X-ray linear dichroic ptychography.

Biominerals such as seashells, coral skeletons, bone, and tooth enamel are optically anisotropic crystalline materials with unique nanoscale and microscale organization that translates into exceptional macroscopic mechanical properties, providing inspiration for engineering new and superior biomimetic structures. Using Seriatopora aculeata coral skeleton as a model, here, we experimentally demonstrate X-ray linear dichroic ptychography and map the c-axis orientations of the aragonite (CaCO3) crystals. Linear dichroic phase imaging at the oxygen K-edge energy shows strong polarization-dependent contrast and reveals the presence of both narrow (<35°) and wide (>35°) c-axis angular spread in the coral samples. These X-ray ptychography results are corroborated by four-dimensional (4D) scanning transmission electron microscopy (STEM) on the same samples. Evidence of co-oriented, but disconnected, corallite subdomains indicates jagged crystal boundaries consistent with formation by amorphous nanoparticle attachment. We expect that the combination of X-ray linear dichroic ptychography and 4D STEM could be an important multimodal tool to study nano-crystallites, interfaces, nucleation, and mineral growth of optically anisotropic materials at multiple length scales.

From particle attachment to space-filling coral skeletons.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space.

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic-related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in-vivo imaging with cryo-electron microscopy and cryo-elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.

Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis - The saccharide moieties and their localization.

To construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.

Breakdown of coral colonial form under reduced pH conditions is initiated in polyps and mediated through apoptosis.

Certain stony corals can alternate between a calcifying colonial form and noncalcifying solitary polyps, supporting the hypothesis that corals have survived through geologic timescale periods of unfavorable calcification conditions. However, the mechanisms enabling this biological plasticity are yet to be identified. Here we show that incubation of two coral species (Pocillopora damicornis and Oculina patagonica) under reduced pH conditions (pH 7.2) simulating past ocean acidification induce tissue-specific apoptosis that leads to the dissociation of polyps from coenosarcs. This in turn leads to the breakdown of the coenosarc and, as a consequence, to loss of coloniality. Our data show that apoptosis is initiated in the polyps and that once dissociation between polyp and coenosarc terminates, apoptosis subsides. After reexposure of the resulting solitary polyps to normal pH (pH 8.2), both coral species regenerated coenosarc tissues and resumed calcification. These results indicate that regulation of coloniality is under the control of the polyp, the basic modular unit of the colony. A mechanistic explanation for several key evolutionarily important phenomena that occurred throughout coral evolution is proposed, including mechanisms that permitted species to survive the third tier of mass extinctions.

Coral biomineralization: A focus on intra-skeletal organic matrix and calcification.

In the recent years several papers and some reviews have dealt with characterization, localization and influence on the precipitation of calcium carbonate, of the organic matrix from scleractinian corals. In fact, it has been well established that coral calcification is a biological controlled process orchestrated in space and time by the organism also trough the secretion of organic matrix molecules because it has been well established that coral calcification is a biological controlled process, and thus is orchestrated in space and time by the organism also through the secretion of organic matrix molecules. In this review is presented a scientific path on the biomineralization of corals having as focusing point the intra-skeletal organic matrix, the molecules that are associated with mineral (aragonite). The review starts with a an overview on coral tissue, skeleton and tissue skeleton interface, describes the intra-skeletal organic matrix putting attention mainly on the proteins associated to aragonite and finally describes the in vivo and in vitro calcium carbonate precipitation experiments carried out aimed to evaluate the role of the organic matrix. The last paragraph reports studies on the role of the organic matrix in controlling calcification when corals are subject ocean acidification effects. The readers are expected to find a source of inspiration for new studies on the biomineralization of corals that are organic matrix addressed and merge diverse scientific disciplines.

Molecular and ultrastructural studies of a fibrillar collagen from octocoral (Cnidaria).

