Microbial community of black band disease on infection ... (Ofri Johan) MICROBIAL COMMUNITY OF BLACK BAND DISEASE ON INFECTION, HEALTHY, AND DEAD PART OF SCLERACTINIAN Montipora sp. COLONY AT SERIBU ISLANDS, INDONESIA Ofri Johan*)#, Dietriech G. Bengen**), Neviaty P. Zamani**), Suharsono***), David Smith****), Angela Mariana Lusiastuti*****), and Michael J. Sweet******) *) Research and Development Institute for Ornamental Fish Culture, Jakarta **) Department of Marine Science and Technology, Faculty of Fisheries and Marine Science, Bogor Agricultural University ***) Research Center for Oceanography, The Indonesian Institute of Science ****) School of Biology, Newcastle University, NE1 7RU, United Kingdom *****) Center for Aquaculture Research and Development ******) Biological Sciences Research Group, University of Derby, Kedleston Road, Derby, DE22 1GB, United Kingdom (Received 19 March 2014; Final revised 12 September 2014; Accepted 10 November 2014) ABSTRACT It is crucial to understand the microbial community associated with the host when attempting to discern the pathogen responsible for disease outbreaks in scleractinian corals. This study determines changes in the bacterial community associated with Montipora sp. in response to black band disease in Indonesian waters. Healthy, diseased, and dead Montipora sp. (n = 3 for each sample type per location) were collected from three different locations (Pari Island, Pramuka Island, and Peteloran Island). DGGE (Denaturing Gradient Gel Electrophoresis) was carried out to identify the bacterial community associated with each sample type and histological analysis was conducted to identify pathogens associated with specific tissues. Various Desulfovibrio species were found as novelty to be associated with infection samples, including Desulfovibrio desulfuricans, Desulfovibrio magneticus, and Desulfovibrio gigas, Bacillus benzoevorans, Bacillus farraginis in genus which previously associated with pathogenicity in corals. Various bacterial species associated with uninfected corals were lost in diseased and dead samples. Unlike healthy samples, coral tissues such as the epidermis, endodermis, zooxanthellae were not present on dead samples under histological observation. Liberated zooxanthellae and cyanobacteria were found in black band diseased Montipora sp. samples. KEYWORDS: bacteria, black band disease, Montipora sp., Seribu Islands INTRODUCTION providing a platform that can be utilised in other Vast research throughout the Caribbean regions of the world. In the mid 1990s, 18 types over the past few decades has provided great of coral disease were recorded, infecting at insight into the epidemiology of coral disease, least 150 species of scleractinian, gorgonian, # Corresponding author. Research and Development Institute for Ornamental Fish Culture Jl. Perikanan No. 13, Pancoran Mas, Depok 16436, Indonesia. Phone: +62 21 7520482 E-mail: ofrijohan@kkp.go.id 1 ofri johan hen.pmd 1 3/16/2015, 4:47 PM  Indonesian Aquaculture Journal Vol. 9 No. 2, 2014 and hydrozoans corals throughout the Carib- by algae. As BBD appears to be activated at bean and Indo-Pacific. Despite this, Koch’s pos- temperatures exceeding 28oC, infections are tulates (to determine the causal agent of a dis- found to increase during summer months and ease) have only been fulfilled for 5 of these 18 dramatically decrease in winter (Cooney et al., diseases (Sutherland et al., 2004). 2002), suggesting the causal agent may be a thermophilic organism. Bacterial infections of corals can lead to devastating large-scale losses on coral reefs, The use of molecular techniques to deter- resulting in reef deterioration and a disturbance mine bacterial causal agents of coral diseases, for the ecosystem as a whole (Harvell et al., (including BBD) has yet to be conducted for 2007). disease cases from Indonesia. Several studies have reported the different in bacterial com- Black Band Disease (BBD) is a classic ex- munities found on corals with different sur- ample of such an infection, where by the dis- rounding environment such as aquaria and ease can reduce and alter the structure of coral nature (Sweet et al., 