A Rickettsiales-like (?) Bacterium Is Responsible For The Tissue Loss Diseases Of Caribbean Acroporid Corals Esther C. Peters, Ph.D. Department of Environmental Science & Policy, George Mason University, Fairfax, Virginia 22030-4444

The Problem Identifying the Pathogen Results Results, Continued Staghorn (Acropora cervicornis), elkhorn (A. palmata), and their To restore lost acroporid populations, coral nurseries are Carolyn Friedman (University of Washing- Tissue sections from acroporid coral samples collected in 1981 hybrid (A. prolifera) coral populations in the Caribbean Sea and culturing branch fragments and replanting them on reefs. For ton) identified these structures (Fig. 2) as from Tague Bay, St. Croix, US Virgin Islands, site of the first tropical western Atlantic Ocean have declined greatly since the this process to be successful, we need to know what causes the the suspect RLOs (WBD-affected A. WBD-I outbreak were re-examined and the results are presented early 1970s when tissue loss due to white-band disease was first tissue loss and whether and how it can be controlled. These cervicornis collected by David Kline, in Table 2. When first studied, coral histology knowledge was reported (Gladfelter 1982; Weil & Rogers 2011). Only a few studies have reported finding a microorganism that might be Bocas del Toro, Panama). More than 30 limited and these microorganisms do not always stain well with studies have used histopathological with microbiological the pathogen causing the tissue loss: histoslides sets prepared since the mid- H&E, possibly also due to variations in fixation and acid techniques to study the affected corals, despite the importance of 1970s have been located and many decalcification. Giemsa staining is necessary to clearly detect . WBDI: bacterial aggregates by histopathology (Peters et doing so to confirm the links between suspected microbial obtained (from International Registry of them (Fig. 2), the cells then stain dark reddish-purple. al. 1983; not seen in Bythell et al. 2002), identified by pathogens and tissue damage (e.g., Bythell et al. 2002; Work et al. Fig. 2. Suspect Coral Pathology, Registry of Tumors in Polson (2007) by molecular analysis as Gram-negative Table 2. Results of histopathological examinations of most of the samples 2008; Williams et al. 2011). RLOs fill muco- Lower Animals, or collectors). Results of Pseudomonas spp. Found in apparently healthy samples and collected in 1981 from Tague Bay, St. Croix. cytes, tentacles. observations to date are in Table 1. not always found in diseased samples. This report presents progress on a histopathology study based A. A. A. A. on recent sample collections and archived tissues and . WPD: Gram-negative Serratia marsecens by micro- Table 1. Results of histopathological examinations of acroporid samples cervicornis cervicornis palmata palmata from the tropical western Atlantic. histoslides to try to improve our understanding of the tissue biology, Koch’s postulates satisfied (Sutherland et al. WBD Healthy WBD Healthy Species and Condition n = 15* n = 4* n = 13* n = 13 loss disease(s). 2011), only found in A. palmata. Location (Year) Collector Species Condition* RLOs** Looe Key, Florida A. cervicornis 20/22† Bacterial aggregates % 100 75 100 100 . WBD-II: Gram-negative Vibrio carchariae/harveyi by Keys (1976) W. Jaap A. palmata AH (91%) Bacterial aggregates SI** 0.7 1.7 2.0 2.9 Four categories of acute to subacute tissue loss (focal, multifocal, microbiology (Ritchie & Smith 1998), Koch’s postulates Biscayne National A. cervicornis Epidermal RLOs % 100 100 100 100 or diffuse), not associated with predation by corallivores, have almost all satisfied, incomplete (Gil-Agudelo et al. 2006). Park (1977–1981) W. Jaap A. palmata AH 13/13† Epidermal RLOs SI 3.0 2.0 4.2 4.7 been identified on Caribbean acroporids, based on rate and pattern Puerto Rico (1983– A. cervicornis Filament RLOs % 100 100 100 100 of tissue loss: . WBD-I: Gram-negative