, 190 microscopy or in a 3% glutaraldehyde solution in Sörensen-sucrose buffer

, ) equipped with polarizing filters. All images 204 were acquired with a digital camera (DS-Ri1, Nikon) at 100x magnification and 40 ms 205 light exposition. Images were analyzed with NIS-element and Image-J software. 206 207 2.3.2. Morphometrical analysis 208 Larval development stage, shell presence and size were determined under light 209 microscopy (n = 100 larvae per tank, 200 larvae per pH condition). Maximal larval 210 length and width of normal larvae were measured in specimens lying on the lateral side, 211 as shown by the dotted black arrows in Figure 1. The product of length * width was 212, Approximately 100 larvae were whole-mounted in 197 a drop of glycerol

. Tukey, Larval size 362 was significantly

, Under control pH conditions, larvae clearly exhibited the characteristic dark cross

, Larvae grown at lower pH (7.6) typically showed a decrease in 370 shell birefringence indicating reduced calcification (Fig. 8D). At 30 hpf, the distribution 371 of larvae into the three categories (i.e. fully mineralized, partially mineralized and less 372 mineralized) was not significantly different between the three pH treatments

, At 48 hpf, the number of fully mineralized 374 larvae was strongly reduced at low pH conditions, while the number of partially and less 375 mineralized larvae was significantly higher (Fig. 9B; Table 4B, p < 0.001), p.0

, At pH 7.6, larval distribution within the three categories was close to

, Figure 1. Trochophore larvae grown in control condition (A) and at pH 7.6 (B), vol.1, p.3

, 534 (unshelled larval phenotype). s: shell, pt: prototrochal ciliary band, at: apical tuft

, Dotted black arrows indicate the length and width measurements

, Morphological variables measured in abalone veliger larvae grown in various 538 pH conditions; the group in brackets refers to morphological groups described in section 539 2.3.2. A: 30 hpf at pH 8.0 (group 1), B: 30 hpf at pH 7

, I: 542 96 hpf at pH 7.6 (group 4). Arrows indicate attributes: eyes (e), foot (f), mantle (m), p.540

, shell (s); arrowheads indicate shell abnormalities (sa) or deformed shell (ds)

, Viability of abalone larvae exposed to different pH conditions at 30 hpf (light 547 grey), 48 hpf (grey) and 96 hpf (dark grey). Errors bars represent standard deviations

, Asterisks denote significant difference between control and low-pH condition (unpaired 549 Student's t-test

, Grey levels represent 553 the morphological groups, according to the developmental stage. A (trochophore 554 larvae): black: normal shelled larvae; dark grey : shelled larvae with abnormalities or 555 delayed development

, larvae with abnormalities or delayed development

B. , C. , and D. Larvae, normal shelled larvae; dark grey larvae with shell malformation(s)/ normal body; light 558 grey : shelled larvae with body abnormalities or delayed development; white: larvae 559 with both shell and body abnormalities

, Scanning electron microscopy (SEM) images of abalone larvae grown under 562 control (A, B) and low pH condition (C, D). Shell surfaces of 48 hpf veliger are shown

, A: lateral view of a 48h-old veliger under control pH (8.0); the protoconch is well 564 developed and covers almost completely the larval body; B: detail of the shell surface 565 boxed in 5A showing a uniform granular texture covered by a very thin organic coating

, 566 C: lateral view of a 48h-old veliger exposed to low pH

, Figure 6. Correlation between length and width for each pH treatment

, Effect of decreased pH on abalone larval growth, expressed as square roots 576 of (length x width) at 20 hpf (A), 30 hpf (B), 48 hpf (C), and 96 hpf (D)

, Shell mineralization of larval abalone determined by polarized light 589 microscopy for each development stage (A: 30hpf, B: 48hpf, C: 96hpf). Grey levels 590 represent the three categories of mineralization: black bars for fully mineralized 591 (birefringence > 90%)

, Differences in 593 larval distribution across pH treatments were tested using a homogeneity ? 2 test by 594 treating birefringence as a categorical factor

, Dissolved Inorganic Carbon (DIC; µmol.kg SW), HCO 3-, CO 3 2-, 601 aragonite saturation state (?ar) and calcite saturation state (?calc) by using the 602 CO 2 SYS software. pH T is the average value logged throughout the 5 days of experiment 603 (every 15 mn) in the two tanks (n = 96/tank). Temperature and salinity were measured 604 daily

, Results of the Spearman's correlation performed to evaluate the nullity of 609 correlation between larval length and width for each pH, vol.2

A. , Results of the repeated measures ANOVA on larval growth index (square root of 614 length * width)

B. , Multiple comparison Tukey tests testing the effects of pH (fixed 615 crossed factor) according to time

, Summary of statistics used to test the differences in shell birefringence

A. , Homogeneity ? 2 test used to test the effect of pH on shell birefringence, for each 620 development stage. B. Pair-wise Wilcoxon rank sum test showing differences between 621 pH groups; Bonferronni adjusted p-values

, Cross-polarized microscopy image of abalone larvae (48 624 hpf) showing the three regions of interest (ROI) selected for the analysis of 625 birefringence intensity. B. Quantification of grey-scale levels

. Roi, The values were averaged to provide a global mean grey level for each larval shell 627 (n = 40 larvae per pH condition), p.628

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