Yu PC et al. (2013), Growth attenuation with developmental schedule ...

ECB-ART-42711

PLoS One 2013 Jan 01;81:e52448. doi: 10.1371/journal.pone.0052448.

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Growth attenuation with developmental schedule progression in embryos and early larvae of Sterechinus neumayeri raised under elevated CO2.

Yu PC , Sewell MA , Matson PG , Rivest EB , Kapsenberg L , Hofmann GE .

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The Southern Ocean, a region that will be an ocean acidification hotspot in the near future, is home to a uniquely adapted fauna that includes a diversity of lightly-calcified invertebrates. We exposed the larvae of the echinoid Sterechinus neumayeri to environmental levels of CO(2) in McMurdo Sound (control: 410 µatm, Ω = 1.35) and mildly elevated pCO(2) levels, both near the level of the aragonite saturation horizon (510 µatm pCO(2), Ω = 1.12), and to under-saturating conditions (730 µatm, Ω = 0.82). Early embryological development was normal under these conditions with the exception of the hatching process, which was slightly delayed. Appearance of the initial calcium carbonate (CaCO(3)) spicule nuclei among the primary mesenchyme cells of the gastrulae was synchronous between control and elevated pCO(2) treatments. However, by prism (7 days after the initial appearance of the spicule nucleus), elongating arm rod spicules were already significantly shorter in the highest CO(2) treatment. Unfed larvae in the 730 µatm pCO(2) treatment remained significantly smaller than unfed control larvae at days 15-30, and larvae in the 510 µatm treatment were significantly smaller at day 20. At day 30, the arm lengths were more differentiated between 730 µatm and control CO(2) treatments than were body lengths as components of total length. Arm length is the most plastic morphological aspect of the echinopluteus, and appears to exhibit the greatest response to high pCO(2)/low pH/low carbonate, even in the absence of food. Thus, while the effects of elevated pCO(2) representative of near future climate scenarios are proportionally minor on these early developmental stages, the longer term effects on these long-lived invertebrates is still unknown.

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Genes referenced: LOC115919910 LOC115925415

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	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).
	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).
	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.

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