Rickettsiales-like bacterium by 1985) A. Szmant A. palmata AH Present Filament RLOs SI 2.4 3.0 3.7 4.5 molecular analysis (90% similarity to uncultured Belize (1986) E. Peters A. cervicornis AH, WBD 6/6 *not same n as in Peters et al. (1983), due to missing histoslides sent to archives Looe Key, Florida K. **SI = severity index based on relative numbers of infected cells: 1 = minimal, 2 = mild, 3 = . White-band disease type I (WBD-I) with sloughing or straight Rickettsiales with BLASTN based on cloning and moderate, 4 = severe. Scores based on H&E staining, will probably change with Giemsa sequencing of bacterial 16S rDNAs) (Casas et al. 2004). Keys (1997) Patterson A. palmata AH, WPD Present tissue loss margin (Fig. 1A), reviewed in Bythell et al. (2008); Broward County B. Vargas- Also in apparently healthy staghorn corals and in other 2A 2B 2C . White-band disease type II (WBD-II) with bleaching at tissue (2000–2013) Angel et al. A. cervicornis AH Present loss margin (Fig. 1B), reviewed in Ritchie and Smith (1998); coral species, conclusion: not the pathogen of WBD-I. Florida Reef Tract, A. cervicornis, . White pox or white patch disease (WPD) (Fig. 1C), reviewed Biscayne to Dry A. palmata, A. AH, WBD, Tortugas (2003) D. Williams prolifera WPD, RTL 15/15 in Patterson-Sutherland & Ritchie (2004); and Should the Rickettsia-like organism (RLO) be dismissed? Key Largo, Florida AH, WBD, . Rapid tissue loss (RTL) (Fig. 1D), Williams & Miller (2005). RLOs are obligate intracellular parasites and known Keys (2005) M. Miller A. palmata WPD 14/14 pathogens of many marine organisms. Could they be Panama (2005) D. Kline A. cervicornis AH, WBD 24/24† harmful to corals without causing gross disease signs? Antigua, Belize, Curaçao (2006–07) N. Fogarty A. prolifera AH 41/41 US Virgin Islands Fig. 2. A. H&E-stained section, poor quality, difficult to see RLO-infected Materials and Methods (2009) C. Woodley A. palmata AH Present cells. B. H&E-stained section, better differentiation of suspect RLOs from Guantanamo Bay, mucus. C. Giemsa-stained section, RLOs are dark purple, mucus paler Freshly collected and archived preserved specimens of A. cer- and frothy. All sections from same sample, A. cervicornis with RTL (11- vicornis, A. palmata, and A. prolifera obtained from sites Cuba (2009–2011) C. Woodley A. palmata AH 71/71 Key Largo, FL Keys M. Miller, AH, WBD, 174), Conch Shallow, Key Largo, FL, 08/03/2011, collected by M. Miller. throughout the region. All specimens immersed in a formal- (2010–2012) E. Peters A. cervicornis RTL 43/43† dehyde-based fixative, decalcified, paraffin-embedded, and Belize (2012) L. Carne A. cervicornis AH, WBD 8/8 Apical polyps have fewer infected mucocytes compared with sectioned (Peters et al. 2005). Tissues stained with hematoxy- Puerto Rico (2012) S. Griffin A. cervicornis AH 24/24 lateral polyps. Mucocytes of the oral disc and tentacle epidermis lin and eosin (H&E) for examination by light microscopy to *AH = apparently healthy, WBD = white-band disease, WPD = white patch disease, 1A usually have the largest bacterial cells within their cytoplasm. John Halas detect pathological changes in the tissues. Additional sections RTL = rapid tissue loss **Prevalence of suspect RLOs in specimens collected in each sample set, Present = Mucocytes of the ciliated actinopharynx epithelium and stained with a Giemsa method, which is used to detect suspect RLOs seen in some of the samples but not all of the samples have been cnidoglandular bands of the mesenterial filaments have smaller rickettsia and other Gram-negative bacteria. examined yet. † = More slides in set to be examined cells; mucocytes of the basal body wall gastrodermis lining Kathryn gastrovascular canals can also be infected. These cells all stain Sutherland with Giemsa, but differ in size and shape, suggesting they are more likely members of the Chlamydiales forming reticulate Conclusions bodies and elementary bodies, and not Rickettsiales (Fig. 3).

