In Situ Quantification of Biological N2 Production Using Naturally Occurring 15N15N.

We describe an approach for determining biological N2 production in soils based on the proportions of naturally occurring 15N15N in N2. Laboratory incubation experiments reveal that biological N2 production, whether by denitrification or anaerobic ammonia oxidation, yields proportions of 15N15N in N2 that are within 1‰ of that predicted for a random distribution of 15N and 14N atoms. This relatively invariant isotopic signature contrasts with that of the atmosphere, which has 15N15N proportions in excess of the random distribution by 19.1 ± 0.1‰. Depth profiles of gases in agricultural soils from the Kellogg Biological Station Long-Term Ecological Research site show biological N2 accumulation that accounts for up to 1.6% of the soil N2. One-dimensional reaction-diffusion modeling of these soil profiles suggests that subsurface N2 pulses leading to surface emission rates as low as 0.3 mmol N2 m-2 d-1 can be detected with current analytical precision, decoupled from N2O production.

Nitrous oxide from chemodenitrification: A possible missing link in the Proterozoic greenhouse and the evolution of aerobic respiration.

The potent greenhouse gas nitrous oxide (N2 O) may have been an important constituent of Earth's atmosphere during Proterozoic (~2.5-0.5 Ga). Here, we tested the hypothesis that chemodenitrification, the rapid reduction of nitric oxide by ferrous iron, would have enhanced the flux of N2 O from ferruginous Proterozoic seas. We empirically derived a rate law, d N 2 O d t = 7.2 × 10 - 5 [ Fe 2 + ] 0.3 [ NO ] 1 , and measured an isotopic site preference of +16‰ for the reaction. Using this empirical rate law, and integrating across an oceanwide oxycline, we found that low nM NO and µM-low mM Fe2+ concentrations could have sustained a sea-air flux of 100-200 Tg N2 O-N year-1 , if N2 fixation rates were near-modern and all fixed N2 was emitted as N2 O. A 1D photochemical model was used to obtain steady-state atmospheric N2 O concentrations as a function of sea-air N2 O flux across the wide range of possible pO2 values (0.001-1 PAL). At 100-200 Tg N2 O-N year-1 and >0.1 PAL O2 , this model yielded low-ppmv N2 O, which would produce several degrees of greenhouse warming at 1.6 ppmv CH4 and 320 ppmv CO2 . These results suggest that enhanced N2 O production in ferruginous seawater via a previously unconsidered chemodenitrification pathway may have helped to fill a Proterozoic "greenhouse gap," reconciling an ice-free Mesoproterozoic Earth with a less luminous early Sun. A particularly notable result was that high N2 O fluxes at intermediate O2 concentrations (0.01-0.1 PAL) would have enhanced ozone screening of solar UV radiation. Due to rapid photolysis in the absence of an ozone shield, N2 O is unlikely to have been an important greenhouse gas if Mesoproterozoic O2 was 0.001 PAL. At low O2 , N2 O might have played a more important role as life's primary terminal electron acceptor during the transition from an anoxic to oxic surface Earth, and correspondingly, from anaerobic to aerobic metabolisms.

Extreme enrichment in atmospheric 15N15N.

Molecular nitrogen (N2) comprises three-quarters of Earth's atmosphere and significant portions of other planetary atmospheres. We report a 19 per mil (‰) excess of 15N15N in air relative to a random distribution of nitrogen isotopes, an enrichment that is 10 times larger than what isotopic equilibration in the atmosphere allows. Biological experiments show that the main sources and sinks of N2 yield much smaller proportions of 15N15N in N2. Electrical discharge experiments, however, establish 15N15N excesses of up to +23‰. We argue that 15N15N accumulates in the atmosphere because of gas-phase chemistry in the thermosphere (>100 km altitude) on time scales comparable to those of biological cycling. The atmospheric 15N15N excess therefore reflects a planetary-scale balance of biogeochemical and atmospheric nitrogen chemistry, one that may also exist on other planets.

Site-specific variation in gene expression from Symbiodinium spp. associated with offshore and inshore Porites astreoides in the lower Florida Keys is lost with bleaching and disease stress.