2011; Sweet et al., 2012). reef communities (Rosenberg & Loya, 2004). This valuable approach can be used to analyse The extent of damage from BBD in Indonesian microbial community structure and species reefs has yet to be reported. composition associated with diseased tissues, through the amplification of ribosomal DNA Changes in the bacterial communities as- (rDNA) using specific 15S rRNA primers, via sociated with corals have been identified in polymerase chain reaction (PCR), followed by various infectious diseases of corals and can sequence analysis of cloned rDNA (Muyzer et be used to determine the causal agent of a al., 1993). given disease, including that of Black Band Disease. BBD can be found in various coral This study aims to identify the prevalence species (Voss, 2006), including, the massive of BBD across three different Indonesian reefs, corals (Frias-Lopez et al., 2004), Faviidae all at different distances from the mainland of (Barneah et al., 2007) and coral foliose (Sato et Java Island. Additionally, the prokaryotic com- al., 2010). The disease has also been found to munity associated with healthy, BBD diseased have a wide geographic distribution, having and dead tissue is also determined, helping to been identified in the Caribbean, Indo-Pacific elucidate the causal agent of this disease at waters, the Red Sea, the Great Barrier Reef (Willis these locations. et al., 2004), as well as Indonesian waters (Haapkylä et al., 2007). MATERIALS AND METHODS Previous research has proposed that the Sample Collection primary cause of Black Band Disease is the bacteria Phormidium corallyticum, but further Coral samples were taken from zone 1 (Pari clarification is necessary to support this claim Island), zone 2 (Pramuka Island) and zone 3 (Cooney et al., 2002). It has also been sug- (Penjaliran Island) using hammer and punch. gested that BBD is caused by a consortium of Three different types of sample were collect- microorganisms which are being dominated by ed, based on disease state, including healthy cyanobacteria. Cyanobacteria are made up of tissue, BBD-affected tissue and dead tissue an array of species, including filamental from the same coral colony. Samples were taken and measured 5 cm2 in 4 replicates of Phormidium corallyticum, heterotrophic bac- each sample. Samples were collected for the teria (Garrett & Ducklow, 1975), marine fungi purposes of identifying associated bacteria (Ramos-Flores, 1983) and bacteria that can oxi- and histology (Figure 1). dizes sulfite (Beggiatoa) and sulphate reduc- ing (Desulfovibrio) (Ducklow & Mitchell, 1979; Upon collection, samples for prokaryotic Richardson, 1996). analysis were placed into separate sterile con- tainers containing 70% ethanol, allowing Black Band Disease is characterized by the samples to be preserved until DNA extraction presence of a progressive black band (width could take place at the Coral Health and Dis- 5-30 mm) on the surface of a coral, leaving a ease laboratory at the Newcastle University bare skeleton with a lack of soft tissue behind. School of Biology, United Kingdom. The rate at which these bands progress can reach up to 2 cm per day (Rutzler et al., 1983), Samples for histological analysis were sus- with exposed skeleton being quickly colonised pended in seawater taken from the reef upon 2 ofri johan hen.pmd 2 3/16/2015, 4:47 PM  Microbial community of black band disease on infection ... (Ofri Johan) riod of weeks, with the 10% EDTA being re- placed for a fresh 10% EDTA in every few days. C The healthy, BBD-affected and dead Montipora sp. samples for histological analysis were pre- pared and sectioned following the same pro- tocol used by Smith et al. (2014). Data Analysis B Similarities in the microbial community as- sociated with the different sample types A (healthy, BBD-affected, and dead) were ana- lyzed using BioNumeric software. This analy- sis identified 18 bands across all sample types, each band representative of a dominant pro- Figure 1. Photograph representing the man- karyotic member of the microbial community. ner in which samples were col- lected for the determination of bac- RESULTS terial communities associated