Multiple-biomarkers show the importance of blue carbon to commercially important fishery species.

Coastal blue carbon ecosystems (BCE) support nearshore food webs and provide habitat for many commercially important fish and crustacean species. However, the complex links between catchment vegetation and the carbon food-base of estuarine systems are difficult to disern. We employed a multi-biomarker approach (stable isotope ratios - δ13C and δ15N, fatty acid trophic markers - FATMs and metabolomics - central carbon metabolism metabolites) to test links between estuarine vegetation and the food sources available to commercially important crabs and fish occurring within the river systems of the near-pristine eastern coastline of the Gulf of Carpentaria, Australia. Stable isotope analysis confirmed the dietary importance of fringing macrophytes to consumer diet, but showed that this is modulated by their dominance along the riverbank. FATMs indicative of specific food sources further confirmed the differences among upper intertidal macrophytes (driven by concentrations of 16: 1ω7, 18:1ω9, 18:2ω6, 18:3ω3 & 22.0) and seagrass (driven by 18:2ω6, 18:3ω3). These dietary patterns were also reflected in the concentration of central carbon metabolism metabolites. Overall, our study demonstrates the congruence of different biomarker approaches to resolve biochemical links between blue carbon ecosystems and important nekton species, and provides fresh insights into the pristine tropical estuaries of northern Australia.

Multi-omics eco-surveillance of complex legacy contamination with a locally adapted estuarine invertebrate.

Complex legacy contamination from human use is a major issue for estuaries globally. In particular, contamination of water and sediments with bioavailable metals/metalloids, in addition to other industrial contaminants, such as hydrocarbons. Yet, understanding of complex toxicity and local adaptation in field exposed, non-model, invertebrate communities is limited. Herein, we apply multi-omics (metabolomics, lipidomics, proteomics) coupled to traditional sediment quality analyses, to better characterise molecular and cellular responses necessary for application to monitoring, as an eco-surveillance tool. Using these approaches, we characterise functional phenotypes of a sediment associated invertebrate (sipunculid), from an estuary exposed to complex legacy contamination (metals: Zn, Hg, Cd, Pb, Cu, As; and polycyclic aromatic hydrocarbons, PAHs). We sampled individuals at a range of exposure sites, highly (NTB5), moderately (NTB13), and lesser-influenced reference sites. Size differences were observed in sampled individuals between sites, with smaller individuals collected from NTB13. Analysis of environmental variables that correlated with change in the metabolite data revealed that the metabolism of smaller individuals at medium exposure NTB13 was highly differentiated by sediment concentrations of Hg, despite higher concentrations at more exposed NTB5. Functional phenotypes of these smaller individuals were characterised by sulphur and aromatic amino acid metabolism, increases in oxidised intermediates, upregulation of protein responses to oxidative stress, and melanin synthesis, and saturation of membrane and storage of lipids; in addition to the metabolism of naphthalene (PAH). Such widespread change was not observed in the metabolite and lipid profiles of larger individuals at high exposure NTB5, suggesting possible differences in effects between sites may also be associated with size (developmental stage, or age) and/or PAH exposure. This study serves to further understanding of differing modes of toxicity and local adaptation to multiple contaminants, and drivers of functional change in a complex estuary environment.

Multi-omics eco-surveillance of bacterial community function in legacy contaminated estuary sediments.