Scleractinian coral are experiencing unprecedented rates of mortality due to increases in sea surface temperatures in response to global climate change. Some coral species however, survive high temperature events due to a reduced susceptibility to bleaching. We investigated the relationship between bleaching susceptibility and expression of five metabolically related genes of Symbiodinium spp. from the coral Porites astreoides originating from an inshore and offshore reef in the Florida Keys. The acclimatization potential of Symbiodinium spp. to changing temperature regimes was also measured via a two-year reciprocal transplant between the sites. Offshore coral fragments displayed significantly higher expression in Symbiodinium spp. genes PCNA, SCP2, G3PDH, PCP and psaE than their inshore counterparts (p<0.05), a pattern consistent with increased bleaching susceptibility in offshore corals. Additionally, gene expression patterns in Symbiodinium spp. from site of origin were conserved throughout the two-year reciprocal transplant, indicating acclimatization did not occur within this multi-season time frame. Further, laboratory experiments were used to investigate the influence of acute high temperature (32°C for eight hours) and disease (lipopolysaccharide of Serratia marcescens) on the five metabolically related symbiont genes from the same offshore and inshore P. astreoides fragments. Gene expression did not differ between reef fragments, or as a consequence of acute exposure to heat or heat and disease, contrasting to results found in the field. Gene expression reported here indicates functional variation in populations of Symbiodinium spp. associated with P. astreoides in the Florida Keys, and is likely a result of localized adaptation. However, gene expression patterns observed in the lab imply that functional variation in zooxanthellae observed under conditions of chronic moderate stress is lost under the acute extreme conditions studied here.

Genetically divergent Symbiodinium sp. display distinct molecular responses to pathogenic Vibrio and thermal stress.

Global climate change and anthropogenic activities are threatening the future survival of coral reef ecosystems. The ability of reef-building zooxanthellate coral to survive these stressors may be determined through fundamental differences within their symbiotic dinoflagellates (Symbiodinium sp.). We define the in vitro apoptotic response of 2 evolutionarily distant Symbiodinium sp., subtypes B2 and C1, to determine the synergistic effects of disease and temperature on cell viability using flow cytometry. The putative yellow band disease (YBD) consortium of Vibrio spp. bacteria and temperature (33°C) had a positive synergistic effect on C1 apoptosis, while B2 displayed increased apoptosis to elevated temperature (29 and 33°C), the Vibrio consortium, and a lone virulent strain of V. alginolyticus, but no synergistic effects. Additionally, heat shock protein 60 expression revealed differential cell-mediated temperature sensitivity between subtypes via western blotting. This result marks the first evidence of Symbiodinium sp. apoptotic variations to YBD pathogens and emphasizes the potential impact of synergistic stress on globally distributed coral-Symbiodinium symbioses.

Ulcerated yellow spot syndrome: implications of aquaculture-related pathogens associated with soft coral Sarcophyton ehrenbergi tissue lesions.

We introduce a new marine syndrome called ulcerated yellow spot, affecting the soft coral Sarcophyton ehrenbergi. To identify bacteria associated with tissue lesions, tissue and mucus samples were taken during a 2009 Indo-Pacific research expedition near the Wakatobi Island chain, Indonesia. Polymerase chain reaction targeting the 16S rDNA gene indicated associations with the known fish-disease-causing bacterium Photobacterium damselae, as well as multiple Vibrio species. Results indicate a shift toward decreasing diversity of bacteria in lesioned samples. Photobacterium damselae ssp. piscicida, formerly known as Pasteurella piscicida, is known as the causative agent of fish pasteurellosis and in this study, was isolated solely in lesioned tissues. Globally, fish pasteurellosis is one of the most damaging fish diseases in marine aquaculture. Vibrio alginolyticus, a putative pathogen associated with yellow band disease in scleractinian coral, was also isolated from lesioned tissues. Lesions appear to be inflicting damage on symbiotic zooxanthellae (Symbiodinium sp.), measurable by decreases in mitotic index, cell density and photosynthetic efficiency. Mitotic index of zooxanthellae within infected tissue samples was decreased by ~80%, while zooxanthellae densities were decreased by ~40% in lesioned tissue samples compared with healthy coral. These results provide evidence for the presence of known aquaculture pathogens in lesioned soft coral and may be a concern with respect to cross-species epizootics in the tropics.

Development of gene expression markers of acute heat-light stress in reef-building corals of the genus Porites.

Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide.