Microbially facilitated nitrogen cycling in tropical corals

Tropical scleractinian corals support a diverse assemblage of microbial symbionts. This ‘microbiome’ possesses the requisite functional diversity to conduct a range of nitrogen (N) transformations including denitrification, nitrification, nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). Very little direct evidence has been presented to date verifying that these processes are active within tropical corals. Here we use a combination of stable isotope techniques, nutrient uptake calculations and captured metagenomics to quantify rates of nitrogen cycling processes in a selection of tropical scleractinian corals. Denitrification activity was detected in all species, albeit with very low rates, signifying limited importance in holobiont N removal. Relatively greater nitrogen fixation activity confirms that corals are net N importers to reef systems. Low net nitrification activity suggests limited N regeneration capacity; however substantial gross nitrification activity may be concealed through nitrate consumption. Based on nrfA gene abundance and measured inorganic N fluxes, we calculated significant DNRA activity in the studied corals, which has important implications for coral reef N cycling and warrants more targeted investigation. Through the quantification and characterisation of all relevant N-cycling processes, this study provides clarity on the subject of tropical coral-associated biogeochemical N-cycling.Subject terms: Stable isotope analysis, Biogeochemistry, Biogeochemistry, Biogeochemistry, Microbial ecology     

Evolutionary dispersal drives the latitudinal diversity gradient of stony corals

The diversity of stony corals displays one of the most exemplary latitudinal gradients on the planet, yet the evolutionary dynamics that produced this pattern remains unclear. Using both paleontological and distributional data, we compare the origination, extinction and immigration levels between low and high latitudes since the earliest proliferation of the group during the mid‐Triassic. Altogether, first and last occurrence localities in the fossil record do not support a positive preference towards either latitudinal bin. Nonetheless, considering past and present scleractinian fauna, the process of extinction has been apparently more pronounced at higher latitudes based on face values and correlation coefficients. Far above these differences, immigration of extant taxa has been substantially higher towards the tropics than to temperate regions. While the net dispersal toward low latitudes persists in all temporal intervals, the gradient of diversity was largely built up during the Cenozoic Era and only becomes significantly steep from the Neogene Period onwards. This dynamic supports the ‘into the tropical museum’ model, which suggests that tropics have historically acted as a center of accumulation for marine biodiversity.     

Characterization of black band disease in Red Sea stony corals

Microbial communities associated with black band disease (BBD) in massive stony corals from the Northern Red Sea (Eilat) were examined for the first time using molecular tools and microscopy. A high microbial diversity was revealed in the affected tissue in comparison with the healthy area of the same colony. Microscopy revealed the penetration of cyanobacteria into the coral mesoglea and adjacent tissues. Cyanobacterial sequences from Red Sea BBD-affected corals formed a cluster with sequences previously identified from black band and red band diseased corals from the Indo-Pacific and Caribbean. In addition, 11 sequences belonging to the genus Vibrio were retrieved. This group was previously documented as pathogenic to corals. Sulfate-reducing bacteria, a group known to be associated with BBD and produce toxic sulfide, were studied using specific primers for the amplification of the dissimilatory sulfite reductase gene (dsrA). This technique facilitated and improved the resolution of the study of diversity of this group. All the sequences obtained were closely related to sequences of the genus Desulfovibrio and 46% showed high homology to Desulfovibrio desulfuricans. The complex nature of BBD and the lack of success in isolating a single causative agent suggest that BBD may be considered a polymicrobial disease.     

Bacteria associated with the rapid tissue necrosis of stony corals

The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.     

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.     

Molecular and morphological supertree of stony corals (Anthozoa: Scleractinia) using matrix representation parsimony

The supertree algorithm matrix representation with parsimony was used to combine existing hypotheses of coral relationships and provide the most comprehensive species-level estimate of scleractinian phylogeny, comprised of 353 species (27% of extant species), 141 genera (63%) and 23 families (92%) from all seven suborders. The resulting supertree offers a guide for future studies in coral systematics by highlighting regions of concordance and conflict in existing source phylogenies. It should also prove useful in formal comparative studies of character evolution. Phylogenetic effort within Scleractinia has been taxonomically uneven, with a third of studies focussing on the Acroporidae or its most diverse genera. Sampling has also been geographically non-uniform, as tropical, reef-forming taxa have been considered twice as often as non-reef species. The supertree indicated that source trees concur on numerous aspects of coral relationships, such as the division between robust versus complex corals and the distant relationship between families in Archaeocoeniina. The supertree also supported the existence of a large, taxonomically diverse and monophyletic group of corals with many Atlantic representatives having exsert corallites. Another large, unanticipated clade consisted entirely of solitary deep-water species from three families. Important areas of ambiguity include the relationship of Astrocoeniidae to Pocilloporidae and the relative positions of several, mostly deep-water genera of Caryophylliidae. Conservative grafting of species at the base of congeneric groups with uncontroversial monophyletic status resulted in a more comprehensive, though less resolved tree of 1016 taxa.     

