Award: EF-1416895

Intellectual Merit: We conducted experiments testing the separate and combined effects of ocean acidification (OA) and hypoxia on behavior, physiology, and gene expression of multiple species of rockfish (genus Sebastes), focusing on the vulnerability juvenile fish to environmental stressors associated with climate change in kelp forests in the California Current Ecosystem. In Year 1, we examined the independent effects of OA by comparing the effects of low pH (elevated pCO2) on behavior, physiology, and patterns of gene expression in juvenile rockfish, integrating responses from the transcriptome to the whole organism. Experiments were conducted simultaneously on two closely related species, to compare high-CO2 tolerance. Copper rockfish exhibited changes in behavioral lateralization (i.e., test of brain function), reduced critical swimming speed, depressed aerobic scope (i.e., measure of the capacity for aerobic activity), changes in metabolic enzyme activity, and increases in the expression of transcription factors and regulatory genes at high pCO2 exposure. Blue rockfish, 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 pCO2, indicating acclimatization potential. In Year 2, we examined the independent and combined effects of OA and hypoxia on juvenile rockfish of 3 species (blue, copper, and gopher rockfish). Findings indicate that for many of the behavioral and physiological responses, fish from the combined pH and DO treatment exhibited a similar response to fish from the low DO treatment alone, such that the magnitude of the behavioral or physiological response is much stronger to reduced oxygen levels than reduced pH levels, but in some instances the interactive effects of low pH and low DO produced the greatest impairment. We also conducted a short-term timecourse experiment in which we examined blue and gopher rockfish gene expression at 12h, 24h and two weeks. Year 3 experiments tested the effects of static vs. fluctuating pH and DO levels, simulating upwelling-relaxation cycles. Experiments included 3 static treatments with combined pH and DO levels reflecting upwelling conditions and 2 fluctuating treatments simulating upwelling and relaxation events over a 7-day cycle. The results indicate that swimming abilities and physiological performance are progressively impaired as pH and DO levels decrease. Fish from the fluctuating upwelling treatment exhibited the same physiological response as fish in the most extreme static treatment. Interestingly, fish from the fluctuating relaxation treatment showed full recovery of physiological performance after 7 days in ambient conditions and these physiological responses were not different from fish held chronically in ambient conditions. The results indicate that short-term exposure to future ocean chemistry with increased upwelling can impair physiological performance while recovery occurs rapidly upon return to ambient or relaxation-type conditions. However, the growth and body condition data indicated that growth and condition were depressed in fluctuating treatments, indicating cumulative negative impacts of future upwelling. Studies of associated changes in gene expression and enzymatic activity were completed in combination with physiological studies in Years 2-5. We evaluated the effects of short and long-term exposure of hypoxia, high pCO2, and combined stressors on gene expression changes in Sebastes mystinus, S. caurinus, S. carnatus and S. melanops. A targeted gene expression panel of 192 genes was also designed from multiple experiments and tested as a potential tool for testing rockfish stress levels in field collected specimens. De novo transcriptome assemblies were constructed for five rockfish species (Sebastes mystinus, S. caurinus, S. carnatus, S. melanops, and S. auriculatus) using RNAseq data. We identified conserved and unique responses to stressors across species and time-scales, finding that exposure duration and type (e.g., static vs. fluctuating) resulted in divergent expression responses. The majority of genes responsive to hypoxia, high pCO2, and combined hypoxia/high pCO2 were unique among treatments, timepoints, and species. Overall, our data suggest that rockfish congeners have evolved specific-specific gene expression responses to cope with hypoxia and high pCO2 over acute and chronic timescales. Broader Impacts: Graduate student training has been provided for 7 MS students conducting thesis research. Over 40 undergraduates from 4 institutions participated on the research, conducting independent projects and presenting results at scientific meetings. Many of the graduate and undergraduate students are women, first generation students, or from underrepresented groups. Tissues samples from our research were used five times in teaching a Capstone Marine Environmental Physiology course at CSU Monterey Bay, with approximately 15 undergraduates per class. The students were provided hands-on training in RNA sequencing and bioinformatics analysis of transcriptomics data. We have engaged with over 300 8th grade students in the Monterey Unified School District with lesson plans and curricula about the effects of climate change and ocean acidification in Monterey Bay. We also worked with the California Ocean Science Trust to develop outreach materials on the impacts of ocean acidification on rockfish and other commercially important or indicator species in the California Current Ecosystem. Last Modified: 12/16/2019 Submitted by: Cheryl Logan