Mucospheres produced by a mixotrophic protist impact ocean carbon cycling

Mixotrophic protists (unicellular eukaryotes) that engage in both phototrophy (photosynthesis) and phago-heterotrophy (engulfment of particles)-are predicted to contribute substantially to energy fluxes and marine biogeochemical cycles. However, their impact remains largely unquantified. Here we describe the sophisticated foraging strategy of a widespread mixotrophic dinoflagellate, involving the production of carbon-rich 'mucospheres' that attract, capture, and immobilise microbial prey facilitating their consumption. We provide a detailed characterisation of this previously undescribed behaviour and reveal that it represents an overlooked, yet quantitatively significant mechanism for oceanic carbon fluxes. Following feeding, the mucospheres laden with surplus prey are discarded and sink, contributing an estimated 0.17-1.24 mg m-2 d-1 of particulate organic carbon, or 0.02-0.15 Gt to the biological pump annually, which represents 0.1-0.7% of the estimated total export from the euphotic zone. These findings demonstrate how the complex foraging behaviour of a single species of mixotrophic protist can disproportionally contribute to the vertical flux of carbon in the ocean

Quantifying inorganic nitrogen assimilation by synechococcus using bulk and single-cell mass spectrometry: A comparative study

Copyright © 2018 Giardina, Cheong, Marjo, Clode, Guagliardo, Pickford, Pernice, Seymour and Raina. Microorganisms drive most of the major biogeochemical cycles in the ocean, but the rates at which individual species assimilate and transform key elements is generally poorly quantified. One of these important elements is nitrogen, with its availability limiting primary production across a large proportion of the ocean. Nitrogen uptake by marine microbes is typically quantified using bulk-scale approaches, such as Elemental Analyzer-Isotope Ratio Mass Spectrometry (EA-IRMS), which averages uptake over entire communities, masking microbial heterogeneity. However, more recent techniques, such as secondary ion mass spectrometry (SIMS), allow for elucidation of assimilation rates at the scale at which they occur: the single-cell level. Here, we combine and compare the application of bulk (EA-IRMS) and single-cell approaches (NanoSIMS and Time-of-Flight-SIMS) for quantifying the assimilation of inorganic nitrogen by the ubiquitous marine primary producer Synechococcus. We aimed to contrast the advantages and disadvantages of these techniques and showcase their complementarity. Our results show that the average assimilation of 15N by Synechococcus differed based on the technique used: values derived from EA-IRMS were consistently higher than those derived from SIMS, likely due to a combination of previously reported systematic depletion as well as differences in sample preparation. However, single-cell approaches offered additional layers of information, whereby NanoSIMS allowed for the quantification of the metabolic heterogeneity among individual cells and ToF-SIMS enabled identification of nitrogen assimilation into peptides. We suggest that this coupling of stable isotope-based approaches has great potential to elucidate the metabolic capacity and heterogeneity of microbial cells in natural environments

Transcriptomic analysis of the response of Acropora millepora to hypo-osmotic stress provides insights into DMSP biosynthesis by corals

BACKGROUND: Dimethylsulfoniopropionate (DMSP) is a small sulphur compound which is produced in prodigious amounts in the oceans and plays a pivotal role in the marine sulfur cycle. Until recently, DMSP was believed to be synthesized exclusively by photosynthetic organisms; however we now know that corals and specific bacteria can also produce this compound. Corals are major sources of DMSP, but the molecular basis for its biosynthesis is unknown in these organisms. RESULTS: Here we used salinity stress, which is known to trigger DMSP production in other organisms, in conjunction with transcriptomics to identify coral genes likely to be involved in DMSP biosynthesis. We focused specifically on both adults and juveniles of the coral Acropora millepora: after 24 h of exposure to hyposaline conditions, DMSP concentrations increased significantly by 2.6 fold in adult corals and 1.2 fold in juveniles. Concomitantly, candidate genes enabling each of the necessary steps leading to DMSP production were up-regulated. CONCLUSIONS: The data presented strongly suggest that corals use an algal-like pathway to generate DMSP from methionine, and are able to rapidly change expression of the corresponding genes in response to environmental stress. However, our data also indicate that DMSP is unlikely to function primarily as an osmolyte in corals, instead potentially serving as a scavenger of ROS and as a molecular sink for excess methionine produced as a consequence of proteolysis and osmolyte catabolism in corals under hypo-osmotic conditions.CA gratefully acknowledges receipt of an AIMS@JCU scholarship, and the work was supported by the Australian Research Council via the ARC Centre of Excellence for Coral Reef Studie