Complex legacy contamination is a major issue for many estuaries, with toxicity affecting change in bacterial communities, and their provision of associated goods and services. Sequencing surveys of bacterial community composition provide inferred function; however, additional insights may be generated by measurement of realised metabolic phenotypes. We apply multi-omics (genomics, lipidomics, and metabolomics), with traditional sediment quality analyses, to characterise sediment-associated bacterial communities in an estuary subject to legacy metal contamination (Zn, Hg, As, Cd, Cu and Pb). Analyses of bacterial composition and inferred function (genomics) are coupled with measurements of realised bacterial phenotype (metabolomics and lipidomics) at multiple industrialised and reference sites. At sites with the highest sediment metal concentrations (NTB), we also observed increased abundances of hydrocarbon and sulphuric acid metabolites, indicating additional sediment contamination. Bacterial phyla across sampled sites were dominated by Proteobacteria and Desulfobacteria. NTB sites were enriched with metabolically versatile, cooperative and biofilm forming phyla including, Zixibacteria, Spirochaetota, SAR324 clade, Proteobacteria, Latescibacterota, Desulfobacterota, Deferrisomtota and Acidobateriota; with inferred functions characterised by sulphur metabolism, pathways associated with the degradation of complex organic molecules, and fermentation. Reference sites were characterised by enhanced vitamin biosynthesis, cell wall, cofactor and carbohydrate biosynthesis, and CO2 fixation. Measured metabolic phenotypes at NTB sites supported predicted functions, with most consistent change observed to naphthalene and aminobenzoate degradation pathways and carbohydrate metabolism (galactose, amino and nucleotide sugar). Change in NTB metabolite profiles was most highly correlated with sediment Hg concentrations, indicative of toxic exposure and potential for Hg methylation. Lipid profiles generated further insight into potential functional (hydroxy fatty acids) and community level change (ceramide phosphoethanolamines, unsaturated glycerides). Multi-omics outputs provided insights into bacterial community functions, modes of contaminant toxicity and expressed mechanisms of adaptation, necessary to better inform management decisions and predictive models in increasingly human-influenced environments.

Colonization and metabolite profiles of homologous, heterologous and experimentally evolved algal symbionts in the sea anemone Exaiptasia diaphana.

The sea anemone, Exaiptasia diaphana, is a model of coral-dinoflagellate (Symbiodiniaceae) symbiosis. However, little is known of its potential to form symbiosis with Cladocopium-a key Indo-Pacific algal symbiont of scleractinian corals, nor the host nutritional consequences of such an association. Aposymbiotic anemones were inoculated with homologous algal symbionts, Breviolum minutum, and seven heterologous strains of Cladocopium C1acro (wild-type and heat-evolved) under ambient conditions. Despite lower initial algal cell density, Cladocopium C1acro-anemeones achieved similar cell densities as B. minutum-anemones by week 77. Wild-type and heat-evolved Cladocopium C1acro showed similar colonization patterns. Targeted LC-MS-based metabolomics revealed that almost all significantly different metabolites in the host and Symbiodiniaceae fractions were due to differences between Cladocopium C1acro and B. minutum, with little difference between heat-evolved and wild-type Cladocopium C1acro at week 9. The algal fraction of Cladocopium C1acro-anemones was enriched in metabolites related to nitrogen storage, while the host fraction of B. minutum-anemones was enriched in sugar-related metabolites. Compared to B. minutum, Cladocopium C1acro is likely slightly less nutritionally beneficial to the host under ambient conditions, but more capable of maintaining its own growth when host nitrogen supply is limited. Our findings demonstrate the value of E. diaphana to study experimentally evolved Cladocopium.

Metabolomics as a tool for in situ study of chronic metal exposure in estuarine invertebrates.

Estuaries are subject to intense human use globally, with impacts from multiple stressors, such as metal contaminants. A key challenge is extending beyond traditional monitoring approaches to understand effects to biota and system function. To explore the metabolic effects of complex metal contaminants to sediment dwelling (benthic) fauna, we apply a multiple-lines-of-evidence approach, coupling environmental monitoring, benthic sampling, total metals analysis and targeted metabolomics. We characterise metabolic signatures of metal exposure in three benthic invertebrate taxa, which differed in distribution across sites and severity of metal exposure: sipunculid (very high), amphipod (high), maldanid polychaete (moderate). We observed sediment and tissue metal loads far exceeding sediment guidelines where toxicity-related adverse effects may be expected, for metals including, As, Cd, Pb, Zn and Hg. Change in site- and taxa-specific metabolite profiles was highly correlated with natural environmental drivers (sediment total organic carbon and water temperature). At the most metal influenced sites, metabolite variation was also correlated with sediment metal loads. Using supervised multivariate regression, taxa-specific metabolic signatures of increased exposure and possibility of toxic effects were characterised against multiple reference sites. Metabolic signatures varied according to each taxon and degree of metal exposure, but primarily indicated altered cysteine and methionine metabolism, metal-binding and elimination (lysosomal) activity, coupled to change in complex biosynthesis pathways, responses to oxidative stress, and cellular damage. This novel multiple-lines-of-evidence approach combining metabolomics with traditional environmental monitoring, enabled detection and characterisation of chronic metal exposure effects in situ in multiple invertebrate taxa. With capacity for application to rapid and effective monitoring of non-model species in complex environments, these approaches are critical for improved assessment and management of systems that are increasingly subject to anthropogenic drivers of change.