with tissue type and histological analy- Bacteria Associated with Coral sis. Black band diseased sample (A), Samples dead sample (B), and healthy sample (C) Based on DGGE analysis of the dominant prokaryotic community, 18 different bacterial species were identified across all three sample collection before further sample preparation types (Figure 2). The diversity of bacteria in was conducted at Newcastle University. BBD-affected corals was higher than in healthy and dead corals samples. Bacteria Identification Healthy Coral Samples Coral samples were crushed with a sterile pestle and mortar and DNA was subsequently Species identified across all healthy extracted using a Blood and Tissue DNA Ex- samples include those represented by the fol- traction Kit (Qiagen). PCR was carried out, fol- lowing bands; 1 (Halomonas anticariensis), 8 lowing the same protocol as Smith et al. (2014) (Pseudoalteromonas denitrificans), 13 (Psychro- using a Hybaid thermal cycler to amplify target monas ingrahamii), 16 (Shewanella benthica), 16S rRNA using the primers 357f-GC-518r 17 (Shewanella loihica), and band 18 (Vibrio (Muyzer et al., 1993). Denaturing Gradient Gel fischeri). Band 13 (Psychromonas ingrahamii) Electrophoresis (DGGE) was utilised to profile was only found in two healthy samples (H1 and the bacterial community based on the separa- H2) (Figure 2). tion of16S rRNA bands, which were subse- Bacteria of the Halomonas genus are halo- quently purified using a Qiagen Gel Extraction philic bacteria, usually isolated from highly Kit. DGGE was conducted according to Smith saline environments. They are gram-negative et al. (2014). Purified samples were sent for bacteria that live at temperatures range of sequencing at Genevision (Newcastle Univer- 20oC-45oC and at pH 6-9 (Martinez-Canovas et sity). Sequence data was analysed by compar- al., 2004). Halomonas anticariensis (Gamma- ing sequences with those in the BLAST data- proteobacteria) was first isolated from highly base (http://blast.ncbi.nlm.nih.gov/) to iden- saline found in peatland environments. Bacte- tify the species of bacteria (Altschul et al., rium belonging to the genus Halomonas have 1997). been found on the coral species Acropora sp. Histology tissue. Pseudoalteromonas denitrificans, be- longs to genus of bacteria often associated Healthy, BBD-affected, and dead coral with a host organism from the marine environ- samples were preserved in 5% paraformalde- ment, such as fish, mollusks, tunicates, hyde (diluted with seawater) for a minimum sponges, and micro and macro algae. Some period of one week, then decalcified using 10% members of the genus Pseudoalteromonas (in- EDTA (dissolved in distilled water) over a pe- cluding P. denitrificans) have been found pro- 3 ofri johan hen.pmd 3 3/16/2015, 4:47 PM  Indonesian Aquaculture Journal Vol. 9 No. 2, 2014 H1 H2 H3 H4 D1 D2 D3 D4 M1 M2 M3 M4 Note: 1. Halomonas anticariensis 2. Burkholderia bryophila 3. Flavobacterium columnare 4. Sulfurihydrogenibium sp. 5. Desulfovibrio desulfuricans 6. Sporobacterium olearium 7. Bacillus farraginis 8. Pseudoalteromonas denitrificans 9. Desulfovibrio gigas 10. Bacillus benzoevorans 11. Desulfovibrio magneticus 12. Pseudoalteromonas peptidolytica 13. Psychromonas ingrahamii 14. Sphingomonas wittichii 15. Thiobacillus denitrificans 16. Shewanella benthica 17. Shewanella loihica 18. Vibrio fischeri Figure 2. Band profile of DGGE result on DNA extraction of health (H1-H4), disease (D1-D4) and dead sample (M1-M4) by gen test using primer 16S rRNA and PCR application ducing active compounds which also being Psychrobacter sp., Ralstonia sp., Silicibacter isolated from seawater samples (Skovhus et sp., Bacillus sp., and Cytophaga sp. (Abdel- al., 2004). Pseudoalteromonas ruthenica has nasser & Ibrahim, 2008). previously been isolated from the coral Acropora sp. (Raina et al., 2009). Black Band Disease Samples Vibrio fischeri (of the family Vibrionaceae) Bacterial bands associated with BBD-af- is a motile, gram-negative, rod-shaped bacte- fected coral samples (Figure 2) i.e., Burkholderia ria found in both seawater and freshwater. bryophila (2), Flavobacterium columnare, (3) Vibrio