Electronic supplemental material- appendix 2
Molecular analyses
Extractions. RNA, DNA, and protein were extracted in sequential fashion from ~50 mg
portions of the six coral nubbins sampled at each of six sampling times (n = 36 total samples).
Briefly, the methods developed by Mayfield et al. (2010) were used for homogenization and
precipitation of the samples, while the protocols described in Putnam et al. (in press) were used
to further purify the RNAs with the GeneMark® Plant Miniprep RNA purification kit (Hopegen
Biotechnology, Taipei, Taiwan). DNAs were extracted as in Mayfield et al. (2009), and
precipitated DNA pellets were dissolved in PCR-A buffer (Axygen Biosciences, Union City, CA,
USA) and purified with the AxyPrep™ PCR clean-up kit as recommended by the manufacturer.
For both the RNA and the DNA, the spin columns were incubated for several minutes in a 60°C
oven prior to the final elution in DEPC-treated water (30 μl) and manufacturer’s eluent (50 μl),
respectively, in order to completely remove residual ethanol.
RNA and DNA quantity and quality were assessed as in Mayfield et al. (2012b). Briefly,
a NanoDrop spectrophotometer (Infinigen, City of Industry, CA, USA) was used to estimate the
concentration of both the RNA and DNA, and native tris-borate-EDTA (TBE) agarose gels (1%)
were run at 100 V for 30 min, post-stained with ethidium bromide, and visualized under an
ultraviolet lamp to visualize the quality of these nucleic acids. The total RNA (μg) from every
sample was divided by the total DNA (μg) from that same sample to calculate the RNA/DNA
ratio, which serves as a proxy for total gene expression (Mayfield et al. 2012b).
Proteins were purified from the same samples from which RNA and DNA were isolated
as in Mayfield et al. (2011) and dissolved in sample buffer (Laemmli 1970). Protein quantity and
quality were assessed as described previously (Mayfield et al. 2011). Briefly, protein quantity
was calculated with the 2-D Quant™ kit (Amersham Biosciences, GE Healthcare, Piscataway,
NJ, USA) according to the manufacturer’s recommendations, and protein quality was assessed
by analysis on 4-12% stacking-separating SDS-PAGE gels that were run at 75 and 150 V
through the stacking and separating gel phases, respectively, and post-stained in SYPRO® Ruby
(Life Technologies, Grand Island, NY, USA). The protein/DNA ratio was calculated for each
sample in an analogous manner as for the RNA/DNA ratio in order to serve as a proxy for total
protein expression.
Gene expression. RNAs (200 ng) were converted to cDNA with 1x Solaris™ RNA spike
(Thermo-Scientific, Lafayette, CO, USA) and the High Capacity™ cDNA synthesis kit (Life
Technologies) as recommended by the respective manufacturers. cDNA was diluted 4-fold prior
to real-time PCR with assays previously developed for the Solaris spike (Putnam et al. in press),
Symbiodinium rbcL (Mayfield et al. 2012a), pgpase (primers published in Crawley et al. 2010,
protocol [mastermix composition and thermocycling conditions] published in Mayfield et al.
2012a), psI (Mayfield et al. 2012a), apx1 (Mayfield et al. 2012a), nrt2 (Mayfield et al. 2013),
“carbonic anhydrase” (primers published in Vidal-Dupiol et al. 2009, assay described below),
hsp70 (Mayfield et al. 2009), and host coral hsp70 (Putnam et al. in press). The gene proposed
by Vidal-Dupiol et al. (2009) to encode a carbonic anhydrase actually appears to represent a
nuclear transport protein (ntf2-like) upon BLAST analysis, though its expression was
nevertheless measured in all samples. For this gene, 500 nM each primer was used in each
reaction (mastermix described below), and a thermocycling protocol of 95°C for 10 min x 1
cycle followed by 40 cycles of 95°C for 15 s and 60°C for 60 s was employed prior to the
melting curve analysis (discussed below).
All reactions (20 μl) for each assay were conducted in triplicate with 1x (10 μl) EZTIME® SYBR® Green mastermix with ROX® passive reference dye (Yeastern Biotechnology,
Taipei, Taiwan) on an Applied Biosystems StepOnePlus™ real-time PCR machine, and serial
dilutions of a randomly chosen sample were run on each plate to estimate the efficiency of the
assays; all were found to be 95-105%. Melt curves, which were conducted after every run,
always demonstrated a single peak, demonstrating an absence of non-specific amplification
products.
Expression of all target genes was first normalized to recovery of the RNA spike to
control for reverse transcription efficiency differences (sensu Bower et al. 2007). Then,
expression of the Symbiodinium and host target genes was normalized to the real-time PCRderived Symbiodinium and host genome copy proportions (GCPs), respectively, as it was
possible that different ratios of nucleic acids may have been extracted between samples due to,
for instance, bleaching. In short, both traditional reverse transcription and biological composition
controls are required to generate meaningful and accurate macromolecular expression data from
endosymbiotic organisms such as reef-building corals (Mayfield et al. 2009).
Genotyping. The same DNAs used for calculation of the Symbiodinium and host GCPs
were used for clade-level genotyping of Symbiodiniumsensu Correa et al. (2009). Briefly, the
same real-time PCR mastermix, reaction volumes, and instrument were used as described above
with the primers and recommended concentrations of Correa et al. (2009). However, only 20 ng,
rather than 100 ng, of template DNA were used in a reaction. As the its2 marker from which the
assays were designed is intragenomically variable (Pochon et al. 2012), these assays can only be
used for presence/absence tests, not to calculate exact ratios of the four clades (A, B C, and D).
“Presence” of a particular Symbiodinium clade was defined a priori to occur when threshold
cycle (Ct) values less than 30 were observed.