Vol. 500: 1–9, 2014 MARINE ECOLOGY PROGRESS SERIES Published March 17 doi: 10.3354/meps10698 Mar Ecol Prog Ser FREEREE ACCESSCCESS FEATURE ARTICLE Microbial aggregates within tissues infect a diversity of corals throughout the Indo-Pacific Thierry M. Work1,2,*, Greta S. Aeby2 1US Geological Survey, National Wildlife Health Center, Honolulu Field Station, PO Box 50167, Honolulu, Hawaii, USA 2Hawaii Institute of Marine Biology, PO Box 1346, Kane‘ohe, Hawaii, USA ABSTRACT: Coral reefs are highly diverse eco - systems where symbioses play a pivotal role. Corals contain cell-associated microbial aggregates (CAMA), yet little is known about how widespread they are among coral species or the nature of the symbio tic re- lationship. Using histology, we found CAMA within 24 species of corals from 6 genera from Hawaii, American Samoa, Palmyra, Johnston Atoll, Guam, and Australia. Prevalence (%) of infection varied among coral genera: Acropora, Porites, and Pocillo- pora were commonly infected whereas Montipora were not. Acropora from the Western Pacific were significantly more likely to be infected with CAMA than those from the Central Pacific, whereas the re- verse was true for Porites. Compared with apparently healthy colonies, tissues from diseased colonies were Microscopic bacteria in tissues of coral polyps are important significantly more likely to have both surface and to coral health, ecology, and possibly evolution. basal body walls infected. The close association of Illustration: Thierry M. Work CAMA with host cells in numerous species of appar- ently healthy corals and lack of associated cell patho- logy reveals an intimate agent− host association. Fur- thermore, CAMA are Gram negative and in some corals may be related to chla mydia or rickettsia. We INTRODUCTION propose that CAMA in adult corals are facultative secondary symbionts that could play an important Coral reefs are highly diverse ecosystems where ecological role in some dominant coral genera in the both ecto- and endo-symbioses play a pivotal role. Indo-Pacific. CAMA are important in the life histories Corals host species-specific mucus-associated bacteria of other animals, and more work is needed to under- (ectosymbionts) that provide antibiotic defenses for stand their role in the distribution, evolution, physio - the host (Ritchie 2006), and endolithic bacteria within logy, and immunology of reef corals. the skeleton are important for photosynthesis (Fine & KEY WORDS: Coral · Microbes · Facultative · Loya 2002). Corals also have unicellular algae (zoo- Symbiont · Cell-associated microbial aggregates · xanthellae) within their cells (endosymbionts) that Histology provide photosynthetically fixed carbon to the host and aid in deposition of the coral skeleton that forms Resale or republication not permitted without written consent of the publisher the foundation of coral reefs (Muscatine 1990, Rowan *Corresponding author: [email protected] © Inter-Research 2014 · www.int-res.com 2 Mar Ecol Prog Ser 500: 1–9, 2014 1998). The coral−zooxanthellae symbiosis is well studied, and disruption of this symbiosis leads to bleaching (expulsion of zooxanthellae from coral tis- sues), potential death of the coral, and subsequent ecological disruption to myriad organisms that depend on corals for shelter and food (Glynn 1996, Brown 1997a,b, Hoegh-Guldberg 1999, Coles & Brown 2003). Cnidaria also have microbial aggregates (bacteria or archaea) found within tissues of 2 species of anemones (McKinstry et al. 1989, Palincsar et al. 1989, Peters & Yevich 1989), 5 species of reef corals in the Caribbean (Peters et al. 1983, Peters 1984), and 4 species of corals in the Indo-Pacific (Ainsworth et al. 2006, Ainsworth & Hoegh-Guldberg 2009, Sudek et al. 2012). In situ hy- bridization studies of microbial aggregates in Acro - pora and Stylophora from Australia revealed these Fig. 1. Regions where corals were sampled for examination organisms to be Gamma proteobacteria (Ainsworth et of cell-associated microbial aggregates (CAMA) al. 2006), but little is known of their identities in other coral species. Furthermore, nothing is known about basal body wall) portions of polyps. When gonads how widespread microbial aggregates are among were evident, they were examined for CAMA to coral species, the nature of the symbiotic relationship, investigate the potential for vertical transmission. or the role they may have in the health, ecology, or CAMA were measured with an ocular scale cali- evolution of corals. brated to a stage micrometer in 1 or 2 dimensions The US Geological Survey has a growing archive depending on whether they were circular or oblong. of histological tissue sections comprising 131 species Area (µm2) was calculated using the formula for a cir- and 36 genera of healthy and diseased scleractinian cle or ellipse for circular or oblong CAMA, respec- corals from across the Indo-Pacific. We examined this tively. archive to determine how