We report here the biochemical, molecular and ultrastructural features of a unique organization of fibrillar collagen extracted from the octocoral Sarcophyton ehrenbergi Collagen, the most abundant protein in the animal kingdom, is often defined as a structural component of extracellular matrices in metazoans. In the present study, collagen fibers were extracted from the mesenteries of S. ehrenbergi polyps. These fibers are organized as filaments and further compacted as coiled fibers. The fibers are uniquely long, reaching an unprecedented length of tens of centimeters. The diameter of these fibers is 9±0.37 µm. The amino acid content of these fibers was identified using chromatography and revealed close similarity in content to mammalian type I and II collagens. The ultrastructural organization of the fibers was characterized by means of high-resolution microscopy and X-ray diffraction. The fibers are composed of fibrils and fibril bundles in the range of 15 to 35 nm. These data indicate a fibrillar collagen possessing structural aspects of both types I and II collagen, a highly interesting and newly described form of fibrillar collagen organization.

Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.

The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral's metabolic pathways.

Gonadal Morphology and Gametogenesis in Japanese Red Coral Corallium japonicum (Octocorallia: Alcyonacea) Collected off Cape Ashizuri, Japan.

Colonies of the Japanese red coral Corallium japonicum Kishinouye, 1903 collected off Cape Ashizuri, Japan were gonochoric and produced gonads in siphonozooids annually, mainly during the spring season. Polyp anatomy, gonadal morphology and gametogenesis in this species were revealed by light and electron microscopy. A siphonozooid had a pharynx with a prominent siphonoglyph and eight mesenteries: two sulcal, two asulcal, and four lateral. A rudimentary retractor was found on one side of each mesoglea of these mesenteries. The retractor arrangement in the siphonozooid was reverse of what was described in the autozooids of octocorals. Gonads initiated as small protrusions on the mesenteries, except in the asulcal ones, and even at an incipient stage they were covered with a sac-shaped thin layer of mesoglea, which was continuous with the mesoglea of mesenteries. Gastrodermis enveloped the complete outer surface of the thin layer of mesoglea throughout gametogenesis in both oocytes and sperm cysts. Oocytes produced many microvilli on their cortical surfaces beneath the thin layer of mesoglea concomitantly with the accumulation of lipid globules in the cells, whereas in sperm cysts spermatocytes and spermatids increased in number without microvilli production, followed by synchronous spermiogenesis involving remarkable changes in the shape and position of organelles. Based on the comparison of patterns in gonadal development between octocorals including C. japonicum, hexacorals and scyphozoans, octocoral and stauromedusa species may be characterized by the fact that gametogenesis never occurs in the matrix of mesoglea, but rather exclusively within the thin sac of mesoglea surrounded by gastrodermis.

The possible role of cyanobacterial filaments in coral black band disease pathology.

Black band disease (BBD), characterized by a black mat or line that migrates across a coral colony leaving behind it a bare skeleton, is a persistent disease affecting massive corals worldwide. Previous microscopic and molecular examination of this disease in faviid corals from the Gulf of Eilat revealed a number of possible pathogens with the most prominent being a cyanobacterium identified as Pseudoscillatoria coralii. We examined diseased coral colonies using histopathological and molecular methods in order to further assess the possible role of this cyanobacterium, its mode of entry, and pathological effects on the coral host tissues. Affected areas of colonies with BBD were sampled for examination using both light and transmission electron microscopies. Results showed that this dominant cyanobacterium was found on the coral surface, at the coral-skeletal interface, and invading the polyp tissues and gastrovascular cavity. Although tissues surrounding the invasive cyanobacterial filaments did not show gross morphological alterations, microscopic examination revealed that the coral cells surrounding the lesion were dissociated, necrotic, and highly vacuolated. No amoebocytes were evident in the mesoglea of affected tissues suggesting a possible repression of the coral immune response. Morphological and molecular similarity of the previously isolated BBD-associated cyanobacterium P. coralii to the current samples strengthens the premise that this species is involved in the disease in this coral. These results indicate that the cyanobacteria may play a pivotal role in this disease and that the mode of entry may be via ingestion, penetrating the coral via the gastrodermis, as well as through the skeletal-tissue interface.

