Award: EF-1416846

The goals of this study were to document the combined impact of elevated temperature, nutritional stress, and ocean acidification (OA) on the development and metamorphosis of the ubiquitous marine gastropod Crepidula fornicata. We have studied the impact of OA on 1) larval survival; 2) relative rates of larval tissue growth and calcareous shell growth; 3) ability of competent larvae to respond to cues for metamorphosis; 4) latent effects of larval exposure to OA on the survival and growth of juveniles after metamorphosis; 5) impact of OA on duration of the pre-competent larval swimming period, during which time larvae are not yet capable of metamorphosing; 6) influence of elevated OA on gene expression patterns in larvae; and 7) influence of OA exposure in the larval stage on gene expression patterns in juveniles. Most larvae and juveniles survived even the most acidic conditions tested, at a pH of 7.5. Although shell growth rates were generally not affected at pH 7.6, both larvae and juveniles grew more slowly when pH level was reduced even further. Moreover, rearing the larvae at pH 7.5 reduced subsequent juvenile growth rates even when the juveniles were reared under control conditions after metamorphosing (pH 8.0); thus, we documented a subtle but potentially important "carry-over effect" resulting from larval experience at the low pH. Rearing larvae at pH 7.6 also reduced subsequent juvenile growth at the control pH, even when larval growth rates were not significantly affected. Larvae reared under reduced pH generally took significantly longer to become competent to metamorphose, even under high food conditions, something that would alter dispersal patterns in the field and increase vulnerability to planktonic predators. Remarkably, rearing larvae at the low pH of 7.5 did not affect their ability to perceive and metamorphose in response to chemical cues released by adults. When larvae were reared at lower pH levels, shell weights were relatively high and tissue weights were relatively low. It appears that with the increasing acidity, more energy was dedicated to building up the shell mass. However, the shells of individuals reared at the lower pH levels were often relatively frail and easily cracked, despite their greater weight. Larvae reared at reduced pH for 4 days showed substantial shifts in patterns of gene expression, predominantly involving the downregulation of genes related to growth and metabolism. Relative to larvae reared under control conditions (pH 8.0), C. fornicata larvae reared at pH 7.6 upregulated 38 genes and downregulated 257 genes, while larvae reared at pH 7.5 differentially expressed only 55 genes (19 were upregulated, and only 36 were downregulated). Notably, most differentially expressed genes at both pH levels (pH 7.5, 65.5%; pH 7.6, 87.1%) were underrepresented relative to those seen in larvae reared under control conditions (pH 8.0). Larvae reared at pH 7.6 mounted a much stronger overall transcriptomic response when compared to those that had been reared at the even more acidic pH of 7.5. We are currently analyzing data for larvae that were exposed to reduced pH for shorter periods of time, to determine how long it takes to see these shifts in gene expression patterns, and which shifts are the first to appear. We are also currently analyzing gene expression data for juveniles reared under the different pH conditions. A particularly unexpected finding was seen when larvae were reared in seawater with a pH of 7.5. Most of those larvae grew slowly and had poor-quality, misshapen shells, but about 10% of the larvae grew at normal rates with healthy shells. Looking at patterns of gene expression, we found that 3204 transcripts were significantly differentially expressed between the two larval phenotypes; 2251 of those transcripts were expressed at higher levels in the unhealthy phenotype larvae while 953 genes were expressed at higher levels in the healthy phenotype larvae. Based on which genes were being upregulated and which were being downregulated, it appears that the poor-quality larvae were experiencing more oxidative stress than the healthy phenotype. Unhealthy larvae may also have been expending more energy attempting to mitigate stressor impact, as shown by increased hexokinase-2 expression. These data indicate that there are clear genetic differences determining tolerance to reduced pH among larvae of this species, suggesting that C. fornicata may be able to evolve a greater tolerance to low pH conditions over time. During the course of this project, I trained two graduate students and three undergraduates. I also created a 4-week section of our Experiments in Ecology course at Tufts, in which undergraduate students conducted experiments with our experimental marine animal, Crepidula fornicata. Last Modified: 08/01/2018 Submitted by: Jan A Pechenik