Physiological and Proteomic Responses of Pacific Abalone (Haliotis discus hannai) under Fluctuating Temperature Stress

Abstract

Novel aspect Quantitative proteomic study of Pacific abalone in response to fluctuating temperature stress.

Introduction Understanding acclimatory adjustment of marine ectotherms in response to temperature stress is a prime interest in managing aquaculture systems. Pacific abalone (Haliotis discus hannai) is an ectothermic gastropod and is one of the major seafood resources in East Asia. The recent climate change in the South Sea of Korea has led to a higher average temperature and larger daily tidal temperature fluctuations, resulting in suppressed growth rate and increased mortality of the abalone. To understand how daily temperature fluctuations contribute to the physiological change of the abalone, a quantitative proteomic study was conducted using isobaric tags for relative and absolute quantification (iTRAQ).

Experimental Eight Pacific abalones were exposed to a semidiurnal temperature change from 20-26°C and one abalone was sampled at each time point of 0, 12, 24, 48, 72, 96, 120, and 144 h (T0-T7). Proteins were extracted from the foot muscle of an abalone sampled at each time point. An equal amount of protein from each sampling time point was digested and labeled using iTRAQ. The labeled peptides were analyzed in technical triplicates using a NanoLC-Q-Exactive Orbitrap mass spectrometer. Proteins were searched against the ORF-based protein database of H. discus hannai. From the reporter ion intensity at time point k (I(Tk)), the protein fold change (I(Tk)/I(T0)) was calculated in each replicate. By applying t-test (p < 0.05) and average fold change cutoff of 1.2, significantly changed proteins were assigned. The gene ontology (GO), metabolic pathway, and hierarchical clustering analyses were conducted upon significantly changed proteins.

Preliminary data From the technical triplicate analysis of abalone foot muscle proteins, 217 proteins were commonly identified and 160 proteins were significantly annotated by BLAST sequence similarity search. Among these, 40 proteins were found to be significantly changed in one sampling time point or more. Twenty proteins were found to be up-regulated, while the rest twenty were down-regulated. Hierarchical clustering analysis of protein fold change over different time points revealed that the largest quantitative change was made at T7 (144 h). From the GO analysis, the down-regulated proteins were highly associated with the catalytic activity, whereas the up-regulated proteins were relevant to the structural molecular activity. The KEGG metabolic pathway analysis reported two proteins responsible for pyruvate catabolism and another two for amino acid catabolism. Among these, glutamate dehydrogenase (GDH), which is responsible for amino acid deamination, was found to be gradually decreasing over exposure time. Moreover, GDH showed a significant negative time-series correlation with four up-regulated muscle microfibril constituents (Spearman’s rho < −0.7). This may imply that the long-term fluctuating temperature stress can suppress the amino acid catabolism, resulting in a decreased protein degradation rate and increased muscle microfibril proteins.