Microstructural evolution and nanoscale crystallography in scleractinian coral spherulites.

One of the most important aspects in the research on reef-building corals is the process by which corals accrete biogenic calcium carbonate. This process leads to the formation of a mineral/organic composite and it is believed that the development of the nano- and microstructure of the mineral phase is highly sensitive to the growth conditions. Transmission electron microscopy (TEM) analysis of large-scale (10×30µm) focused ion beam (FIB) prepared lamellae was performed on adult and juvenile scleractinian coral skeleton specimens. This allowed for the investigation of the nano and microstructure and the crystallographic orientation of the aragonite mineral. We found the following microstructural evolution in the adult Porites lobata specimens: randomly oriented nanocrystals with high porosity, partly aligned nanocrystals with high porosity and areas of dense acicular crystals of several micrometers extension, the latter two areas are aligned close to the [001] direction (Pmcn space group). To the best of our knowledge, for the first time the observed microstructure could be directly correlated with the dark/bright bands characteristic of the diurnal growth cycle. We hypothesize that this mineral structure sequence and alignment in the adult specimen is linked to the photosynthetic diurnal cycle of the zooxanthellea regulating the oxygen levels and organic molecule transport to the calcifying medium. These observations reveal a strong control of crystal morphology by the organism and the correlation of the accretion process. No indication for a self-assembly of nanocrystalline units, i.e., a mesocrystal structure, on the micrometer scale could be found.

Bacterial consortium of Millepora dichotoma exhibiting unusual multifocal lesion event in the Gulf of Eilat, Red Sea.

Colonies of the hydrocoral Millepora dichotoma along the Gulf of Eilat are exhibiting unusual tissue lesions in the form of white spots. The emergence and rapid establishment of these multifocal tissue lesions was the first of its kind reported in this region. A characterization of this morphological anomaly revealed bleached tissues with a significant presence of bacteria in the tissue lesion area. To ascertain possible differences in microbial biota between the lesion area and non-affected tissues, we characterized the bacterial diversity in the two areas of these hydrocorals. Both culture-independent (molecular) and culture-dependent assays showed a shift in bacterial community structure between the healthy and affected tissues. Several 16S rRNA gene sequences retrieved from the affected tissues matched sequences of bacterial clones belonging to Alphaproteobacteria and Bacteroidetes members previously associated with various diseases in scleractinian corals.

A time-domain nuclear magnetic resonance study of Mediterranean scleractinian corals reveals skeletal-porosity sensitivity to environmental changes.

Mediterranean corals are a natural model for studying global warming, as the Mediterranean basin is expected to be one of the most affected regions and the increase in temperature is one of the greatest threats for coral survival. We have analyzed for the first time with time-domain nuclear magnetic resonance (TD-NMR) the porosity and pore-space structure, important aspects of coral skeletons, of two scleractinian corals, Balanophyllia europaea (zooxanthellate) and Leptopsammia pruvoti (nonzooxanthellate), taken from three different sites on the western Italian coast along a temperature gradient. Comparisons have been made with mercury intrusion porosimetry and scanning electron microscopy images. TD-NMR parameters are sensitive to changes in the pore structure of the two coral species. A parameter, related to the porosity, is larger for L. pruvoti than for B. europaea, confirming previous non-NMR results. Another parameter representing the fraction of the pore volume with pore sizes of less than 10-20 µm is inversely related, with a high degree of statistical significance, to the mass of the specimen and, for B. europaea, to the temperature of the growing site. This effect in the zooxanthellate species, which could reduce its resistance to mechanical stresses, may depend on an inhibition of the photosynthetic process at elevated temperatures and could have particular consequences in determining the effects of global warming on these species.

