Nachshon Siboni

Global distribution and diversity of coral‐associated Archaea and their possible role in the coral holobiont nitrogen cycle

Diversity, distribution and genetic comparison of Archaea associated with the surface mucus of corals from three genera, namely Acanthastrea sp., Favia sp. and Fungia sp., from the Gulf of Eilat, Israel and from Heron Island, Australia were studied. Sequencing of the 16S rRNA gene of the coral-associated Archaea revealed dominance of Crenarchaeota (79%, on average). In this phylum, 87% of the sequences were similar (>or= 97%) to the Thermoprotei, with 76% of these being similar (>or= 97%) to the ammonium oxidizer, Nitrosopumilus maritimus. Most of the coral-associated euryarchaeotal sequences (69%) were related to marine group II, while other euryarchaeotal clades were found to be related to anaerobic methanotrophs (8%), anaerobic nitrate reducers (i.e. denitrification, 15%) and marine group III (8%). Most of the crenarchaeotal and euryarchaeotal coral-associated 16S rRNA gene sequences from Heron Island (61%) and from the Gulf of Eilat (71%) were closely related (>or= 97%) to sequences previously derived from corals from the Virgin Islands. Analysis of archaeal amoA sequences obtained from the fungiid coral, Fungia granulosa, divided into three clades, all related to archaeal sequences previously obtained from the marine environment. These sequences were distantly related to amoA sequences previously found in association with other coral species. Preliminary experiments suggest that there is active oxidation of ammonia to nitrite in the mucus of F. granulosa. Thus, coral-associated Archaea may contribute to nitrogen recycling in the holobiont, presumably by acting as a nutritional sink for excess ammonium trapped in the mucus layer, through nitrification and denitrification processes.

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

ABSTRACT To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.

Vibrio sp. as a potentially important member of the Black Band Disease (BBD) consortium in Favia sp. corals

Black Band Disease (BBD) is a well-described disease plaguing corals worldwide. It has been established that ecological and environmental stress factors contribute to the appearance and progression of the disease, believed to be caused by a diverse microbial consortium. We have identified and characterized Vibrio sp. associated with BBD in Eilat reef corals using both culture-dependent and -independent methods. Direct sampling using 16S rRNA gene clone libraries showed seasonal dynamics in the diversity of BBD-associated Vibrios. In the two sampling periods, BBD-associated Vibrio clones showed similarities to different groups: October samples were similar to known pathogens, while December samples were similar to general aquatic Vibrio sp. Cultured bacterial isolates of Vibrio sp. were highly homologous (>or=99%) to previously documented BBD-associated bacteria from the Caribbean, Bahamas and Red Seas, and were similar to several known coral pathogens, such as Vibrio coralliilyticus. The proteolytic activity of Vibrio sp., as measured using casein- and azocasein-based assays, directly correlated with temperature elevation and peaked at 26-28 degrees C, with the microorganisms producing more proteases per bacterial cell or increasing the rate of proteolytic activity of the same proteases (potentially metalloproteases). This activity may promote coral tissue necrosis and aid in ensuing progression of the coral BBD.

Pseudoscillatoria coralii gen. nov., sp. nov., a cyanobacterium associated with coral black band disease (BBD).

Black band disease (BBD) is a widespread coral disease which mainly infects massive framework-building corals. BBD is believed to be caused by a consortium of microorganisms and may not result from the actions of a primary pathogen. The BBD microbial community is dominated, in terms of biomass, by filamentous cyanobacteria. Here we describe a cyanobacterial strain, designated BgP10_4S(T), cultured from a BBD-affected Favia sp. 25 degreesoal from the northern Red Sea (Gulf of Eilat, Israel). This dark-green pigmented cyanobacterium showed optimal growth at salinities of 5.0 to 5.5% (w/v), pH of 7 to 8 and cultivation temperatures of 25 0C. Morphological examination revealed cylindrical, unbranched trichomes with tapering and blunt cells at the ends which leave a thin mucilaginous trail as they glide. No sheath was evident under these conditions. Inclusion bodies and straight thylakoids were clearly discerned by transmission electron microscopy. Pigment analysis revealed absorption spectra for phycocyanin, carotenoid and chlorophyll a. The sequence of the 16S rRNA gene in this cyanobac(t)erium isolate showed high similarity (99%) to cyanobacterial sequences retrieved from BBD-affected corals from different geographical sites (i.e. the Caribbean Sea, Palau and the Red Sea). The BgP10_4ST strain is observed to be a persisten(t) component of the BBD mat of Faviid corals and may thus be an important agent in the disease etiology. On the basis (of its morphological, physiological and phylogenetic distinctiveness, strain BgP10_4ST represents a novel genus and species of Subsection III (formerly Oscillatoriales), for which the name Pseudoscillatoria coralii gen. nov., sp. nov. is proposed.

Increased seawater temperature increases the abundance and alters the structure of natural Vibrio populations associated with the coral Pocillopora damicornis.

