Physiological responses of the scleractinian coral Pocillopora damicornis to bacterial stress from vibrio coralliilyticus

INTRODUCTION

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The Journal of Experimental Biology 214, 1533-1545 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jeb.053165

RESEARCH ARTICLE

Physiological responses of the scleractinian coral Pocillopora damicornis to

bacterial stress from Vibrio coralliilyticus

Jeremie Vidal-Dupiol

1,

_{*, Ophélie Ladrière}

2,

_{*, Anne-Leila Meistertzheim}

1

_{, Laurent Fouré}

3

_{, Mehdi Adjeroud}

4

and Guillaume Mitta

1,†

1_{UMR 5244, CNRS UPVD EPHE, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France,} 2_{Unité d’écologie marine, Laboratoire d’écologie animale et écotoxicologie, Université de Liège, 15 Allée du 6 août, Bat. B6C, 4000}

Liege, Belgium,3_{Aquarium du Cap d’Agde, 11 rue des 2 frères, 34300 Cap d’Agde, France and} 4_{Institut de Recherche pour le} Développement, Unité 227 CoRéUs2 “Biocomplexité des écosystèmes coralliens de l’Indo-Pacifique”, bp A5, 98848 Nouméa

Cedex, Nouvelle-Calédonie *These authors contributed equally to this work

†_{Author for correspondence (mitta@univ-perp.fr)}

Accepted 19 January 2011

SUMMARY

As the effects of climate change have become increasingly visible over the past three decades, coral reefs have suffered from a number of natural and anthropogenic disturbances that have caused a critical decline in coral populations. Among these disturbances are coral diseases, which have appeared with increasing frequency and severity, often in correlation with increases in water temperature. Although the crucial role played by Vibrio species in coral disease has been widely documented, the scientific community does not yet fully understand the infection process of Vibrio or its impact on coral physiology and immunology. Here, we investigated the physiological and transcriptomic responses of a major reef-building coral, Pocillopora damicornis, when exposed to a specific pathogen (Vibrio coralliilyticus) under virulent (increasing water temperature) and non-virulent (constant low temperature) conditions. The infection process was examined by electron microscopy and quantitative reverse-transcription PCR, and coral health was monitored by visual observations and measurements of zooxanthellar density. The results obtained suggest that coral tissue invasion occurs upon increasing water temperature only. Transcriptomic variations were investigated using a suppression–subtractive–hybridization approach, and the expression levels of six candidate immune-related genes were examined during bacterial exposure. These genes correspond to three lectin-like molecules putatively involved in the recognition of pathogens, two metal-binding proteins putatively involved in antibacterial response and one cystein protease inhibitor. The transcription patterns of these selected genes provide new insights into the responses of coral colonies to virulent versusnon-virulent bacteria.

Supplementary material available online at http://jeb.biologists.org/cgi/content/full/214/9/1533/DC1 Key words: Pocillopora damicornis, Vibrio coralliilyticus, biomarker, coral disease, global change, vibriosis.

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Table 1. Combinations of forward and reverse primers used in real-time PCR expression analysis

Gene name Forward primer (5"r 3") Reverse primer (5"r 3")

Concanavalin CATCTCCGTCCCAAAC CAACTGCATAATGAACCCA

Cystatin B ATGGCCTTCCTTCTTTG AGGTGTATGTGGGTGAC

Ferritin AGAATATGCGGGGAGG CCTGGACCAACACGAG

Major basic nuclear protein GGTACAGCAAACTGCG TTGGAAACGTCCGACC

PdC-lectin ATTGGCAGAACGGAAG GGGAGGAGACCTGGTA

P-selectin CAATGTTATCATTACCGACCC GTCTGTTCCAGTCCAGG

Selenium binding protein TACGCCTTCCGGTAAC GTTCTACCCTGACCTGG

60S ribosomal protein L40A CGACTGAGAGGAGGAGC CTCATTTGGACATTCCCGT

60S ribosomal protein L22 TGATGTGTCCATTGATCGTC CATAAGTAGTTTGTGCAGAGG 60S acidic ribosomal phosphoprotein P0 GCTACTGTTGGGTAGCC CTCTCCATTCTCGTATGGT Vibrio coralliilyticus 16S rRNA TTAAGTAGACCGCCTGGG GATGTCAAGAGTAGGTAAGGTTC Pocillopora damicornis 28S rRNA CGGGTGAGAATCCTGT CTGAAGGCCATCCTCAA

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RESULTS

Coral health and infection status

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Fig.
1. Visual monitoring. Photos of Pocillopora damicornis nubbins taken at various time points during the experiment from treatments C (25^C without bacterial balneation), Cb (25^C with bacterial balneation), T (temperature increasing from 25 to 32.5^C without bacterial balneation) and Tb (temperature increasing from 25 to 32.5^C with bacterial balneation).

