Figure 1.
Morphometric measurements of spicule elements and total lengths at prism and pluteus stages.
Scale bar shown in lower right corner of A and B = 100 µm. A) A prism stage larva (15 days) visualized under cross-polarized illumination. The ALA rod length (ALA) was measured from the origin of the triradiate center to the tip of the spicule rod. B and C) A pluteus larva (30 days) visualized under brightfield (B) and cross-polarized illumination (C). Plutei oriented with their oral side facing up were imaged with the postoral arms in the same plane of focus as the aboral tip of the larva. Total length (TL) was measured from the tip of the postoral arm rod to the aboral tip of the body. Postoral arm rod length (POA) was measured from the tip of the postoral arm rod to the origin of the triradiate center, and body rod spicule length (B) was measured from the origin of the triradiate center to the aboral tip of the body rod.
Figure 2.
Summary of seawater chemistry.
Daily measurements of salinity (A) and Total Alkalinity (B) in the three reservoir buckets for the three treatment levels 410, 510 and 730 µatm. Total Alkalinity was not significantly different between treatments over the duration of the experiment (n = 30, ANOVA, p>0.1). Averaged daily measurements of temperature (C) and pHTS (D) in 5 replicate culture vessels per treatment (± SD). Calculated average pCO2 (E) of 5 replicate culture vessels per treatment (± SD).
Table 1.
Developmental schedule (days post-fertilization) of S. neumayeri reared in water from McMurdo Sound – for this study and for data reported by Bosch et al. [23].
Figure 3.
Developmental series of embryo images, including polarized microscopy.
Developmental stages and skeletal development of S. neumayeri under elevated pCO2. A–C) Representative embryos at 2 cell, 16 cell and morula stages reared under ambient (unmanipulated seawater) conditions in a separate culture. D–F) Eight day old early gastrula with vegetal plate indentation where CaCO3 spicule nuclei are first visible. Three spicule nuclei are visible under polarized light (D) from the ventral view of the embryo grown in the control treatment. Polarized (E) and brightfield (F) side views of a gastrula grown in 730 µatm pCO2 treatment. Two spicule nuclei among the mesenchyme cells are visible from this angle. G–I) Ten day old mid-gastrula embryos (also G-inset) from three treatments: control (G), 510 µatm (H) and 730 µatm (I). Triradiate spicules are similar in shape. J–L) Twelve day old late gastrula stage embryos in three treatments. Large well-developed spicules are visible from the side (J, L) and ventral view (K). Aberrant spicule development is readily distinguishable in some individuals in the 730 µatm treatment (L).
Figure 4.
Developmental series of larvae images, including polarized microscopy.
A–C) Fifteen day old prism larvae imaged under brightfield and polarized light. Aberrant skeletal and morphological development is found in the 510 µatm (B) and 730 µatm (C) treatments. D–F) Twenty day old 4-arm plutei in three treatments as imaged for morphometric measurements. G–I) Thirty day old plutei. Spicule nuclei for the developing posterodorsal arms are visible in the polarized light image of control treatment larvae (G-arrow). Smaller larvae with short arms are visible in the 510 µatm and 730 µatm treatments (H, I).
Figure 5.
Developmental progression and synchrony at specific times over the duration of the experiment.
Samples for each specified day (n>200, each treatment) were scored for developmental stage. A) Relative percentages of normally developed 2–16 cell embryos at 1 day post-fertilization. B) Relative percentages of morulas, and early blastulas (E Blas) at Day 2. Some normally developed embryos were missing a fertilization membrane (no FM-E Blas). C) Day 3 and 5 development. Unhatched (UH) blastulas were observed without cilia (UH-no cilia Blas) on day 3, while ciliated blastulas in various stages were observed at Day 5. Earlier-stage blastulas were unhatched but ciliated (UH+cilia Blas-inset left) or hatched (H Blas). More developed mesenchyme blastulas (Mes Blas) with a distinct vegetal plate were also unhatched and hatched (inset right). D) Gastrulation over days 8–15. Note the change in Y-axis scale. Early gastrula (E Gas) mid-gastrula (M Gas) late gastrula (L Gas) and prism were observed in highly synchronous development. E) Pluteus development at days 20 and 30. Prism and early pluteus (E Plu) were observed at day 20. By day 30 the 4-arms of the plutei are well-developed and some have developed epaulettes (+epau). F) Abnormal development across the experimental period. Percentage of unfertilized and abnormally developed embryos and larvae are shown for the three pCO2 treatments.
Figure 6.
Morphometrics of skeletal elements and larval size from late gastrula to 4-arm pluteus (from day 12–30).
Box plots show median, 25th and 75th percentile within the boundaries of the box, 10th and 90th percentiles in the error bars and three lowest and highest outlier values. See Fig. 1 for diagram of measurements. See Tables 1 and 2 for statistics on variances and differences between treatments. A) Anterolateral arm (ALA) rod length (left side) during late gastrulation to prism transition (n = 30). B) Total length in early (day 20) and advanced (day 30) 4-arm plutei. Also shown for day 30 plutei are the postoral arm (POA) rod lengths and body rod lengths (n = 30, except n = 29 for 510 µatm at day 30).
Table 2.
Permutational homogeneity of dispersion (PERMDlSP2), PERMANOVA and permutational pairwise statistics on morphometrics data.
Figure 7.
Allometric comparisons of body and arm lengths at day 30.
Allometry of individual day 30 plutei considering body rod length as an independent factor. A) The relationship between body rod length and postoral arm (POA) rod length for all assayed individuals in 3 pCO2 treatments (n = 30 for 410 and 730 µatm treatments, n = 29 for 510 µatm). POA rod length is correlated significantly to body rod length (r2 = 0.14, p<0.04) only in the control (410 µatm) treatment (solid line; regressions not shown for NS correlations). B) Relationship of body rod length to POA rod:body rod length ratio. There was a significant correlation at 510 µatm (solid line, r2 = 0.29, p<0.003) and at 730 µatm (dashed line, r2 = 0.32, p<0.002). NS correlation for control not shown.