Heat stress reduces the contribution of diazotrophs to coral holobiont nitrogen cycling

Efficient nutrient cycling in the coral-algal symbiosis requires constant but limited nitrogen availability. Coral-associated diazotrophs, i.e., prokaryotes capable of fixing dinitrogen, may thus support productivity in a stable coral-algal symbiosis but could contribute to its breakdown when overstimulated. However, the effects of environmental conditions on diazotroph communities and their interaction with other members of the coral holobiont remain poorly understood. Here we assessed the effects of heat stress on diazotroph diversity and their contribution to holobiont nutrient cycling in the reef-building coral Stylophora pistillata from the central Red Sea. In a stable symbiotic state, we found that nitrogen fixation by coral-associated diazotrophs constitutes a source of nitrogen to the algal symbionts. Heat stress caused an increase in nitrogen fixation concomitant with a change in diazotroph communities. Yet, this additional fixed nitrogen was not assimilated by the coral tissue or the algal symbionts. We conclude that although diazotrophs may support coral holobiont functioning under low nitrogen availability, altered nutrient cycling during heat stress abates the dependence of the coral host and its algal symbionts on diazotroph-derived nitrogen. Consequently, the role of nitrogen fixation in the coral holobiont is strongly dependent on its nutritional status and varies dynamically with environmental conditions.Peer reviewe

DMSP production by coral-associated bacteria

Dimethylsulfoniopropionate (DMSP) is an important molecule in the marine sulfur cycle, produced in large amounts by corals and their dinoflagellate endosymbionts, Symbiodiniaceae. Although corals are known to harbour bacteria that can catabolise DMSP, the recent discovery of bacteria capable of producing DMSP in coastal and deep-sea environments raises the possibility of a bacterial contribution to the DMSP output of corals. Here, 157 bacteria associated with four common coral species were isolated and screened for their ability to produce DMSP by targeting dsyB, a key gene involved in DMSP biosynthesis. Approximately 9% (14 out of 157) of the bacterial isolates harboured dsyB, all being members of the Alphaproteobacteria. The ability of these isolates to produce DMSP was confirmed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) measurements. A dsyB-harbouring strain, Shimia aestuarii AMM-P-2, was selected for genome sequencing. This strain harbours the complete genetic machinery to (i) assimilate sulfate and synthesise the DMSP precursors, cysteine and methionine; (ii) demethylate DMSP and generate methanethiol; (iii) cleave DMSP, generating dimethyl sulfide (DMS) and acrylate; and (iv) utilise or detoxify acrylate. The impacts of varied environmental factors (temperature, salinity, light and UV radiation) on S. aestuarii AMM-P-2 DMSP biosynthesis were characterised. DMSP levels in S. aestuarii AMM-P-2 increased almost two-fold under both hypersaline conditions (40 PSU) and high UV exposure. DMSP catabolism through the cleavage pathway also increased under these conditions, producing the antioxidants DMS and acrylate, a potential response to the oxidative stress generated. Overall, our results reveal that coral-associated bacteria can synthesize DMSP and may therefore contribute to DMSP production by the coral holobiont

Coral-Bacterial Communities before and after a Coral Mass Spawning Event on Ningaloo Reef

