Aposematic coloration does not deter corallivory by fish on the coral <Emphasis Type="Italic">Montastraea cavernosa</Emphasis>

Predation on corals by visual predators is a significant source of partial or total mortality on coral reefs, and corals have evolved strategies, including chemical defenses, to deter predation. One mechanism that organisms use to communicate the presence of chemical defenses is aposematic coloration, or the display of bright coloration as a warning to visual predators such as fish. Corals exhibit multiple colors, and it has been hypothesized that one role for this variability in coloration is as an aposematic warning of adverse palatability. Here, we test green and orange color morphs of the Caribbean coral Montastraea cavernosa for the presence of chemical defenses and whether their differences in coloration elicited different feeding responses. While M. cavernosa is chemically defended, there is no difference in feeding deterrence between color morphs; thus, the different color morphs of this coral species do not appear to represent an example of aposematic coloration.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

Molecular Characterization of Diazotrophic and Denitrifying Bacteria Associated with Mangrove Roots

An analysis of the molecular diversity of N₂ fixers and denitrifiers associated with mangrove roots was performed using terminal restriction length polymorphism (T-RFLP) of nifH (N₂ fixation) and nirS and nirK (denitrification), and the compositions and structures of these communities among three sites were compared. The number of operational taxonomic units (OTU) for nifH was higher than that for nirK or nirS at all three sites. Site 3, which had the highest organic matter and sand content in the rhizosphere sediment, as well as the lowest pore water oxygen concentration, had the highest nifH diversity. Principal component analysis of biogeochemical parameters identified soil texture, organic matter content, pore water oxygen concentration, and salinity as the main variables that differentiated the sites. Nonmetric multidimensional scaling (MDS) analyses of the T-RFLP data using the Bray-Curtis coefficient, group analyses, and pairwise comparisons between the sites clearly separated the OTU of site 3 from those of sites 1 and 2. For nirS, there were statistically significant differences in the composition of OTU among the sites, but the variability was less than for nifH. OTU defined on the basis of nirK were highly similar, and the three sites were not clearly separated on the basis of these sequences. The phylogenetic trees of nifH, nirK, and nirS showed that most of the cloned sequences were more similar to sequences from the rhizosphere isolates than to those from known strains or from other environments.     

Differences in Bacterial Community Structure in Two Color Morphs of the Hawaiian Reef Coral Montipora capitata

Corals harbor diverse bacterial associations that contribute to the health of the host. Using 16S rRNA pyrosequencing, we compared the bacterial communities of red and orange morphs of the Hawaiian coral Montipora capitata. Although both color morphs shared dominant bacterial genera, weighted and unweighted UniFrac analyses showed distinct bacterial communities. A single operational taxonomic unit (OTU), classified as Vibrio, represented the largest driver of differences between the color morphs. This OTU comprised 35.4% (±5.5%) of the orange morph bacterial community yet comprised 1.1% (±0.6%) of the red morph bacterial community. Cultivable bacteria from the two color morphs were also compared and tested for antibacterial activity. Cultured isolates represented 14 genera (7% of the total genera identified from sequencing data), and all but two cultured isolates had a matching OTU from the sequencing data. Half of the isolates tested (8 out of 16) displayed antibacterial activity against other cultured isolates but not against two known bacterial pathogens of M. capitata. The results from this study demonstrate that the specificity of coral-bacterial associations extends beyond the level of coral species. In addition, culture-dependent methods captured bacterial diversity that was representative of both rare and abundant members of the associated bacterial community, as characterized by culture-independent methods.     

Diversity of <Emphasis Type="Italic">nifH</Emphasis> gene amplified from plankton-community DNA in a shallow eutrophic lake (Lake Donghu,Wuhan, China)

Biological nitrogen fixation (BNF) is one of the major nitrogen inputs into the biosphere, and the nitrogenase iron protein (nifH) gene plays important roles in regulating the molecular nitrogen (N₂) fixation process. The nifH gene has also been extensively used to study the diversity and function of nitrogen-fixing microorganisms. In this study, we investigated the diversity of the nifH gene by culture-independent methods to analysis the planktonic nitrogen-fixing organisms in Lake Donghu, Wuhan, the largest urban lake in China. Results indicate that nifH gene sequences cloned from planktonic-community DNA showed high similarity to the uncultured cyanobacterial sequences deposited in the GenBank database. Phylogenetic analysis on the basis of the translated amino acid sequences further showed that most nifH clones were closely related to the reported cyanobacterial nifH gene sequences. Results also indicate that there are similar planktonic nitrogen-fixing organisms in the relatively independent areas of Lake Donghu, even though different regions showed a wide gradient in trophic status. These and other observations led us to believe that studies on nifH gene diversity and expression will increase our ability to understand the ecological function of target nitrogen-fixing groups in aquatic ecosystems.     