Space partitioning by stony corals soft corals and benthic algae on the coral reefs of the northern Gulf of Eilat (Red Sea)

The major faunistic and floristic components occupying space on the coral reefs of the northern Gulf of Eilat (Red Sea) are stony corals, soft corals and benthic algae. The percent living coverage of the three components and the relative abundance of the different species of each component were studied by line transects, on the reef flats and the upper forereef zones of nine localities. A wider and higher range of living coverage values of stony corals were recorded at the upper fore-reef zones (18.30–49.09%) compared with the reef flats (5.50–31.66%) at the different stations. The most abundant stony corals on the reef flats areCyphastrea microphthalma, Stylophora pistillata, Favia favus, Porites lutea, Platygyra lamellina and the hydrozoanMillepora dichotoma. The fire coralM. dichotoma dominates the upper fore-reef zone in most of the stations. The average percent living coverage of soft corals on the reef flats ranged between 0.20 and 17.06%, and on the upper fore-reef zones between 1.68 and 15.13%. Seventy percent of the total living coverage of the soft coral community is contributed by 2 to 3 species. They tend to form large monospecific carpets, such as those composed ofSinularia sp.,Sarcophyton glaucum andLobophytum pauciflorum. The common benthic algae on the coral reef studied occur as turfs or macroscopic noncalcareous algae. They play a significant role in occupying space, especially on the reef flats. The most abundant algae recorded in all localities are the turfsSphacelaria tribuloides, Jania sp. and the macroscopic non-calcareous algaeTurbinaria elatensis andColpomenia sinuosa. Comparison between reef flats and upper fore-reef zones, in terms of average living cover of stony corals, shows that the variation among the reef flats is grater than the variation among the upper fore-reef zones. However, there is no significant variation in the average living coverage of soft corals between these two zones. Annual living-coverage values of algae on the reef flats are significantly higher than those of the upper fore-reef zones. Extremely low tides occurring periodically but unpredictably at Eilat cause mass mortality of the benthic communities on the reef flats reopening new spaces for settlement. The coexistence of stony corals, soft corals and algae on the reef ecosystem is due to different biological properties of each component. Opportunistic life histories of certain stony corals and most algae enable quick colonization of newly opened spaces. Lack of predators, high tolerance against abiotic factors and ability to form large aggregates of colonies are suggested as possible factors supporting the existence of soft corals in shallow water. Biological factors such as competition, predation and grazing pressure play an increasingly important role in controlling space utilization by the components studied with the advancement of succession.     

Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production

Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these "defective" metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (~8-9 genes), addition (~6-7 genes), up- and downexpression (~6-7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in "core" metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.     

Assessment of nitrogen flows into the Cuban landscape

The alteration of the nitrogen (N) cycle by human activities is widespread and has often resulted in increased flows of nitrogen to the marine environment. In this paper we have attempted to know the changes of N fluxes in Cuba by quantifying the N inputs to the landscape from (1) fertilizer applications, (2) atmospheric deposition, (3) biological nitrogen fixation and (4) net import of food and feeds. N-inputs to the country progressively increased until the end of the 20th century, reaching a peak during the 80s when low cost fertilizer imported from the former Soviet Union led to heavy rates of application. This rapid growth represented more than a 5-fold increase with respect to pristine values; higher than the two-fold global increase of anthropogenic N reported by Vitousek et al. (1997 Human alteration of the global nitrogen cycle: sources and consequences. Ecol. Appl. 7:737–750). Inorganic fertilizer was the largest single source of reactive N, followed by atmospheric deposition, biological fixation, and net imports of foods and feedstocks. Nitrogen inputs peaked in 1987 and data expressed on an area basis show that N flux to the Cuban landscape, in the 80s, was one of the highest reported in the literature. During the 90s, there was a dramatic drop in nitrogen inputs mainly associated to a decrease in the use of inorganic fertilizer. Other factors reducing nutrient inflows to Cuba, during the same period, were imports of foodstuff and livestock feeds, a decrease of nitrogen oxide emissions, and a decrease in the sugar cane crop area. Using an empirical relationship (Howarth et al. 1996 Regional nitrogen budgets and riverine N & O fluxes for the drainages to the North Atlantic Ocean: Natural and human influences. Biogeochemistry 35:75–139) we present a very preliminary estimate of N-inputs to coastal waters and discuss the consequences of these changes on the coastal zone.     

