to dark-induced coral bleaching in the reef coralStylophora pistillata
Alice Rouan1, Mélanie Pousse1, Eric
Tambutté2, Nadir Djerbi1, William
Zozaya1, Laura Capasso2,3, Didier
Zoccola2, Sylvie Tambutté2*, Eric
Gilson1,4*
1 Université Côte d’Azur-CNRS-Inserm, IRCAN, Nice,
France.
2 Department of Marine Biology, Centre Scientifique de
Monaco, Monte Carlo, Principality of Monaco.
3 Sorbonne Université, Collège Doctoral, F-75005
Paris, France
4 Department of Medical Genetics, CHU, Nice, France.
* Corresponding authors : Eric.GILSON@univ-cotedazur.fr,
stambutte@centrescientifique.mc
Abstract
Telomere DNA length is a complex trait controlled both by multiple loci
and environmental factors. Even though the use of telomere DNA length
measurement, as a method of assessing stress accumulation and predicting
how this will influence survival, is currently being studied in numerous
human cohort studies, the importance of telomere length for stress
response in ecological studies remains at its infancy. Here, we
investigated the telomere changes occurring in the symbiotic coralStylophora pistillata that has experienced a continuous darkness
over six months. This stress condition led to the loss of its symbionts,
as what is also observed when bleaching occurs in the field at a
large-scale due to climate changes and anthropogenic activities,
threatening the worldwide reef ecosystem. We found that the continuous
darkness condition was associated with telomere DNA length shortening
and a downregulation of the expression of the telomere-associated
protein Pot2. These results pave the way for future studies on the role
of telomere in coral stress response and the importance of telomere
dysregulation in endangered coral species.
Introduction
Scleractinian corals are metazoans that successfully built complex
ecosystems, the coral reefs, which are among the most important
biodiversity hotspots of the planet. However, all over the world, coral
reefs are under threat due to global climate change and anthropogenic
activities (Hughes et al. 2018b). These stressors can cause imbalances
in the community of endosymbiotic partners hosted by corals such as
viruses, bacteria, fungi and most notably, members of the algal
dinoflagellate family Symbiodiniaceae. The breakdown of this
intricate symbiosis leads to the loss of the intracellular symbionts
and/or their pigments, a process that is called bleaching (Downs et al.
2013; Sully et al. 2019; Downs et al. 2009; Danovaro et al. 2008;
Kushmaro et al. 1996; Rosenberg et al. 2009). Corals can either recover
from bleaching by acquiring new symbionts or die in case of prolonged or
repeated stresses. Of note, bleaching can be triggered in the laboratory
under different experimental set-up (increases in temperature or light
intensity, continuous darkness, hyposalinity, chemical pollutants and
pathogen infection) but if food is provided, the corals can survive for
long time periods even though different physiological processes can be
affected (body mass loss, stop of calcification, growth arrest and polyp
mortality) (Lyndby et al. 2020; Gardner et al. 2017).
In order to mitigate coral loss, there is currently an urgent need to
understand the mechanisms involved in the response of corals to stress
and among them, the pathways maintaining genome stability are essential.
An accurate response to DNA damages caused by environmental stressors
(radiation, heat, pollutants, nutrient fluctuations…) as well as
normal metabolic processes (such as replication, respiration and, in the
case of symbiotic corals, photosynthetic activity leading to daily
oxidative switches), requires efficient DNA damage checkpoints, DNA
repair and survival pathways to maintain genome stability and health
(López-Otín and Kroemer, 2021). Therefore, the efficiency of DNA repair
mechanisms is one of the main regulators of stress resistance and
longevity in many organisms (Ma et al.,2016; Tian et al., 2019).
However, aside from antioxidant defenses (Furla et al., 2005) and UV DNA
repair (Reef et al., 2009) and recent report on increased levels of
phosphorylated H2AX (a marker of DNA damage response)
(Rodriguez‐Casariego et al., 2018) and Rad51 (a DNA double strand break
repair protein) (Maor-Landaw and Levy, 2016) during heat stress, our
knowledge on the role of genome maintenance pathways in environmental
tolerance in corals remains limited.