Omics-based ecosurveillance uncovers the influence of estuarine macrophytes on sediment microbial function and metabolic redundancy in a tropical ecosystem.

Vertical zonation within estuarine ecosystems can strongly influence microbial diversity and function by regulating competition, predation, and environmental stability. The degree to which microbial communities exhibit horizontal patterns through an estuary has received comparatively less attention. Here, we take a multi-omics ecosurveillance approach to study environmental gradients created by the transition between dominant vegetation types along a near pristine tropical river system (Wenlock River, Far North Queensland, Australia). The study sites included intertidal mudflats fringed by saltmarsh, mangrove or mixed soft substrata habitats. Collected sediments were analyzed for eukaryotes and prokaryotes using small sub-unit (SSU) rRNA gene amplicons to profile the relative taxonomic composition. Central carbon metabolism metabolites and other associated organic polar metabolites were analyzed using established metabolomics-based approaches, coupled with total heavy metals analysis. Eukaryotic taxonomic information was found to be more informative of habitat type. Bacterial taxonomy and community composition also showed habitat-specificity, with phyla Proteobacteria and Cyanobacteria strongly linked to mangroves and saltmarshes, respectively. In contrast, metabolite profiling was critical for understanding the biochemical pathways and expressed functional outputs in these systems that were tied to predicted microbial gene function (16S rRNA). A high degree of metabolic redundancy was observed in the bacterial communities, with the metabolomics data suggesting varying degrees of metabolic criticality based on habitat type. The predicted functions of the bacterial taxa combined with annotated metabolites accounted for the conservative perspective of microbial community redundancy against the putative metabolic pathway impacts in the metabolomics data. Coupling these data demonstrates that habitat-mediated estuarine gradients drive patterns of community diversity and metabolic function and highlights the real redundancy potential of habitat microbiomes. This information is useful as a point of comparison for these sensitive ecosystems and provides a framework for identifying potentially vulnerable or at-risk systems before they are significantly degraded.

Cryptosporidiosis Modulates the Gut Microbiome and Metabolism in a Murine Infection Model.

Cryptosporidiosis is a major human health concern globally. Despite well-established methods, misdiagnosis remains common. Our understanding of the cryptosporidiosis biochemical mechanism remains limited, compounding the difficulty of clinical diagnosis. Here, we used a systems biology approach to investigate the underlying biochemical interactions in C57BL/6J mice infected with Cryptosporidium parvum. Faecal samples were collected daily following infection. Blood, liver tissues and luminal contents were collected 10 days post infection. High-resolution liquid chromatography and low-resolution gas chromatography coupled with mass spectrometry were used to analyse the proteomes and metabolomes of these samples. Faeces and luminal contents were additionally subjected to 16S rRNA gene sequencing. Univariate and multivariate statistical analysis of the acquired data illustrated altered host and microbial energy pathways during infection. Glycolysis/citrate cycle metabolites were depleted, while short-chain fatty acids and D-amino acids accumulated. An increased abundance of bacteria associated with a stressed gut environment was seen. Host proteins involved in energy pathways and Lactobacillus glyceraldehyde-3-phosphate dehydrogenase were upregulated during cryptosporidiosis. Liver oxalate also increased during infection. Microbiome-parasite relationships were observed to be more influential than the host-parasite association in mediating major biochemical changes in the mouse gut during cryptosporidiosis. Defining this parasite-microbiome interaction is the first step towards building a comprehensive cryptosporidiosis model towards biomarker discovery, and rapid and accurate diagnostics.

