Basic HTML Version
International Journal of Marine Science 2014, Vol.4, No.50, 1-22
http://ijms.biopublisher.ca
13
3.3 Target gene selection
In the
Symbiodinium
compartment, three cellular
processes previously suggested to be thermo-sensitive
(Mayfield et al., 2013b, d) were targeted;
photosynthesis, metabolism, and the stress response.
Regarding the former, three photosynthesis-targeted
genes;
rbcL
,
psI
(subunit III), and
pgpase
were
targeted with real-time PCR (Table 4), as
Symbiodinium
have repeatedly been shown to be
photoinhibited when exposed to foreign temperature
regimes (Warner et al., 1996; 1999). Such
photoinhibition has been found to manifest at the
mRNA level (Crawley et al., 2010). Furthermore,
protein expression of RBCL, which is the rate-limiting
enzyme of the Calvin Cycle, was previously found to
decrease in
Symbiodinium
within coral larvae (Putnam
et al., 2013), as well as diatom symbionts within
foraminiferers (Doo et al., 2012), exposed to elevated
temperatures.
One metabolism-targeted gene,
nrt2
, was additionally
assayed in samples of both studies (Table 4). Briefly,
expression of this gene, whose respective protein may
be involved in the translocation of nitrate within the
holobiont, was previously hypothesized to be
thermo-sensitive (Mayfield et al., 2013a) given that
the nutrient dialogue between
Symbiodinium
and the
host coral could be impaired during periods of
elevated temperature (Mayfield and Gates, 2007).
Finally, expression of two stress-targeted genes,
hsp70
and
apx1
(Table 4), was measured in the
Symbiodinium
compartment. The latter is involved in
the detoxification of reactive oxygen species (ROS),
which have been documented to be produced in
Symbiodinium
within thermally-challenged corals
(Lesser 1996; 1997; 2006). The expression of all six
Symbiodinium
target genes was hypothesized to be
similar between temperature treatments and over time
in the ETE, yet vary between temperature treatments
in the VTE.
Eight genes across three cellular processes- the
cytoskeleton (
n
=4), osmoregulation (
n
=3), and the
stress response (
n
=1)- were targeted in the coral host.
Regarding the former two processes, cell volume
control and the cytoskeleton have been hypothesized
to be significantly compromised by changes in the
abiotic environment of a coral (Mayfield and Gates,
2007). The reason underlying this hypothesis is
because the host coral must theoretically spend a
considerable amount of energy to osmoregulate since
Symbiodinium
occupy the majority of the volume of
its gastrodermal cells (Chen et al., 2012). As
Symbiodinium
are photoinhibited at stress-inducing
conditions (Jones et al., 1998), the consequent
decrease in their translocation of compatible solutes,
such as glycerol, into the host cytoplasm could cause a
drop in osmotic pressure and a consequent collapse of
the cytoskeleton. In fact, a temporary turnover of the
cytoskeleton may occur at night in the gastroderm of
reef-building corals (Mayfield et al., 2010), possibly
due to this photosynthetic hysteresis (Levy et al.,
2004). Specifically, expression of
actb
, the gene
encoding the actin-binding protein tropomyosin,
trp1
,
and
tuba
was found to decrease at night in
S. hystrix
,
and their expression has been previously shown to
also be responsive to temperature changes in other
coral species (DeSalvo et al., 2008). Herein, the
expression of these cytoskeleton-targeted genes was
hypothesized to be similar across temperature
treatments of the ETE, yet vary between the stable and
variable temperature treatments of the VTE.
Expression of a fourth cytoskeleton gene,
ezrin
, whose
respective protein is involved in anchoring the
cytoskeleton to the plasma membrane (
Rasmussen
et
al., 2008), and an osmoregulation-targeted gene,
cplap2
, which can catabolize lipids in order to
generate the osmolyte glycerol upon decreases in
osmotic pressure (Ollivier et al., 2006), was also
measured; as with the target genes listed above, they
were hypothesized to be expressed at similar levels
over time and between treatments in the ETE, yet
display temperature treatment differences in the VTE.
The final two host coral osmoregulation genes,
trcc
and
oatp
, which encode proteins involved in salt and
anion balance, respectively, were also predicted to be
stably expressed in the former study and demonstrate
temperature treatment differences in the latter. Finally,
it was also hypothesized that corals from Houbihu, the
UWS, would express higher levels of each of the 14
candidate genes relative to con-specifics from
Houwan, the NUWS, for reasons discussed in the
Introduction.
3.4 ETE: target gene expression
Twelve genes were targeted in the ETE (Table 4);