Bacterial Diversity Associated with Cinachyra cavernosa and Haliclona pigmentifera,Cohabiting Sponges in the Coral Reef Ecosystem of Gulf of Mannar,Southeast Coast of India

Sponges are abundant, diverse and functionally important organisms of coral reef ecosystems. Sponge-associated microorganisms have been receiving greater attention because of their significant contribution to sponge biomass, biogeochemical cycles and biotechnological potentials. However, our understanding of the sponge microbiome is limited to a few species of sponges from restricted geographical locations. Here, we report for the first time the bacterial diversity of two cohabiting sponges, viz. Cinachyra cavernosa and Haliclona pigmentifera, as well as that in the ambient water from the coral reef ecosystems of the Gulf of Mannar, located along the southeast coast of India. Two hundred and fifty two clones in the 16S rRNA gene library of these sponges were grouped into eight distinct phyla, of which four belonged to the core group that are associated only with sponges. Phylogenetic analysis of the core bacteria showed close affinity to other sponge-associated bacteria from different geographical locations. γ-Proteobacteria, Chloroflexi, Planctomycetes and Deferribacter were the core groups in C. cavernosa while β and δ-Proteobacteria performed this role in H. pigmentifera. We observed greater OTU diversity for C. cavernosa (H^ǀ 2.07) compared to H. pigmentifera (H^ǀ 1.97). UniFrac analysis confirmed the difference in bacterial diversity of the two sponge species and also between the sponges and the reef water (p<0.001). The results of our study restate the existence of a host driven force in shaping the sponge microbiome.     

Parrotfish abundance and selective corallivory on a Belizean coral reef

Parrotfish are important members of coral reef communities because they consume macroalgae that would otherwise outcompete reef-building corals for space. However, some Caribbean parrotfish species also feed directly on live corals, and thus have the potential to negatively impact coral fitness and survival. This study investigates selective grazing by parrotfish on particular coral species, differences in grazing incidence among reef habitats and intraspecific discrimination among colonies of several coral species. We also investigate spatial and temporal patterns of parrotfish species abundance across habitats on the Belize barrier reef, and examine correlations between parrotfish abundance and grazing intensity across reef habitats. We found that members of the Montastraea annularis species complex, major builders of Caribbean reefs, were preferred targets of parrotfish grazing across all reef habitats, while M. cavernosa, Agaricia agaricites, Diploria strigosa, Porites astreoides and Porites porites were not preferred; Siderastrea siderea was preferentially grazed only in the spur and groove habitats. Parrotfish grazing preferences varied across habitats; M. annularis was grazed most often in shallow habitats, whereas M. franksi was consumed more at depth. Although it was not possible to directly observe parrotfish grazing on corals, we did find a positive correlation between Sparisoma aurofrenatum abundance and M. franksi grazing incidence across habitats. Finally, when we compared our results to parrotfish abundances measured by a previous study, we found that Sparisoma viride and Sp. aurofrenatum, two species known to be corallivorous, had increased abundances between 1982 and 2004. In light of escalating threats on Caribbean reef corals, it would be important for future studies to evaluate the impact of parrotfish corallivory on coral survival.     

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.     

Molecular Analysis of Diazotroph Diversity in the Rhizosphere of the Smooth Cordgrass, Spartina alterniflora

N₂ fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, the nifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria (γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. The Spartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.     

Genetic Characterization of Cylindrospermopsis raciborskii (Cyanobacteria) Isolates from Diverse Geographic Origins Based on nifH and cpcBA-IGS Nucleotide Sequence Analysis

Isolates of the toxic, N₂-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.     

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.     

Diazotrophic bacteria associated with Hawaiian Montipora corals: Diversity and abundance in correlation with symbiotic dinoflagellates

The primary productivity of coral-dinoflagellate symbiosis is classically viewed as nitrogen (N)-limited. However, the recent discovery of N-fixing bacteria residing within coral tissues challenges this view. This study investigated the diversity and abundance of N-fixing bacteria associated with Hawaiian corals of the genus Montipora. Phylogenetic analysis of a partial sequence of the nitrogenase gene (nifH), revealed a diverse group of N-fixing bacteria in association with Montipora capitata and Montipora flabellata. These nifH sequences were closely affiliated with known taxa in α-, β-, γ-, and δ-proteobacteria, as well as cyanobacteria. Gamma-proteobacteria were the dominant bacterial class represented, and bacteria closely related to the genus Vibrio were the prevailing group within this class. Quantitative PCR was used to evaluate the abundance of the dominant class of N-fixing bacteria from different parts of four M. capitata colonies. The abundance of the dominant γ-proteobacteria was positively correlated (p = 0.001) with the abundance of symbiotic dinoflagellates, suggesting a physiological relationship between the two coral symbionts. The evidence obtained in this study suggests that N-limitation may not be a major regulator of the productivity of dinoflagellate symbionts of corals, but provides impetus for renewed investigation into N-cycling in this multi-partner symbiosis.     