widespread cell-associated To begin to characterize bacteria in CAMA, we microbial aggregates (CAMA) are within coral spe- used histological stains that routinely identify various cies or genera, the geographic pattern of infections, groups of bacteria on representative tissue slides. the type of relationship with host corals, and whether Stains included Gram or Ziehl-Nielsen acid-fast CAMA are associated with coral health. for mycobacteria (Prophet et al. 1992) or Gimenez for chlamydia- or rickettsia-like bacteria (Gimenez 1964). CAMA were examined in more detail in one MATERIALS AND METHODS coral species, Porites compressa, using electron microscopy (EM). For EM, tissues from P. compressa Archived tissue sections stained with hematoxylin were fixed in Trump’s fixative (McDowell & Trump and eosin originated from 110 grossly healthy and 1976) made with seawater, rinsed in 0.1 M Soren- 386 grossly diseased coral colonies collected be - son’s phosphate buffer, and post-fixed in 2% osmium tween February 1999 and September 2012. The 496 tetroxide. Epoxy-embedded tissues were cut to 1 um colonies comprised 21 genera and 57 species from thickness, stained with toluidine blue, and examined Hawaii (n = 279), American Samoa (n = 98), Palmyra under light microscopy to target areas for EM. Ultra- (n = 58), Johnston Atoll (n = 37), Guam (n = 20), and thin sections were stained with uranyl acetate, post- Australia (n = 4) (Fig. 1). Tissues were prepared for stained with lead citrate, and examined with a Zeiss histology as described in Work & Aeby (2010). EM 109 electron microscope. Briefly, coral fragments were fixed in a solution of 1 part zinc formaldehyde (Z-fix, Anatech) and 4 parts seawater, decalcified, trimmed, embedded in paraf- Data analysis fin, sectioned at 5 µm, and stained with hematoxylin and eosin. We examined tissues under light micro - Health status did not significantly affect prevalence scopy (generally, a single slide per fragment) and of CAMA (χ2 = 0.2126, p = 0.64); therefore, healthy recorded presence/absence of CAMA within surface and diseased corals were pooled for sub sequent ana - (surface body wall) or basal (mesenterial filaments, lysis. For coral genera (Acropora, Montipora, Porites, Work & Aeby: Microbial endosymbionts in Indo-Pacific corals 3 and Pocillopora) and islands (Pal myra, Table 1. Occurrence of cell-associated microbial aggregates (CAMA) within Samoa, Johnston, Hawaii) where we paired lesion and apparently normal fragments from 386 diseased colonies and normal fragments from 110 apparently healthy colonies of various coral species had suitable (>20 per region) sample sampled from across the Indo-Pacific. Data show the number of fragments ex- sizes, we used binomial logistic regres- amined histologically for presence of CAMA and the prevalence of infection (%) sion to assess the effects of coral genus, island, or their interaction on presence Genus Species Diseased Healthy or absence of CAMA. For those factors Lesion Normal Normal or interactions of factors considered sig- (n) (%) (n) (%) (n) (%) nificant in the model, post hoc Pearson’s Acropora A. abrotenoides 8638 63 A. acuminata 2 100 2 100 chi square was used to assess effects of A. austera 2502 50 individual factors or combinations. For A. clathrata 5605 80 A. cytherea 68 37 68 43 12 17 Porites where we had adequate samples A. digitifera 2 0 2 100 (Hawaii), we evaluated the effect of spe- A. elseyi 20 A. gemmifera 10 1 0 10 cies on CAMA prevalence. We also as- A. humilis 1 0 1 100 sessed the effect of health versus disease A. hyacinthus 9569 78 A. listeri 10 1 0 on the number of tissue compartments A. muricata 9679 67 infected. To compare the size of CAMA Acropora sp. 12 58 12 58 Acropora total 120 45 120 53 15 13 among families of corals, we used Astreopora A. listeri 10 1 0 parametric or non-parametric ANOVA A. myriophthalma 10 1 0 10 Astreopora total 2 0 2 0 1 depending on whether data followed a Cyphastrea C. ocellina 10 normal distribution as evaluated by the Diploastrea D. heliopora 20 250 Favia F. stelligera 10 1 0 Shapiro-Wilk test. All analyses were Fungia F. concinna 40 4 0 10 conducted using the R statistical pro- F. scutaria 20 2 0 10 Fungia sp. 1 0 1 0 gram version 2.13.2 (R Development Fungia total 7 0 7 0 2 0 Core Team 2011) with a 0.05 level of Galaxea Galaxea sp. 1 0 Goniastrea G. edwardsi 50 5 0 significance. G. retiformis 10 1 0 Goniastrea sp. 1 0 1 100 Goniastrea total 7 0 7 14 Hydnophora H. microconos 10 1 0 RESULTS Leptastrea L. purpurea 10 Leptoria L. phrygia 10 1 0 Leptoseris Leptoseris sp. 4 0 Of the 57 species of corals comprising Lobophyllia L. hemprichii 10 Lobophyllia sp. 2 50 2 0 21 genera examined, CAMA were found Lobophyllia total 2 50 2 0 1 0 within 24 species from 6 genera and 5 Merulina M. scabricula 10 1 0 Montipora M. aequituberculata 30 3 0 families. Ten species of Acropora (Acro- M. capitata 115 0 115 0 12 0 poridae), 6 species of Po rites (Poritidae), M.