Among the genome stability mechanisms, the chromosome ends, or
telomeres, play a key role in stress response. Telomeres are composed of
DNA repeats, (TTAGGG)n in most metazoans (Traut et al. 2007), bound by a
protective protein complex, the shelterin, which protects the chromosome
against unwanted DNA damage response (de Lange 2005). In humans, the
shelterin complex is made of six proteins (TRF1, TRF2, TIN2, TPP1, POT1
and RAP1) (Giraud-Panis et al. 2013). TRF1 and TRF2 (Telomeric-repeat
binding factor 1 and 2) directly bind the duplex telomeric DNA repeats,
POT1 (Protection of Telomere 1) binds the single stranded telomeric DNA
forming the 3’ overhangs of chromosomal DNA, while TIN2 and TPP1 bridge
the double and single stranded parts of telomeric DNA. In addition, RAP1
is associated with TRF2. The telomerase holoenzyme, comprising the
reverse transcriptase subunit TERT and its RNA template, is able to
maintain telomere length during DNA replication (Gilson and Geli 2007).
Functional telomeres protect chromosomes against instability and
senescence and are also involved in a wide range of important processes
such as inflammation, immunity, energetic metabolism and mitochondrial
integrity, stemness as well as cellular differentiation (Ye et al.,
2014). Changes in telomere structure occur during development, stress
response and aging. In general, exposure to unfavorable environments
leads to accelerated telomere shortening (Young et al., 2017). In birds,
telomere DNA length can predict key life-history traits like growth,
reproduction and lifespan (Monaghan, 2014). Thus, it was proposed that
change in telomere DNA length is an adaptive strategy based on
life-history regulation and environmental adaptation (Young 2018) and
that telomere DNA length is a useful biomarker for past stress (Bateson,
2016).
Here we investigated the impact on telomere structure of continuous
darkness stress (six months) in the model coral Stylophora
pistillata (Esper, 1797). Notably, we observed that this stress
condition leads to telomere DNA length shortening as well as a
downregulation of the expression of a coral ortholog of a shelterin
subunit. Since this stress is associated with the bleaching of the coral
colony (Supplementary Figure 2), we discuss the possibility that the
telomere response to continuous darkness is a consequence of the
bleaching state.
Material and
methods
Sampling
Colonies of the tropical coral S. pistillata were exposed to
long-term darkness in an experimental aquarium setup for a long term
exposure of six months. Briefly, coral fragments were kept in aquaria
supplied with Mediterranean seawater (exchange rate of 70% per hour) at
a salinity of 38 g liter−1, temperature of 25°C,
pHT 7.94 ± 0.02 and for the control condition at an
irradiance of 230 μmol photons
m−2s−1 on a 12:12 photoperiod. Both
dark and control conditions corals were fed daily with frozen rotifers
and twice a week with live artemia nauplii. The first experiment D1 was
conducted from July 2018 to January 2019 were a part of the S1 colony
was separated and put in dark condition, 6 branches of the bleached
colony were sampled and 5 of the control one, the second experiment D2
was conducted from June 2019 to December 2019 were a different S.
pistillata colony was divided in 2 part, each one kept in either of the
two experimental condition, 4 branches from each colony part were
sampled.
DNA
extraction
DNA was extracted on the first 3-4 cm of a colony branch. All steps
until cell lysis were done on ice. The fresh coral tissue was removed
from the skeleton using the “air_brush” technique in 50mL of
extraction buffer (NaCl 550mM, EDTA 0,2M
(Tambutté et al., 2007)) in
a Whirl-Pak bag (Nasco) under the hood, then centrifuged for 10 minutes
at 5 000g (Beckman Coulter, Avanti J-E centrifuge, rotor JA-18) at 4°C.
The tissue pellet was kept on ice to perform high molecular weight DNA
extraction following the Midi Kit Cell and Blood culture (QIAGEN).