Rising seawater temperature associated with global climate change is a significant threat to coral health and is linked to increasing coral disease and pathogen-related bleaching events. We performed heat stress experiments with the coral Pocillopora damicornis, where temperature was increased to 31°C, consistent with the 2–3°C predicted increase in summer sea surface maxima. 16S rRNA amplicon sequencing revealed a large shift in the composition of the bacterial community at 31°C, with a notable increase in Vibrio, including known coral pathogens. To investigate the dynamics of the naturally occurring Vibrio community, we performed quantitative PCR targeting (i) the whole Vibrio community and (ii) the coral pathogen Vibrio coralliilyticus. At 31°C, Vibrio abundance increased by 2–3 orders of magnitude and V. coralliilyticus abundance increased by four orders of magnitude. Using a Vibrio-specific amplicon sequencing assay, we further demonstrated that the community composition shifted dramatically as a consequence of heat stress, with significant increases in the relative abundance of known coral pathogens. Our findings provide quantitative evidence that the abundance of potential coral pathogens increases within natural communities of coral-associated microbes as a consequence of rising seawater temperature and highlight the potential negative impacts of anthropogenic climate change on coral reef ecosystems.

A new Thraustochytrid, strain Fng1, isolated from the surface mucus of the hermatypic coral Fungia granulosa

Recent evidence suggests that there is a dynamic microbial biota living on the surface and in the mucus layer of many hermatypic coral species that plays an essential role in coral well-being. Most of the studies published to date emphasize the importance of prokaryotic communities associated with the coral mucus in coral health and disease. In this study, we report the presence of a protist (Fng1) in the mucus of the hermatypic coral Fungia granulosa from the Gulf of Eilat. This protist was identified morphologically and molecularly as belonging to the family Thraustochytridae (phylum Stramenopile, order Labyrinthulida), a group of heterotrophs widely distributed in the marine environment. Morphological examination of this strain revealed a nonmotile organism c. 35 mum in diameter, which is able to thrive on carbon-deprived media, and whose growth and morphology are inoculum dependent. Its fatty acid production profile revealed an array of polyunsaturated fatty acids. A similar protist was also isolated from the mucus of the coral Favia sp. In light of these findings, its possible contribution to the coral holobiont is discussed.

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.

Stramenopile Microorganisms Associated with the Massive Coral Favia sp.

ABSTRACT. The surfaces of massive corals of the genus Favia from Eilat, Red Sea, and from Heron Island, Great Barrier Reef, are covered by a layer of eukaryotic microorganisms. These microorganisms are embedded in the coral mucus and tissue. In the Gulf of Eilat, the prevalence of corals covered by patches of eukaryotic microorganisms was positively correlated with a decrease in water temperatures (from 25–28 °C in the summer to 20–23 °C in winter). Comparisons carried out using transmission and scanning electron microscopy showed morphological similarities between the microorganisms from the two geographically distant reefs. The microorganisms found on and in the tissues were approximately 5–15 μm in diameter, surrounded by scales in their cell wall, contained a nucleus, and included unique auto‐florescent coccoid bodies of approximately 1 μm. Such morphological characters suggested that these microorganisms are stramenopile protists and in particular thraustochytrids. Molecular analysis, carried out using specific primers for stramenopile 18S rRNA genes, revealed that 90% (111/123) of the clones in the gene libraries were from the Thraustochytriidae. The dominant genera in this family were Aplanochytrium sp., Thraustochytrium sp., and Labyrinthuloides sp. Ten stramenopile strains were isolated and cultured from the corals. Some strains showed ≥97% similarity to clones derived from libraries of mucus‐associated microorganisms retrieved directly from these corals. Fatty acid characterization of one of the prevalent strains revealed a high percentage of polyunsaturated fatty acids, including omega‐3. The possible association of these stramenopiles in the coral holobiont appeared to be a positive one.

Using Bacterial Extract along with Differential Gene Expression in Acropora millepora Larvae to Decouple the Processes of Attachment and Metamorphosis

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0–2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05) in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05) following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that utilized cues, such as crustose coralline algae, biofilms or with GLW-amide neuropeptides that stimulate the entire onset of larval metamorphosis and attachment.

Geographic Specific Coral-Associated Ammonia-Oxidizing Archaea in the Northern Gulf of Eilat (Red Sea)

Coral holobionts are densely populated with microorganisms that are essential for their well-being. Here we compared the diversity of the archaeal ammonia monooxygenase alpha subunit (amoA) gene from three coral genera, Acanthastrea sp., Favia sp., and Fungia granulosa, from the Gulf of Eilat, Red Sea. At 99% similarity, archaeal amoA from the three coral genera shared 71% of their cloned sequences, while the Favia and Acanthastrea presented a few genus-specific clones. In addition, the sequences retrieved in our samples displayed lower similarity to amoA sequences previously found in association with other coral species from different geographic regions. This finding suggests that the populations of ammonia-oxidizing archaea are less host-specific and more geographically dependent.