fischeri are also bioluminescent and are Sulfurihydrogenibium sp. (4), Desulfovibrio often found in symbiosis with marine animals desulfuricans (5), Sporobacterium olearium (6), such as squid (Small & McFall-Ngai, 1999). Vibrio Bacillus farraginis (7), Desulfovibrio gigas (9), fischeri has previously been identified in the Bacillus benzoevorans (10), Desulfovibrio coral Acropora sp. (Raina et al., 2009).Vibrio spp. magneticus (11), Pseudoalteromonas peptido- are often associated with coral pathogenicity, lytica (12), Sphingomonas wittichii (14), and thus it is interesting to see how they are asso- Thiobacillus denitrificans (15). ciated with healthy coral samples in this in- Almost all of the bands associated with BBD stance. samples were found across all BBD samples, Shewanella spp. are gram-negative bac- including bands 2-6, and 9-15. Band 7 was only teria and have previously been identified found in two BBD-affected samples (D1 and D2) across a range of environments, including ma- (Figure 2). rine environments, freshwater rivers and lakes, Desulfovibrio spp. are gram-negative bac- and soil. This genus has previously been dis- teria, generally associated with marine, brack- covered by other researchers on healthy cor- ish and freshwater environments. Their pres- als such as Shewanella sp. (Sekar et al., 2006), ence is also found to flourish as the environ- Bacillus algicola (Sabdono & Radjasa, 2006), ment becomes more anoxic. Desulfovibrio spp. Halomonas sp. (Ojima et al., 2012), and belong to a class of bacteria that reduce sul- Sphingomonas wittichii (Rosenber & Ben-Haim, fate and thus they have a role in the cycling of 2002). sulfur. In corals, this would cause localised There are 9 genera bacteria associating anoxia and increased sulphide levels in host with the coral i.e. Vibrio sp., Alteromonas sp., tissues, which would be harmful to the coral 4 ofri johan hen.pmd 4 3/16/2015, 4:47 PM  Microbial community of black band disease on infection ... (Ofri Johan) and could ultimately result in colony death nus have previously been reported as patho- (Viehman et al., 2006). Further, Viehman et al. gens in fish (Vynne, 2011) and mussels (2006) state that many previous molecular- (Venkateswaran & Dohmoto, 2000). based studies have found several Desulfovibrio Pseudoalteromonas ruthenica has previ- spp. to be associated with black band disease. ously been isolated from the coral Acropora Bacillus spp. are ubiquitous in nature and sp (Raina et al., 2009). this genus is found to include free-living and Sphingomonas wittichii is an alphaproteo pathogenic species. Bacillus spp. are gram- bacteria and was first isolated from river water. positive, rod-shaped bacteria that can be It is known to be a good metabolizer of dibenzo- grown in aerobic and anaerobic conditions. p-dioxin (Yabuuchi et al., 2001). Sphingomonas Spores from Bacillus spp. are resistant to heat spp. are gram-negative, rod-shaped, chemo- (high temperature). These bacteria are known heterotrophic bacteria usually associated with to degrade xylan and carbohydrates, as well an oxygenated environment. These bacteria as catalyse enzymes. Bacillus spp. have previ- can cause disease in humans (especially ously been isolated from the sponge species Sphingomonas paucimobilis). Sphingomonas Petromica citrina and Chelonaplysilla erecta bacteria are ubiquitous in nature, have been (Bastos et al., 2013). isolated from soil, water and plant roots, largely The genus Burkholderia belongs to the these bacteria are able to survive in low nutri- betaproteobacteria group, comprising more ent conditions. To date, these bacteria have than 60 species which can be found in se- not been associated with pathogenicity of veral habitats such as, water, plant roots, or it corals. can be a causal disease in plants or humans Thiobacillus denitrificans are gram-nega- (Santos et al., 2013). Despite this, this is the tive, rod-shaped bacteria, belonging to the first report of bacteria belonging to this genus class betaproteobacteria. These bacteria have in coral reefs and diseased corals. been found in seawater sediments and are able Flavobacterium columnare is a gram-nega- to oxidize