Visualization of sub-daily skeletal growth patterns in massive Porites corals grown in Sr-enriched seawater.

We performed high resolution marking experiments using seawater with elevated Sr concentration to investigate the timing and ultrastructure of skeletal deposition by massive Porites australiensis corals. Corals were cultured in seawater enriched with Sr during day-time only, night-time only or for one full-day. Cross sections of skeletal material were prepared and the Sr incorporated into the newly deposited skeleton analyzed by electron probe microanalysis. These regions of Sr incorporation were then correlated with skeletal ultrastructure. Massive Porites coral skeletons are composed of two types of microstructural elements - the "centers of calcification" and the surrounding fibrous structural region. Within the fibrous structural region, alternative patterns of etch-sensitive growth lines and an etch-resistant fibrous layer were observed. In the full-day samples, high-Sr bands extended across both growth lines and fibrous layers. In day-time samples, high-Sr regions corresponded to the fibrous layer, while in the night-time samples high-Sr regions were associated with an outermost growth line. These distinct growth patterns suggest a daily growth pattern associated with the fibrous region of massive P. australiensis corals, where a pair of narrow growth lines and a larger fibrous layer is seen as a daily growth region.

The Melithaeidae (Cnidaria: Octocorallia) of the Ryukyu Archipelago: molecular and morphological examinations.

The family Melithaeidae (Octocorallia: Alcyonacea) is distributed in the West Pacific, Indian Ocean and the Red Sea. They are most abundant in warmer waters but can also be found in temperate waters. At present six genera are assigned to this family (Melithaea, Mopsella, Clathraria, Acabaria, Wrightella and Asperaxis), however overlapping characteristics make this group's taxonomic identification difficult and their relationships unclear. There are only a few reports from the Ryukyu Archipelago in southern Japan of melithaeids and most other octocorals, despite the islands being an area of high octocoral diversity. To help resolve the taxonomic confusion in this family, samples from various Ryukyu Archipelago locations were collected and DNA sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome oxidase I (COI) were obtained. Additionally, SEM micrographs of the sclerites of specimens were taken to further confirm the molecular results. Three strongly supported clades were recovered from the COI and 28S rDNA analyses, corresponding to Melithaea, Acabaria, and Mopsella, and in most cases clades were clearly related with the sclerite shape reported for each genus. These results show clearly that molecular differences are present between the three genera, and also demonstrates the strong need of other molecular markers for resolving intra-generic phylogenies. Our results provide baseline data for future studies of this octocoral family, not only on taxonomy, but also with regards to their distribution in the Ryukyu Islands.

470-Million-year-old black corals from China.

Phosphatic (possibly secondarily phosphatised) remains of antipatharian coralla, previously unknown in the fossil record, occur abundantly in the early Ordovician Fenxiang Formation in the Hubei Province, southern China. Probably two species (and genera) are represented, which differ in spinosity of branches. The more spinose one, Sinopathes reptans, has its lateral spines bearing regular, longitudinally arranged costellae. The early Floian geological age of this finding, about 470 Ma, supports predictions on the timing of anthozoan phylogeny derived from the molecular phylogenetic evidence. Black corals (Antipatharia) are basal to the scleractinians in the Hexacorallia clade, being more derived than sea anemones and the Zoantharia. Based on calibration of the molecular clock with Mesozoic data, the first split of lineages within the scleractinian hexacorals was proposed to take place approximately 425 million years ago. This implies that the origin of Antipatharia should precede this date. They have not been known in the fossil record because of unmineralised skeleton composed primarily of laminar chitin complexed with a protein. Unlike all recent species, the encrusting basal part of the colony dominated in the Ordovician ones and only occasionally erect branches developed, rather chaotically ramified. This presumably plesiomorphic trait seems consistent with ancient geological age and suggests that some problematic fossils from the Late Cambrian may be their, even less-derived, relatives.

Fine structure analysis of black band disease (BBD) infected coral and coral exposed to the BBD toxins microcystin and sulfide.

