OAT and ODC expression in salinity challenged mussels
Description
Mytilid mussels (Mytilus spp.) are dominant members of coastal communities in North America and distributions of closely related species are often linked to differential tolerance to environmental stressors, such as salinity. As osmoconformers, mussels initiate cellular and molecular level changes during periods of salinity variation and previous studies of transcriptomic responses to hypoosmotic exposure in blue mussels have suggested differential utilization of the amino acid ornithine. Ornithine catabolism is used to generate glutamate or proline through the activity of ornithine aminotransferase (OAT), or to create putrescine and other polyamines through activity of ornithine decarboxylase (ODC). This study examined species-specific variation in the expression of genes involved in ornithine metabolic pathways for the euryhaline mussel M. trossulus compared to the congeners M. edulis and M. galloprovincialis, which are less tolerant to salinity variation. We found a consistent, small decrease in the expression of ODC during hypoosmotic exposure in all three species, but strong, species-specific increases in OAT expression. During hyperosmotic stress, the patterns of expression of these genes reversed. These patterns suggest that proline or glutamate synthesis is important during low salinity exposure, while polyamine synthesis may be more important during hyperosmotic exposure, which is an indication of differential osmotic stress. These responses were most pronounced in M. galloprovincialis, which showed nearly 18-fold increase in OAT expression during low salinity and a 2-fold increase in ODC expression during high salinity exposure.
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Juvenile mussels (Mytilus edulis, M. trossulus, and M. galloprovincialis) were used in a series of studies to evaluate changes in gene expression in response to hypo- and hyperosmotic exposure. Animals were collected subtidally: M. edulis (Walpole, Maine), Mytilus galloprovincialis (Catalina Sea Ranch, CA), and M. trossulus (Newport, OR) and then acclimated to control conditions (12 – 13.5C, 30 – 32 ppt) in a recirculating system at the University of Maine, Orono and fed a 3% daily ration of Shellfish Diet 1800 (Reed Mariculture, Inc.) of estimated dry tissue weight. Mussels were exposed to low salinity (20 ppt) or control (30 ppt) at 13.5°C for 4, 24, and 48 h in 1 l beakers following the experimental design described in May et al. (2017) and then the gill tissues was dissected and flash frozen. In another set of experiments, mussels were exposed to control (30 ppt) or high salinity (40 ppt) at 13.5°C for 4 or 24 hours. Gill tissue was sampled and flash frozen. Total RNA was purified from 50 mg (wet weight) using PureLink® RNA Mini Kit (Invitrogen) following the manufacturer’s protocol. We constructed double-stranded cDNA strands from the RNA extracts using the iScript™ cDNA Synthesis Kit (BioRad). Each reaction included 50 ng of total RNA and followed the manufacturer’s protocol.We used RT-qPCR to monitor the expression of two genes involved in ornithine metabolism, ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC). qPCR assays were run in 15 ul reactions in 96-well PCR plates; each well contained 7.5 ul iTaq™ Universal SYBR Green Supermix (BioRad), 300 nM of the forward and reverse primers, 2 ug of cDNA, and nuclease-free water. We ran the assays for two normalizing genes (40S and EF1a) and both of our target genes (OAT and ODC) using 3 technical replicates for each sample. The qPCR reactions were run on a CFX96 Touch™ Real-Time PCR Detection System (BioRad). The cycling protocol involves of 3 m at 95°C, 40 cycles of 10 s at 95°C, 20 s at 60°C, and 30 s at 72°C, an additional 60 s at 95°C, followed by a melt-curve analysis. Our melt-curve analysis was performed by running 30 s cycles beginning at 60°C and increasing by 1°C until reaching 95°C. Each plate included the qPCR reactions for 10 cDNA templates (i.e., the control and treatment samples from one time point; n = 5 each), a no-template control for each gene, and a normalizing control consisting of pooled cDNA amplified with a-tubulin to ensure that there was limited variability in Ct values for a common sample among plates across experiments. Following qPCR amplification, we set the threshold for every plate to 250 RFU (relative fluorescence units) in the CFX Manager software. Expression of OAT and ODC was normalized to 40S ribosomal protein (40S) and elongation factor 1a (EF1a) and analyzed using the common base method (Ganger et al. 2017), which uses efficiency-weighted Ct values for each sample as a modification of the DCt method (Bustin et al. 2009).