Geographic differences in vertical connectivity in the Caribbean coral Montastraea cavernosa despite high levels of horizontal connectivity at shallow depths

The deep reef refugia hypothesis proposes that deep reefs can act as local recruitment sources for shallow reefs following disturbance. To test this hypothesis, nine polymorphic DNA microsatellite loci were developed and used to assess vertical connectivity in 583 coral colonies of the Caribbean depth‐generalist coral Montastraea cavernosa. Samples were collected from three depth zones (≤10, 15–20 and ≥25 m) at sites in Florida (within the Upper Keys, Lower Keys and Dry Tortugas), Bermuda, and the U.S. Virgin Islands. Migration rates were estimated to determine the probability of coral larval migration from shallow to deep and from deep to shallow. Finally, algal symbiont (Symbiodinium spp.) diversity and distribution were assessed in a subset of corals to test whether symbiont depth zonation might indicate limited vertical connectivity. Overall, analyses revealed significant genetic differentiation by depth in Florida, but not in Bermuda or the U.S. Virgin Islands, despite high levels of horizontal connectivity between these geographic locations at shallow depths. Within Florida, greater vertical connectivity was observed in the Dry Tortugas compared to the Lower or Upper Keys. However, at all sites, and regardless of the extent of vertical connectivity, migration occurred asymmetrically, with greater likelihood of migration from shallow to intermediate/deep habitats. Finally, most colonies hosted a single Symbiodinium type (C3), ruling out symbiont depth zonation of the dominant symbiont type as a structuring factor. Together, these findings suggest that the potential for shallow reefs to recover from deep‐water refugia in M. cavernosa is location‐specific, varying among and within geographic locations likely as a consequence of local hydrology.     

Microbial bioindicators of Stony Coral Tissue Loss Disease identified in corals and overlying waters using a rapid field-based sequencing approach

Stony Coral Tissue Loss Disease (SCTLD) is a devastating disease. Since 2014, it has spread along the entire Florida Reef Tract and into the greater Caribbean. It was first detected in the United States Virgin Islands in January 2019. To more quickly identify microbial bioindicators of disease, we developed a rapid pipeline for microbiome sequencing. Over a span of 10 days we collected, processed and sequenced coral and near-coral seawater microbiomes from diseased and apparently healthy Colpophyllia natans, Montastraea cavernosa, Meandrina meandrites and Orbicella franksi. Analysis of bacterial and archaeal 16S ribosomal RNA gene sequences revealed 25 bioindicator amplicon sequence variants (ASVs) enriched in diseased corals. These bioindicator ASVs were additionally recovered in near-coral seawater (<5 cm of coral surface), a potential reservoir for pathogens. Phylogenetic analysis of microbial bioindicators with sequences from the Coral Microbiome Database revealed that Vibrio, Arcobacter, Rhizobiaceae and Rhodobacteraceae sequences were related to disease-associated coral bacteria and lineages novel to corals. Additionally, four ASVs (Algicola, Cohaesibacter, Thalassobius and Vibrio) were matches to microbes previously associated with SCTLD that should be targets for future research. Overall, this work suggests that a rapid sequencing framework paired with specialized databases facilitates identification of microbial disease bioindicators.     

Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines and Thailand

Most community molecular studies of thermophilic cyanobacterial mats to date have focused on Synechococcus occurring at temperatures of ~50–65°C. These reveal that molecular diversity exceeds that indicated by morphology, and that phylogeographic lineages exist. The moderately thermophilic and generally filamentous cyanobacterial mat communities occurring at lower temperatures have not previously been investigated at the community molecular level. Here we report community diversity in mats of 42–53°C recovered from previously unstudied geothermal locations. Separation of 16S rRNA gene-defined genotypes from community DNA was achieved by DGGE. Genotypic diversity was greater than morphotype diversity in all mats sampled, although genotypes generally corresponded to observed morphotypes. Thirty-six sequences were recovered from DGGE bands. Phylogenetic analyses revealed these to form novel thermophilic lineages distinct from their mesophilic counterparts, within Calothrix, Cyanothece, Fischerella, Phormidium, Pleurocapsa, Oscillatoria and Synechococcus. Where filamentous cyanobacterial sequences belonging to the same genus were recovered from the same site, these were generally closely affiliated. Location-specific sequences were observed for some genotypes recovered from geochemically similar yet spatially separated sites, thus providing evidence for phylogeographic lineages that evolve in isolation. Other genotypes were more closely affiliated to geographically remote counterparts from similar habitats suggesting that adaptation to certain niches is also important.     

Epilithic Cyanobacterial Communities of a Marine Tropical Beach Rock (Heron Island, Great Barrier Reef): Diversity and Diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification “microbialite” origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

N2 fixation,and the relative contribution of fixed N,in corals from Curaçao and Hawaii

Coral Reefs - Corals from Hawaii (Montipora capitata) and the Caribbean (brown and orange morphs of Montastraea cavernosa) have previously been shown to harbor symbiotic bacteria capable of fixing...