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.