Use of a Free Ocean CO₂ Enrichment (FOCE) System to Evaluate the Effects of Ocean Acidification on the Foraging Behavior of a Deep-Sea Urchin

The influence of ocean acidification in deep-sea ecosystems is poorly understood but is expected to be large because of the presumed low tolerance of deep-sea taxa to environmental change. We used a newly developed deep-sea free ocean CO₂ enrichment (dp-FOCE) system to evaluate the potential consequences of future ocean acidification on the feeding behavior of a deep-sea echinoid, the sea urchin, <i>Strongylocentrotus fragilis</i>. The dp-FOCE system simulated future ocean acidification inside an experimental enclosure where observations of feeding behavior were performed. We measured the average movement (speed) of urchins as well as the time required (foraging time) for <i>S. fragilis</i> to approach its preferred food (giant kelp) in the dp-FOCE chamber (−0.46 pH units) and a control chamber (ambient pH). Measurements were performed during each of 4 trials (days −2, 2, 24, 27 after CO₂ injection) during the month-long period when groups of urchins were continuously exposed to low pH or control conditions. Although urchin speed did not vary significantly in relation to pH or time exposed, foraging time was significantly longer for urchins in the low-pH treatment. This first deep-sea FOCE experiment demonstrated the utility of the FOCE system approach and suggests that the chemosensory behavior of a deep-sea urchin may be impaired by ocean acidification

Species-Specific Responses of Juvenile Rockfish to Elevated <i>p</i>CO₂: From Behavior to Genomics

<div><p>In the California Current ecosystem, global climate change is predicted to trigger large-scale changes in ocean chemistry within this century. Ocean acidification—which occurs when increased levels of atmospheric CO₂ dissolve into the ocean—is one of the biggest potential threats to marine life. In a coastal upwelling system, we compared the effects of chronic exposure to low pH (elevated <i>p</i>CO₂) at four treatment levels (i.e., <i>p</i>CO₂ = ambient [500], moderate [750], high [1900], and extreme [2800 μatm]) on behavior, physiology, and patterns of gene expression in white muscle tissue of juvenile rockfish (genus <i>Sebastes</i>), integrating responses from the transcriptome to the whole organism level. Experiments were conducted simultaneously on two closely related species that both inhabit kelp forests, yet differ in early life history traits, to compare high-CO₂ tolerance among species. Our findings indicate that these congeners express different sensitivities to elevated CO₂ levels. Copper rockfish (<i>S</i>. <i>caurinus</i>) exhibited changes in behavioral lateralization, reduced critical swimming speed, depressed aerobic scope, changes in metabolic enzyme activity, and increases in the expression of transcription factors and regulatory genes at high <i>p</i>CO₂ exposure. Blue rockfish (<i>S</i>. <i>mystinus</i>), in contrast, showed no significant changes in behavior, swimming physiology, or aerobic capacity, but did exhibit significant changes in the expression of muscle structural genes as a function of <i>p</i>CO₂, indicating acclimatization potential. The capacity of long-lived, late to mature, commercially important fish to acclimatize and adapt to changing ocean chemistry over the next 50–100 years is likely dependent on species-specific physiological tolerances.</p></div

Deletion profiles of <i>uvrD</i> in the PCR products of vesicomyid clam symbionts.

<p>Vertical red lines on Cpac_S and Ifos_S indicate PCR primers redesigned for the second PCR. Gene arrangement and the direction of <i>uvrD</i> and neighboring genes of <i>Ca</i>. Ruthia magnifica are shown at the top, including Rmag_0319, hypothetical protein gene; Rmag_0320, <i>uvrD</i>; Rmag_0321, hypothetical protein gene; Rmag_3022, Lysine 2, 3-aminomutase YodO family protein gene. Bidirectional arrows in columns indicate the consensus helicase motifs. Symbols and demarcations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.g001" target="_blank">Fig 1</a>.</p

Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams

<div><p>Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; <i>uvrA</i>, <i>uvrB</i>, <i>uvrC</i>, <i>uvrD</i>, <i>uvrD</i> paralog [<i>uvrD</i>p] and <i>mfd</i>) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While <i>uvrA</i>, <i>uvrD</i>p and <i>mfd</i> were conserved in all symbionts, <i>uvrB</i> and <i>uvrC</i> were degraded in all clade I symbionts but were apparently intact in clade II symbionts. <i>UvrD</i> was disrupted in two clade II symbionts. Among the intact genes in <i>Ca</i>. Vesicomyosocius okutanii (clade I), expressions of <i>uvrD</i> and <i>mfd</i> were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of <i>uvrA</i> and <i>uvrDp</i> were not. In contrast, all intact genes were expressed in the symbiont of <i>Calyptogena pacifica</i> (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from <i>Bathymodiolus septemdierum</i> were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and <i>mutY</i> in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.</p></div

Summary of the range of exposure duration, acclimation time, time per trial, recovery period, and sample size for copper and blue rockfish used to test behavioral and physiological responses to elevated <i>p</i>CO₂.

<p>Note: Individual fish were used successively in the different trials to enable tracking of performance measures. Data from fish that did not behave normally in a particular trial were excluded (e.g., refusal to swim in the U_crit test). In addition, 2 of 12 blue rockfish individuals that were sequenced had low quality reads and were subsequently excluded from the differential gene expression analysis.</p

Comparison of genetic distances from the symbiont of <i>Bathymodiolus septemdierum</i>, GC contents and repeat sequence densities for ten genes among clade I, II symbionts and <i>Ca</i>. <i>Ruthia magnifica</i> (Rma).

<p>A, Averaged genetic distances from the <i>B</i>. <i>septemdierum</i> symbiont were calculated for genes in clade I symbionts (symbionts of <i>A</i>. <i>kawamurai</i>, <i>C</i>. <i>laubieri</i>, <i>P</i>. <i>kilmeri</i>, <i>P</i>. <i>soyoae</i> and <i>P</i>. <i>okutanii</i>) and clade II symbionts (symbionts of <i>C</i>. <i>pacifica</i>, <i>C</i>. <i>fausta</i>, <i>C</i>. <i>nautilei</i>, <i>P</i>. <i>steansii</i>, <i>C</i>. <i>magnifica</i>, <i>I</i>. <i>fossajaponicum</i> and <i>A</i>. <i>phaseoliformis</i>). B. Average GC contents (%) were calculated from the intact gene ORFs or their corresponding degraded regions in clades I and II. C. Moving average of densities of 5 bp repeat in a window of 200 bp DNA fragments was calculated with 10 bp shift of the window in the examined genes of clade I and II symbionts. Black filled columns indicate clade I symbionts; open columns represent all clade II symbionts; gray columns indicate Rma. Standard deviations indicated by crossed lines at top of bars. * on bracket indicates statistically significant difference (p<0.05).</p

Changes in behavioral lateralization, critical swimming speed, and aerobic scope of juvenile copper and blue rockfish as a function of <i>p</i>CO₂ treatment exposure history.

<p>(A, B) Behavioral lateralization is measured using the relative lateralization index (negative values = right turn bias in a detour test). (C,D) Critical swimming speed (U_crit) is the maximum sustained speed in body lengths per second. (E,F) Aerobic scope represents the difference between maximum and resting metabolic rates (measured as oxygen consumption) and is a proxy for the capacity for aerobic activity. Bars are mean values (± SE). Letters over bars represent results of Tukey HSD post-hoc tests; significantly different means do not share letters in common. Note: Due to logistical constraints all behavioral and physiological trials occurred in control seawater (<i>p</i>CO₂ ~550 μatms).</p

Enzyme activity ratios for lactate dehydrogenase and citrate synthase (LDH:CS) in white muscle tissue of (A) copper and (B) blue rockfish following chronic exposure to extreme <i>p</i>CO₂ (~2800 μatms) or control (~550 μatms) treatments.