Bacteria associated with three coral species, Acropora tenuis, Pocillopora damicornis and Tubastrea faulkneri, were assessed before and after coral mass spawning on Ningaloo Reef in Western Australia. Two colonies of each species were sampled before and after the mass spawning event and two additional samples were collected for P. damicornis after planulation. A variable 470 bp region of the 16 S rRNA gene was selected for pyrosequencing to provide an understanding of potential variations in coral-associated bacterial diversity and community structure. Bacterial diversity increased for all coral species after spawning as assessed by Chao1 diversity indicators. Minimal changes in community structure were observed at the class level and data at the taxonomical level of genus incorporated into a PCA analysis indicated that despite bacterial diversity increasing after spawning, coral-associated community structure did not shift greatly with samples grouped according to species. However, interesting changes could be detected from the dataset; for example, α-Proteobacteria increased in relative abundance after coral spawning and particularly the Roseobacter clade was found to be prominent in all coral species, indicating that this group may be important in coral reproduction

Subcellular tracking reveals the location of dimethylsulfoniopropionate in microalgae and visualises its uptake by marine bacteria

Phytoplankton-bacteria interactions drive the surface ocean sulfur cycle and local climatic processes through the production and exchange of a key compound: dimethylsulfoniopropionate (DMSP). Despite their large-scale implications, these interactions remain unquantified at the cellular-scale. Here we use secondary-ion mass spectrometry to provide the first visualization of DMSP at sub-cellular levels, tracking the fate of a stable sulfur isotope (34S) from its incorporation by microalgae as inorganic sulfate to its biosynthesis and exudation as DMSP, and finally its uptake and degradation by bacteria. Our results identify for the first time the storage locations of DMSP in microalgae, with high enrichments present in vacuoles, cytoplasm and chloroplasts. In addition, we quantify DMSP incorporation at the single-cell level, with DMSP-degrading bacteria containing seven times more 34S than the control strain. This study provides an unprecedented methodology to label, retain, and image small diffusible molecules, which can be transposable to other symbiotic systems.This work was supported by ANNiMS (Australian Government, Department of Education, Employment and Workplace Relations), the AMMRF Centre for Microscopy, Characterisation and Analysis (UWA) and by Australian Research Council Grant DE160100636

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Production and fate of dimethylsulfoniopropionate (DMSP) in reef-building corals and its integral role in coral health