Metabolic Profiling from an Asymptomatic Ferret Model of SARS-CoV-2 Infection.

Coronavirus disease (COVID-19) is a contagious respiratory disease that is causing significant global morbidity and mortality. Understanding the impact of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection on the host metabolism is still in its infancy but of great importance. Herein, we investigated the metabolic response during viral shedding and post-shedding in an asymptomatic SARS-CoV-2 ferret model (n = 6) challenged with two SARS-CoV-2 isolates. Virological and metabolic analyses were performed on (minimally invasive) collected oral swabs, rectal swabs, and nasal washes. Fragments of SARS-CoV-2 RNA were only found in the nasal wash samples in four of the six ferrets, and in the samples collected 3 to 9 days post-infection (referred to as viral shedding). Central carbon metabolism metabolites were analyzed during viral shedding and post-shedding periods using a dynamic Multiple Reaction Monitoring (dMRM) database and method. Subsequent untargeted metabolomics and lipidomics of the same samples were performed using a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) methodology, building upon the identified differentiated central carbon metabolism metabolites. Multivariate analysis of the acquired data identified 29 significant metabolites and three lipids that were subjected to pathway enrichment and impact analysis. The presence of viral shedding coincided with the challenge dose administered and significant changes in the citric acid cycle, purine metabolism, and pentose phosphate pathways, amongst others, in the host nasal wash samples. An elevated immune response in the host was also observed between the two isolates studied. These results support other metabolomic-based findings in clinical observational studies and indicate the utility of metabolomics applied to ferrets for further COVID-19 research that advances early diagnosis of asymptomatic and mild clinical COVID-19 infections, in addition to assessing the effectiveness of new or repurposed drug therapies.

Functional analysis of pristine estuarine marine sediments.

Traditional environmental monitoring techniques are well suited to resolving acute exposure effects but lack resolution in determining subtle shifts in ecosystem functions resulting from chronic exposure(s). Surveillance with sensitive omics-based technologies could bridge this gap but, to date, most omics-based environmental studies have focused on previously degraded environments, identifying key metabolic differences resulting from anthropogenic perturbations. Here, we apply omics-based approaches to pristine environments to establish blueprints of microbial functionality within healthy estuarine sediment communities. We collected surface sediments (n = 50) from four pristine estuaries along the Western Cape York Peninsula of Far North Queensland, Australia. Sediment microbiomes were analyzed for 16S rRNA amplicon sequences, central carbon metabolism metabolites and associated secondary metabolites via targeted and untargeted metabolic profiling methods. Multivariate statistical analyses indicated heterogeneity among all the sampled estuaries, however, taxa-function relationships could be established that predicted community metabolism potential. Twenty-four correlated gene-metabolite pathways were identified and used to establish sediment microbial blueprints of essential carbon metabolism and amino acid biosynthesis that were positively correlated with community metabolic function outputs (2-oxisocapraote, tryptophan, histidine citrulline and succinic acid). In addition, an increase in the 125 KEGG genes related to metal homeostasis and metal resistance was observed, although, none of the detected metabolites related to these specific genes upon integration. However, there was a correlation between metal abundance and functional genes related to Fe and Zn metabolism. Our results establish a baseline microbial blueprint for the pristine sediment microbiome, one that drives important ecosystem services and to which future ecosurveillance monitoring can be compared.

A multi-platform metabolomics approach to identify possible biomarkers for human faecal contamination in Greenshell™ mussels (Perna canaliculus).

