Quantifying Environmental Multi‐Stress Response in Mussels via Respirometry and qPCR
Authors: Dilly Geoffrey , Senger Christiana , Algoso Krisha , Brazier Garrick 
Published: June 2021

Rocky intertidal organisms, such as the sessile West Coast mussel Mytilus californianus (M.c.), experience a dramatic set of acute and chronic environmental stressors. Mussels transition daily from submerged to emerged conditions, experiencing seasonal temperature changes in water, daily shifts in air temperature, and fluctuating durations of exposure based on tidal periods. M.c. must therefore tolerate both gradual and rapid shifts in environmental conditions. To better quantify differential individual outcomes of M.c. to simultaneous acute and chronic stressors, we employ both phenotypic and genotypic methods. We couple respirometric rates to differential gene expression on a suite of genes implicated in stress response: Heat Shock Protein 70 (HSP70; chaperone), Hypoxia‐Induced Factor 1α (HIF‐1α; hypoxia), Glutathione Peroxidase (GPX; oxidative stress), Thioredoxin Reductase (TRR; oxidative stress), and Caspase‐8 (CASP8; apoptosis), referenced to Elongation Factor 1α (EF‐1α; reference).Mussels were collected from Ventura, CA and acclimated for three weeks prior to study. Our full‐factorial experiment subjected M.c. to a range of conditions: water temperatures (14°C, 19°C, 24°C), air temperatures (7°C, 18°C, 35°C), and emersion times (4 hours, 8 hours) using 12 mussels per treatment. Two‐hour in vivo respirometry trials were completed using lab‐designed Chambers for Organismal Response to Environmental Stressors (CORES) and a NeoFox spectrophotometer (Ocean Optics, Dunedin, FL). Gills were excised and divided evenly for dry‐weight respirometry measurements and RNA extraction. Extracted RNA was converted to cDNA for quantitative PCR in technical triplicate using a CFX96 RT‐PCR System (BioRad, Hercules, CA). We quantify gene expression of HSP70, HIF‐1α, GPX, TRR, and CASP8 across all 18 experimental conditions and pair these results with corresponding respirometric rates.Mytilus californianus show a strong acute response when exposed to high air temperature (35°C) across all water conditions, with HSP70 expression elevated by 750x −2460x over 18°C air treatments. However, HSP70 is slightly downregulated (2.5–6.3x) as water temperature increases with highest expression found at 14°C. This may be due to the differential response to acute (air) versus chronic (water) stressors. When considering genes implicated in longer term organismal response, such as CASP8 apoptotic repair mechanisms, the response rate is different, however. We see slight rate changes (1–4x) between air temperature conditions, but a significant downregulation (11–59x) of CASP8 at 19°C water temperature when compared to 14°C and 24°C treatments. We also see a characteristic increase in respiration rates (2–3x) with increasing water temperature (14 to 24°C) which is broadly in line with the Q10 hypothesis. Notably, M.c. exhibit a wide range of individual respirometric responses to the most stressful condition set (24°C water, 35°C air, 8‐hour emersion). Variability also increases in larger deviations of HSP70 expression rates at higher temperatures, suggesting that individual variation may play a large role in determining outcomes under extreme multi‐stress conditions.Support or Funding InformationThis work is supported by CSU Channel Islands and the Santa Rosa Island Research Station.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.