sulfur to sulfate, a process in line tive, rod-shaped bacteria which has previously with the absorption of oxygen and nitrate re- been found to cause disease in freshwater fish. duction (Aminuddin, 1979). Thiobacillus denitri- This species can produce an enzyme that de- ficans is also able to reduce nitrate and nitrite grades chondroitin sulfate (Figueiredo et al., (Haaijer et al., 2006). This bacterium has not 2005). This is the first report of this species in been reported to infect corals. a diseased coral. Out of the bacteria found in BBD-affected Sulfurihydrogenibium spp. are motile, gram- samples, many of them belong to the group negative, rod-shaped bacteria capable of oxi- Cyanobacteria, such as Desulfovibrio desul- dizing sulphur, such that they are believed to furicans, Desulfovibrio gigas, Desulfovibrio play an important role in the sulfur cycle magneticus, Bacillus farraginis, Bacillus (Nakagawa et al., 2005). These bacteria have benzoevorans. Cyanobacteria are a major cause previously been associated with pathogenic- of black band disease in corals (Richardson, ity in corals. 1998). Sporobacterium olearium bacteria can da- Dead Coral Samples mage methoxylated aromatic compounds and syringate in significant amounts in the pre- Few bands were found on dead coral sence of sulfide. These bacteria can be found samples and of those bands identified, none in both freshwater sediments and the marine were found consistently across all dead environment (Lomans et al., 2001), although samples (M1-M4). there are no previous reports associating it It is evident that Black Band disease be- with corals and pathogenicity in corals. gins with high abundance of cyanobacteria, Pseudoalteromonas peptidolytica are mo- known as cyanobacterial patches (CP). CPs tile, gram-negative bacteria belonging to the were found on several colonies of Montipora class Gammaproteobacteria. This bacteria is spp. in shallow waters at Pramuka Island which associated with aerobic environments and has were overgrown with algae a few days later, previously been isolated from seawater in the indicating those coral colonies had died. Tis- Sea of Japan. Interestingly, bacteria of this ge- sue damage between CP and BBD can be eas- 5 ofri johan hen.pmd 5 3/16/2015, 4:47 PM  Indonesian Aquaculture Journal Vol. 9 No. 2, 2014 ily distinguished in the field, as CPs are not Bacterial communities were grouped and associated with a black belt. Colonies found undergo a treatment in accordance with the to have a CP did not always develop BBD (Fig- samples between healthy (H) and diseased (D) ure 3). These observations are similar to that coral. Excluding the samples of dead coral that reported by Sato et al. (2010) in the Great Bar- 2M2 is not clustered either to 3M1 and 2M2 rier Reef. samples. The test results explained that the bacteria live on the healthy coral is different CPs identified in Montipora sp. colonies with diseased and dead sample (Figure 5). (Figure 3A) were found to grow in size after 2 weeks (Figure 3B). Exposed skeleton was then DISCUSSION subsequently colonised by algae (Figure 3C). Microscopic observations of CPs revealed a There are 3 species of Desulfovibrio spp. large numbers of cyanobacteria filaments. found on infected coral samples in the Seribu Islands i.e. Desulfovibrio magneticus, Desulfo- Histology Observation vibrio gigas, and Desulfovibrio desulfuricans. Desulfovibrio spp. are pathogenic because It was easy to indicate epidermis layer, they produce and accumulate sulfide in high gastrodermis, as well as zooxanthellae in concentrations in black band disease, giving healthy Montipora sp. samples, whereas these rise to anoxic conditions, which ultimately tissue structures were found to be broken leads to coral mortality (Richardson et al., 1997). down in BBD samples. Long, rod-shaped struc- tures resembling filamental cyanobacteria Another type of bacteria found in BBD in- were identified in abundance in BBD samples fected samples at Seribu Islands were Bacillus (Figure 4B). These cyanobacteria were not spp. (Bacillus benzoevorans and Bacillus found in dead coral samples and zooxanthel- farraginis). Although these species