Briefly, cell lysis was carried out for 2 hours at 50°C after adding 19
mL of G2 buffer (800 mM guanidine HCl ; 30 mM Tris·Cl, pH 8.0 ; 30 mM
EDTA, pH 8.0 ; 5% Tween-20 ; 0,5% Triton X-100, QIAGEN) with 190 mL
RNase A (ThermoFisher, ref : 12091039 , 20mg/mL) and 400 mL of protease
(QIAGEN, 1 UA/mL) to the tissue pellets. Supernatant was poured in the
anionic column after a 5 min centrifuge at 5 000g and 4°C, columns were
washed following the kit instructions, DNA was eluted with 5mL of warm
QF buffer (1.25 M NaCl ; 50 mM Tris·Cl, pH 8.5; 15% isopropanol,
QIAGEN). Precipitation was performed adding 3.5mL of 2-propanol (Sigma),
after mixing by inverting, tubes were stored overnight at 4°C. Two
washing steps were performed adding 1mL of 70% ethanol (sigma) to DNA
pellets after 30 min centrifuge at 15 000g and 4°C before adding 200 mL
of TE buffer (Tris-Cl pH8 10mM, EDTA 1mM) for long-term storage. Pellets
were resuspended at 50°C for 1 hour. To test DNA quality and quantity
both Nanodrop (ThermoFisher) and agarose gel were used, respectively 1
mL of DNA and 1,5 mL of loading buffer (New England Biolabs, 6X) in a
final volume of 10 mL were loaded in a 1.2% agarose gel run for 30
minutes at 100V in Tris-Borate-EDTA (TBE) 1X. Agarose gel was incubated
for 10 minutes in 4 mg/mL ethidium bromide and DNA was revealed under UV
(GelDoc Transilluminator, BioRad), the average of three measurements was
used. A diluted aliquot was prepared for enzymatic digestion before
storing the stock DNA at -20°C.
Telomere Restriction Fragment
assay:
Dilution and DNA
digestion
To digest non telomeric DNA sequences, we added 60U HinfI (R0155M, New
England Biolabs) and 60U RsaI (R4374, Promega) to of 2,5 mg of DNA
diluted in mqH2O and “CutSmart Buffer” 1X (New England Biolabs) in a
final volume of 40mL, left overnight at 37°C.
Southern
Blot
We measured telomere length using the Telomeric Restriction Fragment
assay (TRF) using the Southern blotting procedure
(Herbert et al., 2003)
digested DNA was loaded on a 1,2% agarose gel for a 3h30 run in TBE
0.5X at 9 volts/cm in a CHEF-DR-II (BioRad). The run timing makes it
suitable for 2 runs a day. After a 5 min wash in mQ H2O agarose gel was
stained in Ethidium bromide bath (4 mg/mL) under agitation for 30min and
imaged the gel (Typhoon, GE Healthcare, Fluorescence, Method=Alexa Fluor
555, Laser=532nm, PMT=ch.1,700V, Resolution = 50 µm). Denaturation was
done in (1 M NaCl ; 0.5 M NaOH) for two incubations of 20 min followed
by a neutralization step with two 20 min incubation in 1M ammonium
acetate. The DNA was transferred to a Hybond N+membrane (GE Healthcare) overnight in SSC (20X). DNA was cross-linked on
the membrane using the Ultraviolet Crosslinker (UVP) at 1200 x 100µ
Joules. Membrane was gently washed in mQH2O before
storage at room temperature for (12h up to 48h) before 1h
pre-hybridization in Denhardt’s buffer (10X) at 40°C. We prepared stocks
of Denhardt’s 100X buffer (2% Ficoll 400, 2% Bovine Serum Albumine,
2% Polyvinylpyrrolidone) filtered (0.2µm) and stored at -20°C.
Probe
synthesis
We prepared probe template 40uM stocks annealing two primers per probe.
Probe targeting (TTAGGG) sequence will be called T2 and the one
targeting (TTTAGGG) sequence will be referred to as T3. We mixed 25 of
Primer F and 25 of Primer R (Primer T2AG3_F :
GGGTTAGGGTTAGGGTTAGGGAAA and T2AG3_R : TTTCCCTAACCCTAA, PrimerT3AG3_F : GGGTTTAGGGTTTAGGGTTTAGGGAAA and T3AG3_R :
TTTCCCTAAACCCTAAA) with STE 5X buffer (Tris 50mM pH8, NaCl 250mM, EDTA
5mM) and heat it to 95°C to therefore gradually let it cool down to room
temperature before being stored at -20°C. We mixed on ice 2.5 of dATP
(10 mM) and 2.5 of dTTP (10mM, New England Biolabs, R0192) with 3of NEB
2 buffer (10X), 15of mQH2O, 5 of gamma-dCTP32 and 1 Klenow
(3’–>5’ exo-) (M0212S, NEB) incubated in a thermocycler
(ThermoFisher Scientific) for a (30min 25°C, 5min 98°C, 5min 25°C)
program. Then we purified the probes following the ProbeQuant G50-micro
columns (GE, Healthcare) instructions. Probes were denatured at 95°C for
5 min and used or stored at -20°C.
