial pressure of CO 2. Values are presented as mean ± SD. Table S2 Results of the regression analyses Parameter Regression R 2 df F p Protease activity Activity vs. TPF ph 8.0 f= /(1+e (-(x )/1.2117) ) , < ph 7.6 f= /(1+e (-(x )/1.0429) ) , < ph 7.2 f= /(1+e (-(x )/0.9778) ) , < Activity vs. BL ph 8.0 f= /(1+e (-(x )/ ) ) , < ph 7.6 f= /(1+e (-(x )/ ) ) , < ph 7.2 f= /(1+e (-(x )/ ) ) , < Feeding rates Feeding rate vs. TPF ph 8.0 f=0.001*e (0.1066*x) , < ph 7.6 f=0.0013*e (0.0691*x) , < ph 7.2 f=0.0005*e (0.1143*x) , < Feeding vs. BL ph 8.0 f=0.0001*e (0.0106*x) , < ph 7.6 f=0.0002*e (0.0080*x) , < ph 7.2 f=0.0001*e (0.0083*x) , < Results of the regression analyses of measured parameters in S. droebachiensis larvae raised under control (ph 8.0) intermediate (ph 7.6) and low ph conditions (ph 7.2): total protease activities and feeding rates relative to time post-fertilization (TPF) and body length (BL). References 1 Cole, A. G., Rizzo, F., Martinez, P., Fernandez-Serra, M. & Arnone, M. I. Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut. Development 136, (2009). 2 Stumpp, M. et al. Acidified seawater impacts sea urchin larvae ph regulatory systems relevant for calcification. Proc Nat Acad Sci USA 109, (2012). 3 Guillard, R. R. L. & Ryther, J. H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustdtand, Detonula confervacea Cleve. Can J Microbiol 8, (1962). 4 Mullin, M. M., Sloan, P. R. & Eppley, R. W. Relationship between carbon content, cell volume and area in phytoplankton. Limnol Oceanogr 11, (1966).

8 5 Dupont, S. T., Dorey, N., Stumpp, M., Melzner, F. & Thorndyke, M. C. Long-term and translife-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis. Mar Biol DOI /s x (2012). 6 Lewis, E. & Wallace, D. Program developed for CO2 system calculations. (Oak Ridge National Laboratory ORNL/CDIAC-105, 1998). 7 Mehrbach, C., Culberso, C. H., Hawley, J. E. & Pytkowicz, R. M. Measurement of apparent dissociation constants of carbonic acid in seawater at atmospheric pressure. Limnol Oceanogr 18, (1973). 8 Dickson, A. G. & Millero, F. J. A comparison of the equilibrium-constants for the dissociation of carbonic-acid in seawater media. Deep-Sea Res 34, (1987).