Constructal pattern formation in stony corals, bacterial colonies and plant roots under different hydrodynamics conditions

This paper explores a new application of the constructal theory, namely in describing and predicting the formation of dissimilar patterns inside elements of the same species under different hydrodynamics conditions. Our study proposes an explanation for the differences found in morphology of stony corals, bacterial colonies and plant roots. It specially provides an answer to the following question: have their shapes (architecture) been developed by chance, or do they represent the optimum structure serving their ultimate purpose? We show that in order to persist in time, these systems must evolve in such a way that an easy access to nutrients is ensured: their shapes develop in such a way as to minimize the time to reach the nutrient source. Moreover, it is also shown that it is the combination of a dispersive (diffusive) and a convective mechanism that allows for the maximization of nutrient transfer through use of the best of these mechanisms at a specific time. In the light of this outcome, it is straightforward to conclude why the existence of an optimal architecture makes sense: it is because there is an overriding natural tendency and because the system has the freedom to morph its shape in the search for an optimal attainment of this goal within a set of constraints imposed by the situation.     

Detection and quantification of microorganisms in anaerobic bioreactors

The presence of sulfate in anaerobic reactors can trigger competitive and syntrophic interactions between various groups of microorganisms, such as sulfate reducers, methanogens and acetogens. In order to steer the reactor process in the direction of sulfidogenesis or methanogenesis, it is essential to get insight into the population dynamics of these groups of microorganisms upon changes in the reactor operating conditions. Several methods exist to characterize and quantify the microbial sludge composition. Combining classical microbiological and modern molecular-based sludge characterization methods has proven to be a powerful approach to study the microbial composition of the anaerobic sludge.     

Transplantation and immunoincompatibility reactions among reef-building corals

Intracolony or syngeneic transplants of pieces of living coral, either occurring naturally on reefs or produced experimentally, were consistently compatible. In contrast, intercolony allografts performed extensively inAcropora formosa and inPorites andrewsii were incompatible, as were allografts occurring naturally in additional species observed in reef communities. Diverse interspecific combinations of coral xenografts generally displayed greater incompatibilities. Four levels of immuno-reactivity, with manifestations ranging from mild to severe, were distinguishable as follows: a) contact avoidance reactions, b) allogeneic contact incompatibility, c) chronic xenogeneic incompatibility, and d) acute interspecific aggression.Allogeneic incompatibility, most studied inA. formosa, was characterized by soft tissue contact avoidance wherever possible; absence of tissue death despite extensive enforced contact (18–20 days); and late interfacial cementation terminating allogeneic soft tissue contact. The properties ofchronic xenogeneic incompatibility as found in diverse species combinations, are slow onset (>7 days), bidirectional or unidirectional killing in contact zones, and short-range or localized effectiveness.Acute interspecific aggression initiated byFungia fungites as a dominant “aggressor ” is distinguished by early occurrence (2–7 days) and unidirectional contact killing. The presence or absence of these acute xenogeneic reactions sharply discriminated between species within each of the generaAcropora, Pocillopora, andPorites. No hierarchy of aggressive interactions was evident at the generic level.     

Percent recent mortality (PRM) of stony corals as an ecological indicator of coral reef condition