Bivalve molluscs have the potential to bioaccumulate microbial pathogens including noroviruses from aquatic environments and as such, there is a need for a rapid and cheap in-situ method for their detection. Here, we characterise the tissue-specific response of New Zealand Greenshell™ mussels (Perna canaliculus) to faecal contamination from two different sources (municipal sewage and human faeces). This is done with the view to identify potential biomarkers that could be further developed into low cost, rapid and sensitive in-situ biosensors for human faecal contamination detection of mussels in growing areas. Tissue-specific metabolic profiles from gills, haemolymph and digestive glands were analysed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Clear differentiation of metabolic profiles was observed among treatments in each tissue type. Overall, energy pathways such as glycolysis, citrate cycle and oxidative phosphorylation were downregulated across the three mussel tissues studied following simulated contamination events. Conversely, considerable sterol upregulation in the gills was observed after exposure to contamination. Additionally, free pools of nucleotide phosphates and the antioxidant glutathione declined considerably post-exposure to contamination in gills. These results provide important insights into the tissue-specific metabolic effects of human faecal contamination in mussels. This study demonstrates the utility of metabolomics as a tool for identifying potential biomarkers in mussels.

Artificial light at night interacts with predatory threat to alter reef fish metabolite profiles.

Light cycles and predatory threat define activity patterns (e.g. feeding/sleeping, activity/rest) in most diurnal fish species. Artificial light at night (ALAN) may disrupt natural cycles and biochemical processes, a mismatch which can eventually reduce condition and fitness. We evaluate the separate and joint effects of ALAN and predator threat on metabolism within brain, liver and muscle tissue of a common, wild caught damselfish, blue green chromis (Chromis viridis). The effects of ALAN varied according to tissue type and predator exposure. In all tissues we observed changes in metabolic pathways associated with increased activity under continuous light (despite provision of shelter), specifically those associated with energy metabolism, cell signalling, responses to oxidative stress and markers of cellular damage. In both the brain and liver tissues, predator threat served to moderate the influence of ALAN on metabolic change, likely due to increased sheltering behaviour. However, no interaction of predator threat with ALAN was observed in metabolism of the muscle tissue. Our results highlight complex sub-acute effects of ALAN exposure on tissue specific and whole organism energy metabolism. Collectively these effects indicate that ALAN has significant scope to reduce fitness of coastal fishes and potentially threaten ecosystem services, but that these changes are highly complex and may be altered by biotic drivers of activity.

Partner switching and metabolic flux in a model cnidarian-dinoflagellate symbiosis.

Metabolite exchange is fundamental to the viability of the cnidarian-Symbiodiniaceae symbiosis and survival of coral reefs. Coral holobiont tolerance to environmental change might be achieved through changes in Symbiodiniaceae species composition, but differences in the metabolites supplied by different Symbiodiniaceae species could influence holobiont fitness. Using 13C stable-isotope labelling coupled to gas chromatography-mass spectrometry, we characterized newly fixed carbon fate in the model cnidarian Exaiptasia pallida (Aiptasia) when experimentally colonized with either native Breviolum minutum or non-native Durusdinium trenchii Relative to anemones containing B. minutum, D. trenchii-colonized hosts exhibited a 4.5-fold reduction in 13C-labelled glucose and reduced abundance and diversity of 13C-labelled carbohydrates and lipogenesis precursors, indicating symbiont species-specific modifications to carbohydrate availability and lipid storage. Mapping carbon fate also revealed significant alterations to host molecular signalling pathways. In particular, D. trenchii-colonized hosts exhibited a 40-fold reduction in 13C-labelled scyllo-inositol, a potential interpartner signalling molecule in symbiosis specificity. 13C-labelling also highlighted differential antioxidant- and ammonium-producing pathway activities, suggesting physiological responses to different symbiont species. Such differences in symbiont metabolite contribution and host utilization may limit the proliferation of stress-driven symbioses; this contributes valuable information towards future scenarios that select in favour of less-competent symbionts in response to environmental change.

Mapping carbon fate during bleaching in a model cnidarian symbiosis: the application of 13 C metabolomics.

