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Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis

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Abstract

Aquatic organisms experience environmental hypoxia as a result of eutrophication and naturally occurring tidal cycles. Mytilus galloprovincialis, being an anoxic/hypoxic-tolerant bivalve, provides an excellent model to investigate the molecular mechanisms regulating oxygen sensing. Across the animal kingdom, inadequacy in oxygen supply is signalled predominantly by hypoxia-inducible factors (HIF) and Hif-prolyl hydroxylases (PHD). In this study, hif-α 5′-end and partial phd mRNA sequences from M. galloprovincialis were obtained. Phylogenetic and molecular characterization of both HIF-α and PHD putative proteins showed shared key features with the respective orthologues from animals strongly suggesting their crucial involvement in the highly conserved oxygen sensing pathway. Both transcripts displayed a tissue-specific distribution with prominent expression in gills. Quantitative gene expression analysis of hif-α and phd mRNAs from gills of M. galloprovincialis demonstrated that both these key sensors are transcriptionally modulated by oxygen availability during the short-time air exposure and subsequent re-oxygenation treatments proving that they are critical players of oxygen-sensing mechanisms in mussels. Remarkably, hif-α gene expression showed a prompt and transient response suggesting the precocious implication of this transcription factor in the early phase of the adaptive response to hypoxia in Mytilus. HIF-α and PHD proteins were modulated in a time-dependent manner with trends comparable to mRNA expression patterns, thus suggesting a central role of their transcriptional regulation in the hypoxia tolerance strategies in marine bivalves. These results provide molecular information about the effects of oxygen deficiency and identify hypoxia-responsive biomarker genes in mussels applicable in ecotoxicological studies of natural marine areas.

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Acknowledgments

This research was supported by a National Interest Research Project, PRIN 2010–2011 (prot. 2010ARBLT7_001/008).

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  1. Department of Biological and Environmental Sciences, University of Messina, 98166, Messina, Italy

    Alessia Giannetto, Maria Maisano, Tiziana Cappello, Sabrina Oliva, Vincenzo Parrino, Antonino Natalotto, Giuseppe De Marco, Chiara Barberi, Orazio Romeo, Angela Mauceri & Salvatore Fasulo

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  1. Alessia Giannetto
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Supp. 1

Hits from BLAST search against nucleotide collection (BLASTN) using the 308 bp hif-α cDNA (GenBank accession number JN595864) (a), the 761 bp hif-α cDNA (GenBank accession number KP185351) (b) and phd cDNA (GenBank accession number KP185352) (c) as queries. Score, query cover, E value, identity and accession numbers are showed (GIF 280 kb)

High Resolution Image (TIFF 16669 kb)

Supp. 2

Minimum Evolution (ME) phylogeny analysis of the HIF-α proteins based on the Dayhoff distance. The phylogenetic tree was constructed by MEGA.4 using the ME method. The bootstrap values from 1000 replicates are given at each branch node and only values higher than 50 % are shown. Abbreviations and sequence accession numbers are shown in Table 2 (GIF 20 kb)

High Resolution Image (TIFF 10504 kb)

Supp. 3

Minimum Evolution (ME) phylogeny analysis of the PHD proteins based on the Dayhoff distance. The phylogenetic tree was constructed by MEGA.4 using the ME method. The bootstrap values from 1000 replicates are given at each branch node and only values higher than 50 % are shown. Abbreviations and sequence accession numbers are shown in Table 2 (GIF 21 kb)

High Resolution Image (TIFF 10503 kb)

Supp. 4

Expression level of HIF-α and PHD proteins analyzed with the image analysis Alliance LD2 87WL software (UVITEC Ltd, Cambridge, UK). Data are presented as Optical density (mean ± SD, n = 6). All densitometry values were normalized to the endogenous Actin protein. Different letters indicate significant differences between time points in the treatment group, different numbers refer to significant differences between time points in the control group. Stars represent significant differences between treatment and control group at the same time point (P < 0.05) (GIF 28 kb)

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Giannetto, A., Maisano, M., Cappello, T. et al. Hypoxia-Inducible Factor α and Hif-prolyl Hydroxylase Characterization and Gene Expression in Short-Time Air-Exposed Mytilus galloprovincialis . Mar Biotechnol 17, 768–781 (2015). https://doi.org/10.1007/s10126-015-9655-7

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