<p>Copper rockfish exhibited a significant increase in aerobic enzyme activity relative to anaerobic activity (i.e., lower LDH:CS ratio) from the control to extreme high <i>p</i>CO₂ treatments (two-way ANOVA; Species×Treatment: <i>F</i>_{<i>1</i>,<i>24</i>} = 4.46, <i>P</i> = 0.045), whereas blue rockfish did not differ in LDH:CS activity among treatments.</p

Gene expression profiles (A) and Gene Ontology (GO) functional categories (B) for copper and blue rockfish muscle tissue as a function of <i>p</i>CO₂ treatment.

<p>(A) Heatmaps display significant differential gene expression (DE) for copper (<i>n</i> = 147) and blue (<i>n</i> = 358) rockfish (FDR<0.001) among <i>p</i>CO₂ treatments; green = up-regulation, red = down-regulation. Each column represents an individual fish (<i>n</i> = 15 copper rockfish and <i>n</i> = 10 blue rockfish). Genes are ordered by similarity in gene expression profile and differ in both order and identity between the two species (only 14 DE genes were in common between the two species). Hierarchical (Euclidean) clustering was used to group similar gene expression profiles, labeled along the right side of each heatmap and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169670#pone.0169670.s002" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169670#pone.0169670.s003" target="_blank">S3</a> Tables. (B) GO categories show relative differences between copper and blue rockfish in the percentage of annotated genes that were differentially expressed, as classified by GO molecular function or biological process. Broken out pie wedges highlight GO categories that were more expressed in one species than the other. Copper rockfish show significant up-regulation of genes involved in transcription and biological regulation at high <i>p</i>CO₂ and down-regulation at low <i>p</i>CO₂. In contrast, blue rockfish differentially express muscle structural genes across <i>p</i>CO₂ treatments.</p

Deletion profiles of <i>uvrB</i> in the PCR products of vesicomyid clam symbionts.

<p>ORFs of Rma are shown at the top: Rmag_0425, aminotransferase gene; Rmag_0427, Radical SAM domain protein. Red bars in the columns indicate stop codons. Horizontal dashed lines indicate the lost portion of the fragmented gene. The additional vertical red line on the right side of the column of Cfau_S indicates the position of the redesigned PCR primer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171274#pone.0171274.s002" target="_blank">S2 Table</a>). Lengths for PCR products are shown on the right side of each column. ORFs of <i>uvrB</i> in clade I symbionts are highly fragmented. Bidirectional arrows in columns indicate the consensus helicase motifs. Color gradient of the bidirectional arrows indicate the identity to that of <i>E</i>. <i>coli</i>. Symbionts are abbreviated as follows: Akaw_S, <i>A</i>. <i>kawamurai</i> symbiont; Clau_S, <i>C</i>. <i>laubieri</i> symbiont; Pkil_S, <i>P</i>. <i>kilmeri</i> symbiont; Psoy_S, <i>P</i>. <i>soyoae</i> symbiont; Vok, <i>Ca</i>. Vesicomyosocius okutanii (<i>P</i>. <i>okutanii</i> symbiont); Cpac_S, <i>C</i>. <i>pacifica</i> symbiont; Cfau_S, <i>C</i>. <i>fausta</i> symbiont; Cnau_S, <i>C</i>. <i>nautilei</i> symbiont; Pste_S, <i>P</i>. <i>stearnsii</i> symbiont; Rma, <i>Ca</i>. Ruthia magnifica (<i>C</i>. <i>magnifica</i> symbiont); Ifos_S, <i>I</i>. <i>fossajaponicum</i> symbiont; Apha_S, <i>A</i>. <i>phaseoliformis</i> symbiont; Bsep_S, <i>Bathymodiolus septemdierum</i> symbiont.</p