have not lae were also can not be found along with any been reported to be pathogenic to corals in healthy tissue structure. the past, Bacillus spp. have been found to have A B C D Figure 3. Montipora sp. with CP (A, B) after overgrown with algae (C) and cyanobacteria patches (CP) under microscope observation 40x cyanobacterial filaments (D) 6 ofri johan hen.pmd 6 3/16/2015, 4:47 PM  Microbial community of black band disease on infection ... (Ofri Johan) A B c e a e b c d C Figure 4. Histological sections of healthy (A), Black Band Disease (B), and dead (C) Montipora sp. tissue under light microscopy (magnification x 40), stained with toluidine blue. Specific coral tissues are labelled, including epider- mis (a), endodermis (b), and zooxanthellae (c). Infected coral samples (B); damaged coral tissue (d) and cyanobacteria (e) 2M2 2D Stress: 0.01 Sample type M D H 3M2 3H1 Similarity 3H2 1H2 3M1 60 1H1 80 3D2 1D2 1D1 3D1 Figure 5. The similarity rate of bacteria on healthy, infected, and dead sample according to different sites by using Primer 6 programme antibacterial properties against other bacteria Sulfurihydrogenibium sp. is a bacteria that and their influx could disrupt the native coral canoxidize hydrogen and sulfur (Hirayama et microbiota (Sabdono & Radjasa, 2006). This al., 2005), but there is no report that describes could potentially having a negative impact it as a pathogenic bacteria in corals. upon symbioses between the host coral and microbial members of the holobiont if key spe- Shewanella sp. was found to be associated cies are reduced/lost. with healthy Montipora sp. samples in the 7 ofri johan hen.pmd 7 3/16/2015, 4:47 PM  Indonesian Aquaculture Journal Vol. 9 No. 2, 2014 present study, coinciding with previous re- sp., Bacillus algicola, and Shewanella sp. are all ports that associate this bacteria with healthy associated with the healthy Montipora sp. corals (Frias-Lopez et al., 2002). Despite this, holobiont in this region. Following histological other studies have also found these bacteria analysis, there are marked differences be- to be associated with infected corals (Sekar et tween healthy, BBD-affected and dead Monti- al., 2006), thus there is the potential that pora sp. tissues. In BBD-affected samples, there healthy samples harbouring this bacterial spe- is mass breakdown in host tissue, zooxanthel- cies may become diseased following an influx lae are lost and filament-shaped bacteria are associated with favourable environmental con- present. These filament-shaped bacteria are ditions (Frias-Lopez et al., 2002; and Rohwer lost in dead samples, thus clearly demonstrat- et al., 2002). ing their presence is strongly associated with BBD tissues. Sphingomonas sp., found in BBD samples in the present study, have previously been ACKNOWLEDGEMENTS associated with healthy coral (Rosenberg & Ben-Haim, 2002). That said, members of this The authors would like to thank the Minis- genus have also been associated with corals try of Marine Affairs and Fisheries for provid- suffering from White Plague in Florida, demon- ing study fees and the fellowship from The strating their pathogenic potential (Ravindran World Bank, tied with the Robert S. McNamara et al., 1998). Fellowships Program (RSM), Coral Health and Although Black Band Disease (BBD) has Disease Laboratory, Newcastle University (UK) been known for decades (since 1970), the main in assisting in analyzing bacteria associated pathogenic bacteria of the disease has yet to with coral samples. This research was also sup- be identified. Previously, it was considered that ported by funding from the Center for Aqua- BBD was caused by the bacteria Phormidium culture Research and Development, and Bogor corallyticum. But, the use of molecular tech- Agriculture University. The authors would also niques to identify the microbial community like to thank those who helped with data col- associated with this disease has led to the dis- lection, Jhon Bythell who help in process to covery of a consortium of bacteria associated get funding in this analysis as well as the with BBD (Frias-Lopez et al., 2004). 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