Bacteria play crucial roles in most biogeochemical cycles in the oceans because of their high abundance and metabolic capabilities. Each square centimetre of coral surface harbours between 10⁶ and 10⁸ bacterial cells, and significantly, bacterial assemblages tend to be highly specific to their coral host. Although the phylogenetic diversity and dynamics of coral-associated bacterial communities have been studied for more than a decade, their ecological and functional roles in the coral holobiont are still poorly understood. The taxonomic composition of these bacterial communities is likely to be greatly influenced by chemicals produced by coral hosts, as well as by their endosymbiotic algae Symbiodinium. Dimethylsulfoniopropionate (DMSP) is a ubiquitous compound found within reefbuilding corals and is a central molecule in the marine sulfur cycle, particularly as a precursor to the climate-regulating gas dimethylsulfide (DMS). Marine bacteria are the primary organisms that degrade DMSP into DMS, and consequently play a critical role in linking the marine environment and the atmosphere in the global sulfur cycle. To date, the role of these organic sulfur compounds in the metabolism of coral-associated bacteria has not been investigated. Consequently, this thesis aims to provide new insights into the roles of DMSP in corals, and more specifically in coral-bacterial associations, with a particular focus on the production and metabolism of this sulfur molecule.\ud \ud To investigate the roles of DMSP in corals, I developed a new direct approach to accurately and rapidly quantify DMSP and one of its breakdown products, acrylate, based on quantitative nuclear magnetic resonance (qNMR) spectroscopy (Chapter 2). This method overcomes inaccuracies associated with indirect methods that convert DMSP to DMS and measure this volatile molecule. The method was tested on a range of coral genera, and enabled simultaneous and direct quantification of multiple molecules from the same extract, as well as rapid processing with high reproducibility. Thus large numbers of samples can be processed in short time periods. The method was successfully applied to environmental samples and provides the first baseline information on diel variation of DMSP and acrylate concentrations in the coral Acropora millepora. The lack of diel variation found raises questions about the role of endosymbiotic dinoflagellates in DMSP biosynthesis in corals.\ud \ud Reef-building corals are among the most prolific DMSP producers in the ocean, but their DMSP production has been attributed entirely to the activities of their algal symbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, I show that coral juveniles from the genus Acropora produce DMSP in the absence of associated microalgae (Chapter 3). DMSP levels increased through time (by up to 54% over 6 days) in coral juveniles raised without access to photosynthetic symbionts. Increased DMSP levels in juvenile and adult corals exposed to experimentally elevated temperature treatments suggest a role for DMSP in thermal stress responses. Discovery of coral orthologs of two algal genes recently identified in DMSP biosynthesis suggests that corals possess the enzymatic machinery necessary for DMSP production. My findings overturn the current paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight a direct role for corals in climate regulation.\ud \ud In order to investigate the influence of DMSP and DMS on coral-associated bacteria, the bacterial communities of two coral species, Acropora millepora and Montipora aequituberculata, were characterized by both culture-dependent and molecular techniques (Chapter 4). Three genera, Roseobacter, Spongiobacter, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of bacterial communities in these two corals based on both clone library and pyrosequencing approaches. Bacteria capable of degrading DMSP represented 37% of the communities in Montipora and between 67 and 92% in Acropora. These results demonstrate that DMSP and potentially DMS act as nutrient sources for coral-associated bacteria, and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals. Exploration of the publically available metagenome databases revealed that genes implicated in DMSP metabolism are abundant in the viral component of coral-reef-derived metagenomes, indicating that viruses can act as a reservoir for such genes (Chapter 4).\ud \ud The metabolic potential of bacteria in pure culture does not necessarily reflect their metabolic activities within the coral holobiont, therefore I used state-of-the-art imaging techniques (NanoSIMS), coupled with analytical chemistry approaches, to determine linkages between DMSP-synthesising Symbiodinium and DMSP-degrading bacteria (Chapter 5). DMSP-degrading bacteria were coincubated with Symbiodinium cells previously grown in a medium with isotopically labelled sulfate as sole sulfur source. This experiment confirmed that the sulfur used for DMSP biosynthesis comes from sulfate assimilation in Symbiodinium and enabled visualization of sulfur isotope hotspots adjacent to Symbiodinium cells that correlated with the location of bacteria observed with electron microscopy. These results confirm the role of coral-associated bacteria in the sulfur cycle and constitute the first empirical evidence of the bacterial assimilation of Symbiodinium secondary metabolites in vivo.\ud \ud Bacterial communities associated with healthy corals have been suspected to produce antimicrobial compounds that inhibit the colonization and growth of invasive microbes and potential pathogens; however, antimicrobial molecules derived from coral-associated bacteria have not been identified. In chapter 6, I describe the isolation of an antimicrobial compound produced by Pseudovibrio sp., a bacterium commonly associated with reef-building corals and able to degrade dimethylsulfoniopropionate (DMSP). Bioassay-guided fractionation and spectroscopic techniques, including NMR and mass spectrometry (MS), identified the antimicrobial as tropodithietic acid (TDA), a sulfur-containing compound likely derived from DMSP metabolism. TDA was produced in large quantities by Pseudovibrio spp. and prevented the growth of two known coral pathogens, V. coralliilyticus and V. owensii, at very low concentrations (0.5 μg/mL) in agar diffusion assays. Its production was significantly reduced at temperatures causing thermal stress in corals, indicating a role for DMSP-metabolizing bacterial communities in coral disease prevention under ambient temperatures and the potential disruption of this protection during thermal stress events.\ud \ud In summary, this thesis presents novel information on the production and fate of DMSP in reef-building corals. It identifies the coral animal as a DMSP producer, provides corroborative evidence of the important role of DMSP for numerous coral-associated bacteria using both in vitro and in vivo approaches, and isolates an antimicrobial compound likely derived from DMSP metabolism. Together, these results constitute the first comprehensive study of DMSP in reef-building corals and underscore the remarkable contribution of this molecule to coral health