Hybridization
Membranes were pr hybridized for 1h at 40°C in hybridization Denhardt’s
buffer (10X). We added 5µL of denatured T3 probes to 15mL of
hybridization buffer for an overnight hybridization at 40°C. Washing
steps included a 2x20min wash in 2X SSC, a 30min step in (2X SSC, SDS
0,2%) and a final H2O washing step. Membrane was carefully wrapped in
plastic sarran film and applied to a phosphor-screen (brand) for 60h. We
imaged the phospho-screen on the Typhoon (GE Healthcare,
Phosphorimaging, Method=[Phosphor], Laser=635nm, PMT=ch.1,1000V,
Resolution = 50 µm), the screen was flashed (brand of device). Membrane
was stripped with NaOH 0,4M for 30min at 42°C, then for 30min in (SDS
1%, SSC 0.1X, Tris-HCl pH7.4 1M) to remove the T3 probe. To control the
stripping, membrane we exposed the membrane to a flashed phosphor-screen
for 12h. The screen was imaged as described above, in the absence of
signal the membrane was pre hybridized for 1h and hybridized overnight
at 45°C with the T2 probe, otherwise the membrane was stripped again
until no signal was imaged after 12h of exposure. Washing steps and
imaging were carried out as described above with a 24h exposure of the
phosphor-screen.
Image
analysis
Telomere Restriction Fragment (TRF) images signal was extracted using
ImageQuant (GE Healthcare) 1D gel analysis mode and manual lane
creation. Ladders signal intensity were extracted from Ethidium bromide
gel images, setting the lane upper limit at the gel wells bottom. Pixel
position of ladder peaks were manually reported. Host and symbiont
telomere signals were extracted from the phospho screen, exposed to
radioactive labelled membrane, images setting sample lane upper limit at
the top of the membrane. Efficiency of stripping step between the two
probes hybridization was assessed imaging phospho screens overnight
exposed to stripped membranes.
Telomere Length
measurements
Single lane intensity files were fused in RStudio. Ladder peaks exact
position was extracted in R, searching for the maximum intensity in a
10-pixel perimeter around the manually reported peaks. Fitted linear
model coefficients (a,b) of log2 ladder size (kb) against peak pixel
position were calculated for high molecular weight (48.5-15kb) and low
molecular weight (10-1kb) using the lm function of “stats” R
package. Coefficients were used to transform samples intensity scale
from pixel to base pair (bp) () in Excel using the high molecular weight
coefficient for the upper part and the low molecular coefficient for the
lower one. Depending on their position on the membrane samples were
divided in left, right and middle to be scaled to the closest ladder.
Intensity signals were imported in R using the read_excel from
“readxl” package, background correction was automatically computed to
level the signal by subtracting to each position the minimal intensity.
Intensity was normalized by the size to avoid probe number hybridization
bias, intensity above 2 kb were discarded to avoid genomic noise,
interstitial telomeric sequence noise and normalization bias
(<1kb). Ponderate telomere mean was calculated as in
(Li and de Lange 2003),
median, quartiles, intensity signal, smoothed signal and telomere
measurements were plotted for each sample using the ggplotfunction from “ggplot2” package and measurements were saved ascsv files.
Transcriptome
RNA
extraction
Coral pieces of approximately 2 cm were sampled just below the coral
piece dedicated to DNA extraction, at least 4 cm from the apex of the
branch. Samples were flash frozen in liquid nitrogen in WhirlPack bags,
and then crushed in small pieces with a press and fragments were put in
600uL of lysis buffer RLT in PowerBeads Glass 0.1mm (Qiagen), samples
were lysed in Bead Beating instrument for 4 minutes with 30 cycles/s.
Samples were spin at max speed for 3 minutes and the 600uL of
supernatant was transferred to a fresh tube. Add 600uL (1V) of cold EtOH
70% to the column (Qiagen, RNeasy Mini Kit), gently mixed by pipetting,
immediately apply 600uL on the column and centrifuge 15s at 8 000g (10
000 rpm), discard the flow-through, repeat this step. Add 350 uL of RWI
buffer to the column and centrifuge 15s at 8 000g (10 000 rpm), discard
the flow-through. Take a DNAse, Rnase free, (Qiagen, 79254) stored at
-20°C, add 70ul of buffer RDD 4°C and apply to the column. Leave the
column at RT for 15min before adding 350 uL of RWI buffer and centrifuge
15s at 8 000g (10 000 rpm), discard the flow-through and add 500uL of
RPE buffer, repeat the centrifugation, add again 500uL of RPE buffer and
make a 2min at 8 000g (10 000 rpm) centrifuge before discarding the
flow-through and putting the column in a clean 1.5ml tube. Add 30ul of
RNase free water to the column and centrifuge for 1min at 8 000g (10 000
rpm), keep the tube on ice for further experiment and store at -80°C.