Tropical western Atlantic acroporid corals have been infected since the 1970s with an obligate intracellular bacterium that has affinities 3A to the primitive bacterial groups Rickettsiales (molecular) or Chlamydiales (morphology) (e.g., Fryer & Lannan 1994). The microorganism EB RB 1C 1B Andrew Bruckner infects polyp mucocytes, proliferates, and kills them. Additional molecular and ultrastructural studies to determine its identity are in progress.

1B Koch’s Postulates require modifications for determining the pathogenicity of nonculturableFigure 5.microorganisms You can use connector and lines other and arrowsstressors to visually (USEPA guide viewers2000). through your results. Making logical points this way is much, much better than Experimental exposures to Serratia marcescens (Patterson et al. 2002) and Vibrio harveyimaking(Gil it in -theAgudelo text section.et Theseal. 2006) lines canresulted help viewers in tissue read your loss, poster but coral condition was not determined by histology. The suspect RLO/CLO is the primaryeven when pathogen, you’re not continuallypresent. killing cells. If the coral cannot obtain adequate nutrition to replace these cells and continue vital metabolic functions, it becomes highly susceptible to other agents. Probable secondary pathogens include: other microorganisms; temperature, turbidity, sedimentation stress; or toxicant exposure. Different 3B 3C patterns of tissue loss are probably due to the intensity and duration of infections, and the identity of the secondary stressor(s) that trigger them. EB The weakened condition of present populations of acroporids explains the intermittent outbreaks, which may occur during transient exposure to additional stressors but then disappear, and inconsistent transmission of tissue loss. Studies are underway to determine whether larvae are infected, when settled planulae become infected, or if any acroporid genotypes resist infection, and to further explore this challenging enigma. RB 1D

Literature Cited Peters EC, Price KL, Borsay Horowitz DJ (2005) Histological preparation of invertebrates for evaluating contaminant effects. In: Fig. 3. A. Small coccoid cells similar to chlamydia-like organisms 1D Bythell J, Barer MR, Cooney RP, Guest JR, O’Donnell AG, Pantos O, Le Tissier MDA (2002) Histopathological methods for the Ostrander GK (ed) Techniques in Aquatic Toxicology Vol 2, pp 653–686. Taylor & Francis, Boca Raton investigation of microbial communities associated with disease lesions in reef corals. Lett Appl Microbiol 34(5):359–364 Polson SW (2007) Comparative Analysis of Microbial Community Structure Associated with Acroporid Corals During a Disease (CLOs), the infective stage elementary bodies (EB), that develop into Bythell J, Pantos O, Richardson L (2004) White plague, white band, and other “white” diseases. In: Rosenberg E, Loya Y (eds) Coral Outbreak in the Florida Reef Tract. PhD Diss, Medical University of South Carolina, Charleston, SC larger reticulate bodies (RB), as seen in this section of cnidoglandular Health and Disease, pp 351–365. Springer-Verlag, Berlin Ritchie KB, Smith GW (1998) Type II white-band disease. Rev Biol Trop 46 (Supl 5):199–203. Fig. 1. A. WBD-I on A. palmata, Grecian Rocks, Florida Keys, upper Casas V, Kline DI, Wegley L, Yu YN, Breitbart M, Rohwer F (2004) Widespread association of a Rickettsiales-like bacterium with Sutherland KP, Shaban S, Joyner JL, Porter JW, Lipp EK (2011) Human pathogen shown to casue disease in the threatened elkhorn band epithelium. B. Section