Basic HTML Version
International Journal of Marine Science 2014, Vol.4, No.50, 1-22
http://ijms.biopublisher.ca
11
developed. A greater knowledge of the sub-cellular
basis of coral phenotypic plasticity, as well as their
acclimatization to altered environments, will allow for
the establishment of hypotheses as to their response to
the increases in temperature and
p
CO
2
that are
predicted to characterize the oceans in the coming
decades as a result of GCC (Ogawa et al., 2013).
3 Materials and Methods
3.1 Experiments
3.1.1 ETE
The details of both the elevated (i.e., ETE; Mayfield et
al., 2011) and variable temperature (i.e., VTE;
Mayfield et al., 2012a) experiments are described in
the respective manuscripts and summarized in Table 1.
Briefly, specimens were collected only from the UWS,
Houbihu, in the former. This coral reef (Figure 1)
is characterized by dramatic temperature fluctuations
as a consequence of spring-tide upwelling events
(Mayfield et al., 2012a); indeed, changes of up to 9
℃
can occur over the course of a single day in the
summer months. Triplicate tanks containing three
S.
hystrix
colonies collected from the UWS were
incubated at either the control (27
℃
) or elevated
(30
℃
) temperature for 48 h, and ~50-mg branches (~2
mm radius x ~7 mm length = ~100 mm
2
surface area)
were taken from each of the 18 colonies after 0, 6, 12,
24, and 48 h (
n
=90 samples). An effort was made to
avoid sampling from the same section of the colony
multiple times in order to ensure that sampling stress
did not bias results. Data from the time 0 samples are
not presented herein.
3.1.2 VTE
In the VTE, coral nubbins fragmented from colonies
originally from the UWS (Houbihu) and the NUWS
(Houwan) were exposed to either a stable temperature
treatment (26
℃
,
n
=3 tanks for corals of each site of
origin) or one that fluctuated from 23 to 29
℃
over a
5-h period (
n
=3 tanks for corals of each site of origin).
Houwan, which is a small bay (Figure 1) located
approximately 15 km north of Houbihu on the western
side of the Hengchun Peninsula, does not experience
upwelling events, and temperature rarely fluctuates by
more than 1
℃
on any given day of the year (Mayfield
et al., 2012a). In this study, two nubbins from each of
the 12 tanks were sampled at the beginning of the
experiment, as well as after seven days of treatment
exposure; however, only the latter 24 samples were
analyzed for molecular response variables.
3.2 RNA, DNA, and protein extractions
RNA, DNA, and protein were extracted in sequential
fashion from each of the 90 and 24 samples of the
ETE and VTE, respectively. Briefly, RNA was reverse
transcribed to complementary DNA (cDNA) in both
studies, and the cDNAs were used as template in
real-time PCRs for target gene analysis. As the reverse
transcription input RNA amounts and, with one
exception, real-time PCR primer concentrations were
identical for both studies, gene expression results
could be directly compared for all but one gene
(discussed in Table 4). The DNAs were used for
normalization of gene expression data (
sensu
Mayfield
et al., 2009),
Symbiodinium
genotyping (
sensu
Correa
et al., 2009), and host genotyping (
sensu
Underwood
et al., 2006; VTE samples only); all of these
procedures are described in detail below. The proteins
were quantified as described previously (Mayfield et
al., 2011) and used to calculate a protein/DNA ratio,
which serves as a proxy for total protein expression.
3.2.1 ETE: extractions
Prior to cDNA synthesis (200 ng RNA/reaction),
RNAs from the three pseudo-replicated samples from
each of the six tanks were mixed, re-extracted with
the GeneMark® Plant RNA Miniprep purification kit
(Hopegen Biotechnology) according to the
manufacturer’s recommendations (including DNase
treatment), and the 24 samples (
n
=3 for each treatment
at each sampling time) from times 6, 12, 24, and 48 h
were re-quantified and assessed for quality as in
Mayfield et al. (2012b). In a similar manner, the
triplicate DNAs from each tank and time were pooled
and re-purified with the AxyPrep™ PCR cleanup kit
(Axygen; manufacturer’s recommendations) prior to
their use in real-time PCRs (20 ng/reaction), and the
24 DNAs from corals sampled after 6, 12, 24, and 48
h (
n
=3 for each treatment-sampling time) were
analyzed.
3.2.2 VTE: extractions
RNA and DNA were re-extracted from coral tissues
that had been frozen in 500 µl TRI-Reagent® (Life
Technologies) at -80
℃
as described in Mayfield et al.
(2012a), and the duplicate, pseudo-replicated RNAs
and DNAs within each tank were left un-pooled prior
to cDNA synthesis (described below) and real-time
PCRs, respectively.