Will threatened staghorn coral be able to adapt to higher temperatures with climate change?

Methods

Summary

Fragments from 20 genetically distinct staghorn coral colonies were obtained from the Coral Restoration Foundation and transported to Keys Marine Laboratory. Coral fragments were maintained in a shaded, flow-through seawater system for 48 hours to acclimate to ambient conditions prior to the experiment. Collected fragments were divided between three control (ambient temperature, ~27 – 29^oC) and three elevated temperature (31 – 33^oC) tanks, with one fragment per genotype suspended in each tank. Coral fragments in the elevated temperature tanks were then exposed to a lethal thermal stress and mortality following lethal temperature exposure was recorded over the course of 26 days. Mortality was determined by a loss of photosynthetic efficiency as measured by pulse amplitude modulated (PAM) fluorometry. The phenotype of time to death of each fragment is being used along with genotypic data to estimate heritability, or amount of genetic variation present, in thermal tolerance in this species. After 12 hours of high temperature exposure, tissue samples were removed from all fragments and preserved for investigating differences in levels of known molecular biomarkers under sub-lethal stress. For the biomarker assays, the tissue samples were flash frozen and an airbrush with 50 mmol/L phosphate buffer and 0.05 mmol/L Dithiothreitol (DTT) (pH 7.72) was used to extract the coral tissue from the calcium carbonate skeleton into solution. Once the tissue was in solution, it was lyophilized (freeze dried). The lyophilized tissue will be resuspended in solution prior to biomarker assays.

The biomarkers that will be investigated include the heat shock protein hsp70, ferritin, ubiquitin, 4-Hydroxynonenal production, and catalase activity. Each of these biomarkers has been shown to change expression in response to oxidative stress in coral species (Barshis et al., 2010; Csaszar et al., 2009; Seneca et al., 2010; Ross et al., 2013). A combination of both protein assays and gene expression assays will be used whenever possible for comparison of transcript expression to actual protein concentration. For protein assays (Hsp70, ubiquitin, 4HNE, and catalase), total protein concentration will be measured using the PierceTM BCA Protein Assay Kit. Heat shock protein hsp70, ubiquitin, and 4HNE levels will first be detected in the samples through immunoblot assays following the methods of Barshis et al. (2010), and then quantified through ELISA. Catalase concentration within coral tissue will be measured using the Amplex Red Catalase Assay Kit, and total protein concentration will be used to calculate catalase concentration per total protein concentration (U/mg). For gene expression assays (hsp70, ferritin, ubiquitin, 4HNE, and catalase), primers will be developed with the help of Dr. Mauricio Rodriguez-Lanetty. The transcriptome of staghorn coral individuals in response to white band disease has already been developed by Libro et al (2009). Current datamining and bioinformatics techniques will be used to identify the sequence for these select genes in this species in order to develop both forward and reverse primers for qRT-PCR analysis. The developed primers will be ordered and used to quantify mRNA transcript expression within coral tissues following the methods of Csaszar et al (2009). 

Pre Analysis Plan

In order to determine how expression patterns of stress biomarkers correlate to survival under thermal stress, a Model II Linear Regression analysis using SPSS will be used to compare the mean expression levels of each biomarker to the mean time of death for each colony. If there is a clear correlation between cellular biomarker expression and lifespan following thermal stress exposure, then we should see a pattern of increased expression of these markers among genotypes that exhibit longer lifespans. There are two possible expectations: either there will be higher expression of these biomarkers among tolerant genotypes within the heated treatments if the coral response is more reactionary, or there will be higher expression within the control treatments of tolerant genotypes if these biomarkers are frontloaded (higher expression) during ambient conditions. If there is no correlation found between expression of hsp70, ferritin, Ubiquitin, catalase, or 4HNE expression and lifespan under thermally stressed conditions, then these biomarkers would not be considered good indicators of thermal tolerance or susceptibility. There may be other biomarkers that are differentially expressed between thermal tolerant and susceptible colonies that have not been tested here that may act as better indicators for coral managers.