Coral bleaching is a major threat to the persistence of coral reefs. Yet we lack detailed knowledge of the metabolic interactions that determine symbiosis function and bleaching-induced change. We mapped autotrophic carbon fate within the free metabolite pools of both partners of a model cnidarian-dinoflagellate symbiosis (Aiptasia-Symbiodinium) during exposure to thermal stress via the stable isotope tracer (13 C bicarbonate), coupled to GC-MS. Symbiont photodamage and pronounced bleaching coincided with substantial increases in the turnover of non13 C-labelled pools in the dinoflagellate (lipid and starch store catabolism). However, 13 C enrichment of multiple compounds associated with ongoing carbon fixation and de novo biosynthesis pathways was maintained (glucose, fatty acid and lipogenesis intermediates). Minimal change was also observed in host pools of 13 C-enriched glucose (a major symbiont-derived mobile product). However, host pathways downstream showed altered carbon fate and/or pool composition, with accumulation of compatible solutes and nonenzymic antioxidant precursors. In hospite symbionts continue to provide mobile products to the host, but at a significant cost to themselves, necessitating the mobilization of energy stores. These data highlight the need to further elucidate the role of metabolic interactions between symbiotic partners, during the process of thermal acclimation and coral bleaching.

13C metabolomics reveals widespread change in carbon fate during coral bleaching.

INTRODUCTION: Rising seawater temperatures are threatening the persistence of coral reefs; where above critical thresholds, thermal stress results in a breakdown of the coral-dinoflagellate symbiosis and the loss of algal symbionts (coral bleaching). As symbiont-derived organic products typically form a major portion of host energy budgets, this has major implications for the fitness and persistence of symbiotic corals. OBJECTIVES: We aimed to determine change in autotrophic carbon fate within individual compounds and downstream metabolic pathways in a coral symbiosis exposed to varying degrees of thermal stress and bleaching. METHODS: We applied gas chromatography-mass spectrometry coupled to a stable isotope tracer (13C), to track change in autotrophic carbon fate, in symbiont and host individually, following exposure to elevated water temperature. RESULTS: Thermal stress resulted in partner-specific changes in carbon fate, which progressed with heat stress duration. We detected modifications to carbohydrate and fatty acid metabolism, lipogenesis, and homeostatic responses to thermal, oxidative and osmotic stress. Despite pronounced photodamage, remaining in hospite symbionts continued to produce organic products de novo and translocate to the coral host. However as bleaching progressed, we observed minimal 13C enrichment of symbiont long-chain fatty acids, also reflected in 13C enrichment of host fatty acid pools. CONCLUSION: These data have major implications for our understanding of coral symbiosis function during bleaching. Our findings suggest that during early stage bleaching, remaining symbionts continue to effectively translocate a variety of organic products to the host, however under prolonged thermal stress there is likely a reduction in the quality of these products.

Metabolite profiling of symbiont and host during thermal stress and bleaching in a model cnidarian-dinoflagellate symbiosis.

Bleaching (dinoflagellate symbiont loss) is one of the greatest threats facing coral reefs. The functional cnidarian-dinoflagellate symbiosis, which forms coral reefs, is based on the bi-directional exchange of nutrients. During thermal stress this exchange breaks down; however, major gaps remain in our understanding of the roles of free metabolite pools in symbiosis and homeostasis. In this study we applied gas chromatography-mass spectrometry (GC-MS) to explore thermally induced changes in intracellular pools of amino and non-amino organic acids in each partner of the model sea anemone Aiptasia sp. and its dinoflagellate symbiont. Elevated temperatures (32 °C for 6 days) resulted in symbiont photoinhibition and bleaching. Thermal stress induced distinct changes in the metabolite profiles of both partners, associated with alterations to central metabolism, oxidative state, cell structure, biosynthesis and signalling. Principally, we detected elevated pools of polyunsaturated fatty acids (PUFAs) in the symbiont, indicative of modifications to lipogenesis/lysis, membrane structure and nitrogen assimilation. In contrast, reductions of multiple PUFAs were detected in host pools, indicative of increased metabolism, peroxidation and/or reduced translocation of these groups. Accumulations of glycolysis intermediates were also observed in both partners, associated with photoinhibition and downstream reductions in carbohydrate metabolism. Correspondingly, we detected accumulations of amino acids and intermediate groups in both partners, with roles in gluconeogenesis and acclimation responses to oxidative stress. These data further our understanding of cellular responses to thermal stress in the symbiosis and generate hypotheses relating to the secondary roles of a number of compounds in homeostasis and heat-stress resistance.