RNA quantity was measured on a microplate spectrophotometer Epoch
(Bioteck) and the quality was assessed by running a 1% agarose gel
labelled with Ethidium Bromide and imaged under UV-exposure in a Fusion
Fx7.
RNA
sequencing
Paired-end sequencing (read length: 2x150bp) was performed by NovoGene
company using HiSeq sequencer, raw data are available with accession
number GSE171268
(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE171268).
Three biological replicates per condition were performed with roughly 45
million reads per sample. Reads were trimmed using fastp software (Chen
et al., 2018). Reads were mapped on the S.pistillatatranscriptome from NCBI (GCF_002571385.1) using Salmon software
(v0.11.4) (Patro et al., 2017). The differential genes expression
analysis was performed using DESeq2 R package (Love et al., 2014). A
cutoff of 0.05 was applied on adjusted p-values, obtained using
Benjamini and Hochberg correction.
Ontology and pathway
analysis
Gene Ontology (GO) analysis was performed using the “topGO” R package
(Alexa et al., 2006). For the cnidarian analysis, all GO terms
identified in S.pistillata were used as background. Ingenuity
Pathway Analysis (Krämer et al. 2014)
(QIAGEN,https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis)
was performed on human ortholog genes. Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway analysis was performed using “gage” and
“KEGGREST” R packages (Kanehisa, 2000). The human ortholog genes
associated with the S.pistillata log2FoldChange values were used
to conduct the analysis. Human orthologs were identified using the
protein sequences. Blastp (Altschul et al. 1990) on UniProtKB/Swiss-Prot
database restricted to Homo sapiens species was performed with a
1e-5 e-value threshold. For each protein, the best hit was selected,
based on e-value and bit-score.
Phylogeny
Ortholog sequences of S.pistillata shelterin proteins were
curated using PSI-BLAST (Altschul et al, 1997) on the non-redundant (Nr)
protein database from the NCBI. The search was restricted to Metazoa
group. The retrieved sequences were aligned using MAFFT v7.310 with
L-INS-I algorithm (Katoh and Standley, 2013). The multiple alignment was
trimmed with TrimAl v1.4 with the gt 0.6 option
(Capella-Gutierrez et al., 2009). Then the best substitution matrix was
assessed using ModelTest (Darriba et al., 2020). Finally, the trees were
built using raxmlGUI 2.0, a graphical interface for the implementation
of RAxML Next Generation and the transfer bootstrap expectation branch
support (Edler et al., 2020). Tree visualization was done with iToL v6.1
(Letunic and Bork, 2019). Multiple alignment for POT1 and POT2 were
wrapped using Alignment Annotator (Gille et al., 2014).
Quantitative Polymerase Chain
Reaction
(PCR)
Primers were designed using PrimerBlast (Ye et al., 2012) and sequences
are available in (Supplementary Table 3). Reverse transcription was
carried for 1ug of RNA samples using the Applied Biosystems reverse
transcription kit for 1h at 37°C followed by 5min of denaturation at
95°C. qPCRs were made on cDNAs obtained using ROCHE’s Fast universal
Sybr Greenmaster (ROX) on a StepOne plus thermocycler (Applied
Biosystems). Each sample was triplicated on the 96 wells PCR plate
containing the interest gene primers and the two control genes. In
addition to the samples from the RNAseq, samples from the same
experiment were added to the qPCR, two additional control samples (S1n2
and S1n4) as well as two treated samples (S1Bn2 and S1Bn6).
Statistics
Telomere length measurements were averaged from two independent
Telomeric Restriction Fragment assay, homoscedasticity and normal
distribution were tested on R using (fligner.test andshapiro.test functions) and significant differences was
calculated using the t_test from the “rstatix” packages with a
Bonferroni pvalue adjustement. Significant differences are reported as
stars ( (ns) non significant, (*) P<0.05, (**)
P<0.01, (***)
P<0.001.
Results