Telomere DNA length shortening in bleached samples
Southern blotting is considered the gold standard technique for telomere
DNA length measurement (Aubert, Hills and Lansdorp, 2012). To measure
the telomere DNA length of the coral host and its symbiont
simultaneously, we adapted the Southern blotting procedure to coral
tissue samples and performed sequential hybridizations, first with a
probe recognizing the symbiont telomere sequence (TTTAGGG)n and then
with a probe recognizing the animal host telomere sequence (TTAGGG)n.
Consistent with previous publications on other coral species (Sinclair
et al., 2007; Tsuta et al. 2014; Tsuta and Hidaka 2013; Zielke and
Bodnar, 2010), colonies of S. pistillata revealed terminal DNA
fragments hybridizing with a host telomere DNA probe of a mean varying
from 3.3 kb to 5.7 kb and with the symbionts telomere DNA probe of a
mean varying from 3kb to 6.2 kb (Figure 2A).
We induced stress by putting coral colonies in constant darkness
(Douglas, A.E., 2003), in two duplicated experiments referred to as D1
and D2 (Supplementary Figure 2). In both experiments S.
pistillata colonies were fully bleached after long-term treatment (six
months) (Supplementary Figure 2). The telomere analysis was performed on
branches of the same colony cut in two and maintained either in control
or dark conditions for six months: for D1 (respectively D2), six
(respectively four) branches from the bleached half colony and three
(respectively two) branches from the control half colony (Figure 2A)
were successfully measured twice in Telomere Restriction Fragment assay.
The host telomere DNA length was measured reporting the smear mean,
median, first quartile (Q1), third quartile (Q3) and interquartile
distance (IQ) (Figure 2B, Supplementary Table 1). In both experiments
bleached colonies were experiencing shorter telomeres with significantly
shorter telomere DNA length mean, median, IQ and Q3 in the host and an
absence of signal for the symbionts due to bleached state. However, Q1
measurements of the host telomere DNA length were not significantly
different from those of control samples. Thus, the stress triggered by a
six month dark incubation of S. pistillata shifted the host
telomere DNA length distribution towards less long telomeres (Q3)
without increasing the short telomeres (Q1) proportion.