Black band disease (BBD) of corals is a complex pathogenic polymicrobial mat community that lyses coral tissue as it migrates over an infected colony. Two known toxins are produced by BBD microorganisms - sulfide, produced by sulfate-reducing bacteria, and microcystin, produced by cyanobacteria. Experiments were carried out to determine the effects of exposing healthy coral fragments to variable concentrations of purified microcystin, sulfide at a concentration known to exist in BBD, and a combination of the two. Healthy fragments of the coral Montastraea annularis were placed into experimental chambers with known toxin/s for 18-22.5 h. Fine structural analysis using scanning electron microscopy (SEM) showed that toxin exposure resulted in thinning or removal of the coral epidermal layer coupled with degradation of the gastrodermis. These effects were exacerbated when both toxins were used in combination. Exposure to sulfide and the highest concentration of microcystin caused zooxanthellae to dissociate from the coral tissue and to form clusters on the coral surface. Examination of coral fragments infected with BBD was carried out for comparison. It was determined that the effects of exposure to sulfide and microcystin on coral fine structure were consistent, both quantitatively and qualitatively, with the effects of artificially induced and naturally occurring BBD on M. annularis.

Skeletal structure and progression of growth anomalies in Porites australiensis in Okinawa, Japan.

Growth anomalies (GAs), one of the diseases recently reported for scleractinian corals, are characterized by an abnormal skeletal structure and reduced zooxanthella density. The pathological characteristics of GAs were studied in colonies of Porites australiensis on a reef in Kayo, Okinawa, Japan. Corallites in the GA region lost the skeletal architecture characteristic of P. australiensis, and polyp density had decreased in the GAs due to enlargement of both calices and the coenosteum. The gross productivity of isolated GA samples was lower than in healthy samples and decreased to almost 0 within 11 d after isolation. However, when GA samples were brought into contact with healthy-looking samples from the same colony, they fused and both the GA and healthy regions grew. Healthy samples fused with GA samples grew more slowly than those fused with healthy samples. For in situ GAs surrounded by healthy tissue, tissue death usually started at the center of the GA, probably due to a deficiency in the translocated energy supply from the surrounding tissue. The total area of the GA region and the dead area increased at a rate of 5.3 ± 2.9 cm2 yr-1. These results suggest that GA regions are maintained by energy supplies from surrounding healthy tissues and that GAs may have a negative impact on host corals.

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

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 versus non-virulent bacteria.

Fine-structural analysis of black band disease-infected coral reveals boring cyanobacteria and novel bacteria.

Examination of coral fragments infected with black band disease (BBD) at the fine- and ultrastructural levels using scanning (SEM) and transmission electron microscopy (TEM) revealed novel features of the disease. SEM images of the skeleton from the host coral investigated (Montastraea annularis species complex) revealed extensive boring underneath the BBD mat, with cyanobacterial filaments present within some of the bore holes. Cyanobacteria were observed to penetrate into the overlying coral tissue from within the skeleton and were present throughout the mesoglea between tissue layers (coral epidermis and gastrodermis). A population of novel, as yet unidentified, small filamentous bacteria was found at the leading edge of the migrating band. This population increased in number within the band and was present within degrading coral epithelium, suggesting a role in disease etiology. In coral tissue in front of the leading edge of the band, cyanobacterial filaments were observed to be emerging from bundles of sloughed-off epidermal tissue. Degraded gastrodermis that contained actively dividing zooxanthellae was observed using both TEM and SEM. The BBD mat contained cyanobacterial filaments that were twisted, characteristic of negative-tactic responses. Some evidence of boring was found in apparently healthy control coral fragments; however, unlike in BBD-infected fragments, there were no associated cyanobacteria. These results suggest the coral skeleton as a possible source of pathogenic BBD cyanobacteria. Additionally, SEM revealed the presence of a potentially important group of small, filamentous BBD-associated bacteria yet to be identified.