of cnidoglandular band epithelium showing right: straight margin on A. cervicornis, Broward County, Florida; reef-building corals. Environ. Microbiol 6: 1137–1148 coral, A. palmata. PLoS ONE DOI: 10.1371/journal.pone. 0023468 more of the small-cell and large-cell bacteria. The pale-pink cells are Fryer JL, Lannan CN (1994) Rickettsial and chlamydial infections of freshwater and marine fishes, bivalves, and crustaceans. Zool USEPA (2000) Stressor identification guidance document. EPA/822/B-00/025. US Environmental Protection Agency, Washington bottom right: sloughing tissue on A. palmata, Tague Bay, St. Croix, Stud 33(2):95–107 Weil E, Rogers CS (2011) Coral reef diseases in the Atlantic and Caribbean. In: Dubinsky Z, Stambler N, Coral Reefs: An Ecosystem acidophilic granular gland cells that secrete enzymes to digest food. C. US Virgin Islands. B. WBD-II on A. cervicornis, Broward County, Gil-Agudelo DL, Smith GW, Weil E (2006) The white band disease type II pathogen in Puerto Rico. Rev Biol Trop 54:59–67 in Transition, pp 465–491. Springer Science+Business Media, New York Section of tentacle epidermis with largest bacterial cells, the pale pink Gladfelter WB (1982) White-band disease in Acropora palmata – implications for the structure and growth of shallow reefs. Bull Mar Williams DE, Miller MW (2005) Coral disease outbreak: pattern, prevalence and transmission in Acropora cervicornis. Mar Ecol Prog Florida. C. WPD on A. palmata, Puerto Rico, inset, WPD on A. Sci 32:639–643 Ser 301:119–128. structures here are spirocysts. All sections from same sample, palmata, Looe Key, Florida. D. RTL on A. cervicornis, Broward Patterson Sutherland K, Ritchie K (2004) White pox disease of the Caribbean elkhorn coral, Acropora palmata. In: Rosenberg E, Loya Williams GJ, Work TM, Aeby GS, Knapp IS, Davy SK (2011) Gross and microscopic morphology of lesions in Cnidaria from Palmyra apparently healthy A. cervicornis (11-167), Coral Restoration Y (eds) Coral Health and Disease, pp 289–300. Springer-Verlag, Berlin Atoll, Central Pacific. J Invertbr Pathol 106(2):165–173 County, Florida. Peters EC, Oprandy JJ Yevich PP (1983) Possible causal agent of "white band disease" in Caribbean scleractinian corals. J Invertebr Work TM, Richardson LL, Reynolds TL, Willis BL (2008) Biomedical and veterinary science can increase our understanding of coral Foundation nursery, Key Largo, FL, 06/23/2011, collected by E. Peters. Pathol 41:394–396 disease. J Exp Mar Biol Ecol 362:63–70

Acknowledgements Course offered For further information Funding: NOAA’s Office of Protected Resources “Understanding Corals from the Inside Out: Comparative Please contact [email protected] for more information on Histopathology” at Nova Southeastern University Collaborators: Margaret Miller, Jennifer Moore, Cheryl Woodley, Ken Nedimyer, Carolyn Friedman, David Kline, Patrick Gillevet, this and related projects. Oceanographic Center, May 12–August 1, 2014; 3 Robert Jonas, Patricia Blackwelder, Katy Lohr, Staci Lewis, Katheryn Patterson, David Allen, Adrienne Gillevet, Songhee Kang, Patrick Do you know of other collections of formaldehyde- graduate credit hours or may be audited, online and one Pansoy, William Norfolk, Doranne Borsay Horowitz, Kathy Price, Abraham Smith, Jan Landsberg, Yasu Kiryu, Kathryn Sutherland, Les preserved Caribbean acroporid samples? Please let me lab week at NSUOC (July 14–18). Kaufman, Lisa Carne, Walter Jaap, Dan and Stephanie Clark, Nicole Fogarty, Sean Griffin, Alina Szmant, Judith Lang know!