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Fig.
2. Enlargement of photos of P. damicornis nubbins subjected to (A) thermal bleaching (T treatment) or (B) bacterial lysis (Tb treatment). (A)
Nubbins 3 days after the end of the T treatment (day 24). Water temperature was decreased to 25^C on day 21, which led the polyps to reopen. A bleached open polyp is shown (surrounded by a circle). Corallites (e.g. skeleton structure containing a polyp) are indicated by bold arrows. (B)
Nubbins at day 20 after the Tb experiment, when coral tissues were quasi-totally lysed. Empty corallites are indicated by arrows. Asterisks mark a portion of bare skeleton; sharps indicates tissue rests undergoing lysis.

Fig.
3. Electron microscopic monitoring. (A)
Thin section of P. damicornis sampled from the Tb treatment on day 9. (B)
Thin section of P. damicornis sampled from the Tb treatment on day 12. (C)
Enlargement of B. (D)
Enlargement of C. B, bacterial aggregate; b, bacteria; arrows, host cell membrane; cross, tissue disorganization; star, area of strong tissue disorganization and lysis; open arrow, double membrane (bacterial cell wall and cytoplasmic membrane). Scale bars: (A) 1
m, (B) 500
nm, (C) 250
nm, (D) 125
nm.

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Infection duration (days)

9 12 15 18 0 3 6 1 10 100 –10–5 0 3 6 9 12 15 18 5

*

*

*

*

Fig.
4. qRT-PCR quantitation of Vibrio coralliilyticus in P. damicornis tissues. The relative expression of V. coralliilyticus 16S rRNA was normalized with respect to that of P. damicornis 28S rRNA. Open and filled histograms correspond to the non-virulent and non-virulent experiments, respectively.

Table 2. General characteristics of the BI, BR, TBI and TBR libraries

BI BR TBI TBR

Sequenced clone 384 384 384 384

Analysed cDNA 279 276 275 229

Mean EST size (bp) 335.9 332.1 367.3 302.7

Contigs 63 59 38 23

EST in contigs 156 127 91 63

Singletons 123 149 184 166

Redundancy* (%) 55.9 46.0 33.1 27.5

BI, bacteria-induced; BR, bacteria-repressed; EST, expressed sequence tag; TBI, thermo-bacteria-induced; TBR, thermo-bacteria-repressed.

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2% 2% TBI 78% 20% 2% 88% 3% 9% TBR

Bi

Bii

78% 18% 83% 14% Apoptosis Cellular homeostasis Cellular metabolism Chromatin remodeling Cytoskeletal organization Immune responses Intracellular protein transport Photosynthetic processes Protein metabolism Stress responses System development Unknown gene Unknown function Signal transduction RNA metabolism 78% 20% 2% 88% 3% 9% 78% 20% 7% 7% 4% 18% 2% 45% 2%_4% 2% 5% 78% 20% 88% 9% 88% 9% 5% 16% 5% 5% 21% 16% 11% 5% 5% 11% 78% 18% 83% 14% 78% 18% 78% 4% 18% 3% 11% 17% 3% 34% 14% 6% 3% 3% 3% 3% 83% 14% 83% 3% 14% 4% 25% 7% 4% 21% 10% 7% 4% 4% 4% 10% BI BR

Ai

Aii

Fig.
5. Functional classification of the sequences obtained from the bacteria-induced (BI), bacteria-repressed (BR), thermo-bacteria-induced (TBI) and thermo-bacteria-repressed (TBR) libraries. Expressed sequence tags presenting similarities with genes of known function were clustered into 13 categories according to their putative biological functions. (A)
Genes putatively upregulated (Ai) or downregulated (Aii) in P. damicornis exposed to V. coralliilyticus at a constant low temperature. (B)
Genes putatively upregulated (Bi) or downregulated (Bii) in coral exposed to V. coralliilyticus under increasing seawater temperature (which triggers virulence).