The reef communities of the Florida coral reef tract play a major role in supporting the regional economy but are threatened by increased exploitation and environmental factors. Coral reef ecosystem services are vital to the economy of SE Florida where revenue and jobs depend on the status of reef resources. Here, we used an extensive, reef monitoring database collected by the Florida Reef Resilience (FRRP, 2003–2011) and the Atlantic and Gulf Rapid Reef Assessment (AGRRA, 1997–2011) programs to evaluate percent recent mortality (PRM) as a robust ecological indicator of coral colony and coral reef status. PRM, the proportion of a coral colony that has experienced recent tissue mortality so that corallite structures in the non-living parts of the coral are still intact and identifiable to species, can be attributed to disturbances taking place within days to a few months preceding the surveys.Based on data from >50,000 colonies from 11 coral species and nearly 1400 sites, we propose a benchmark level of <1% PRM and <5% prevalence of partial mortality for Florida reefs during periods of background, low-stress environmental conditions. PRM levels >1.0% and prevalence levels >5% can be used as early warning indicators of degrading conditions. Average PRM values >2% are indicative of increasingly stressful conditions as those experienced during temperature anomalies and major hurricanes. Finally, PRM values considerably >2% are reflective of significantly stressful conditions and warning signals of potential major coral mortality as evidenced by mean PRM levels of >10% recorded in Florida as a consequence of the 2010 extreme cold-water event. PRM and prevalence values from Florida reefs compared favorably with those recorded in the Caribbean and the Mesoamerican region where a benchmark of 2% for background levels of PRM under low-level, chronic stress was proposed. The status of this indicator can be easily communicated to stakeholders and will benefit managers by providing: (1) a baseline to assess the status of coral populations; and (2) early-warning indicators of unfavorable conditions that may trigger management actions such as temporary closures or the establishment of more permanent protection such as MPAs.     

Interactions among nitrogen fixation and soil phosphorus acquisition strategies in lowland tropical rain forests

Paradoxically, symbiotic dinitrogen (N₂) fixers are abundant in nitrogen (N)‐rich, phosphorus (P)‐poor lowland tropical rain forests. One hypothesis to explain this pattern states that N₂ fixers have an advantage in acquiring soil P by producing more N‐rich enzymes (phosphatases) that mineralise organic P than non‐N₂ fixers. We assessed soil and root phosphatase activity between fixers and non‐fixers in two lowland tropical rain forest sites, but also addressed the hypothesis that arbuscular mycorrhizal (AM) colonisation (another P acquisition strategy) is greater on fixers than non‐fixers. Root phosphatase activity and AM colonisation were higher for fixers than non‐fixers, and strong correlations between AM colonisation and N₂ fixation at both sites suggest that the N–P interactions mediated by fixers may generally apply across tropical forests. We suggest that phosphatase enzymes and AM fungi enhance the capacity of N₂ fixers to acquire soil P, thus contributing to their high abundance in tropical forests.     

Decline in symbiont densities of tropical and subtropical scleractinian corals under ocean acidification

Coral Reefs - Ocean acidification changes the carbonate chemistry of seawater in a manner that reduces the biomineralisation rate of reef-building corals. Other effects of acidification on coral...     

Effects of fermented soybean meal on carbon and nitrogen metabolisms in large intestine of piglets

Fermented soybean meal (FSM), which has lower anti-nutritional factors and higher active enzyme, probiotic and oligosaccharide contents than its unfermented form, has been reported to improve the feeding value of soybean meal, and hence, the growth performance of piglets. However, whether FSM can affect the bacterial and metabolites in the large intestine of piglets remains unknown. This study supplemented wet-FSM (WFSM) or dry-FSM (DFSM) (5% dry matter basis) in the diet of piglets and investigated its effects on carbon and nitrogen metabolism in the piglets’ large intestines. A total of 75 41-day-old Duroc×Landrace×Yorkshire piglets with an initial BW of 13.14±0.22 kg were used in a 4-week feeding trial. Our results showed that the average daily gain of piglets in the WFSM and DFSM groups increased by 27.08% and 14.58% and that the feed conversion ratio improved by 18.18% and 7.27%, respectively, compared with the control group. Data from the prediction gene function of Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) based on 16S ribosomal RNA (rRNA) sequencing showed that carbohydrate metabolism function families in the WFSM and DFSM groups increased by 3.46% and 2.68% and that the amino acid metabolism function families decreased by 1.74% and 0.82%, respectively, compared with the control group. These results were consistent with those of other metabolism studies, which showed that dietary supplementation with WFSM and DFSM increased the level of carbohydrate-related metabolites (e.g. 4-aminobutanoate, 5-aminopentanoate, lactic acid, mannitol, threitol and β-alanine) and decreased the levels of those related to protein catabolism (e.g. 1,3-diaminopropane, creatine, glycine and inosine). In conclusion, supplementation with the two forms of FSM improved growth performance, increased metabolites of carbohydrate and reduced metabolites of protein in the large intestine of piglets, and WFSM exhibited a stronger effect than DFSM.