Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

A genome-centric view of the role of the Acropora kenti microbiome in coral health and resilience.

Microbial diversity has been extensively explored in reef-building corals. However, the functional roles of coral-associated microorganisms remain poorly elucidated. Here, we recover 191 bacterial and 10 archaeal metagenome-assembled genomes (MAGs) from the coral Acropora kenti (formerly A. tenuis) and adjacent seawater, to identify microbial functions and metabolic interactions within the holobiont. We show that 82 MAGs were specific to the A. kenti holobiont, including members of the Pseudomonadota, Bacteroidota, and Desulfobacterota. A. kenti-specific MAGs displayed significant differences in their genomic features and functional potential relative to seawater-specific MAGs, with a higher prevalence of genes involved in host immune system evasion, nitrogen and carbon fixation, and synthesis of five essential B-vitamins. We find a diversity of A. kenti-specific MAGs encode the biosynthesis of essential amino acids, such as tryptophan, histidine, and lysine, which cannot be de novo synthesised by the host or Symbiodiniaceae. Across a water quality gradient spanning 2° of latitude, A. kenti microbial community composition is correlated to increased temperature and dissolved inorganic nitrogen, with corresponding enrichment in molecular chaperones, nitrate reductases, and a heat-shock protein. We reveal mechanisms of A. kenti-microbiome-symbiosis on the Great Barrier Reef, highlighting the interactions underpinning the health of this keystone holobiont.

Symbiodiniaceae diversity varies by host and environment across thermally distinct reefs.

Endosymbiotic dinoflagellates (Symbiodiniaceae) influence coral thermal tolerance at both local and regional scales. In isolation, the effects of host genetics, environment, and thermal disturbances on symbiont communities are well understood, yet their combined effects remain poorly resolved. Here, we investigate Symbiodiniaceae across 1300 km in Australia's Coral Sea Marine Park to disentangle these interactive effects. We identified Symbiodiniaceae to species-level resolution for three coral species (Acropora cf humilis, Pocillopora verrucosa, and Pocillopora meandrina) by sequencing two genetic markers of the symbiont (ITS2 and psbAncr), paired with genotype-by-sequencing of the coral host (DArT-seq). Our samples predominantly returned sequences from the genus Cladocopium, where Acropora cf humilis affiliated with C3k, Pocillopora verrucosa with C. pacificum, and Pocillopora meandrina with C. latusorum. Multivariate analyses revealed that Acropora symbionts were driven strongly by local environment and thermal disturbances. In contrast, Pocillopora symbiont communities were both partitioned 2.5-fold more by host genetic structure than by environmental structure. Among the two Pocillopora species, the effects of environment and host genetics explained four times more variation in symbionts for P. meandrina than P. verrucosa. The concurrent bleaching event in 2020 had variable impacts on symbiont communities, consistent with patterns in P. verrucosa and A. cf humilis, but not P. meandrina. Our findings demonstrate how symbiont macroscale community structure responses to environmental gradients depend on host species and their respective population structure. Integrating host, symbiont, and environmental data will help forecast the adaptive potential of corals and their symbionts amidst a rapidly changing environment.

The coral microbiome in sickness, in health and in a changing world.

Stony corals, the engines and engineers of reef ecosystems, face unprecedented threats from anthropogenic environmental change. Corals are holobionts that comprise the cnidarian animal host and a diverse community of bacteria, archaea, viruses and eukaryotic microorganisms. Recent research shows that the bacterial microbiome has a pivotal role in coral biology. A healthy bacterial assemblage contributes to nutrient cycling and stress resilience, but pollution, overfishing and climate change can break down these symbiotic relationships, which results in disease, bleaching and, ultimately, coral death. Although progress has been made in characterizing the spatial-temporal diversity of bacteria, we are only beginning to appreciate their functional contribution. In this Review, we summarize the ecological and metabolic interactions between bacteria and other holobiont members, highlight the biotic and abiotic factors influencing the structure of bacterial communities and discuss the impact of climate change on these communities and their coral hosts. We emphasize how microbiome-based interventions can help to decipher key mechanisms underpinning coral health and promote reef resilience. Finally, we explore how recent technological developments may be harnessed to address some of the most pressing challenges in coral microbiology, providing a road map for future research in this field.

The road forward to incorporate seawater microbes in predictive reef monitoring.

Marine bacterioplankton underpin the health and function of coral reefs and respond in a rapid and sensitive manner to environmental changes that affect reef ecosystem stability. Numerous meta-omics surveys over recent years have documented persistent associations of opportunistic seawater microbial taxa, and their associated functions, with metrics of environmental stress and poor reef health (e.g. elevated temperature, nutrient loads and macroalgae cover). Through positive feedback mechanisms, disturbance-triggered heterotrophic activity of seawater microbes is hypothesised to drive keystone benthic organisms towards the limit of their resilience and translate into shifts in biogeochemical cycles which influence marine food webs, ultimately affecting entire reef ecosystems. However, despite nearly two decades of work in this space, a major limitation to using seawater microbes in reef monitoring is a lack of a unified and focused approach that would move beyond the indicator discovery phase and towards the development of rapid microbial indicator assays for (near) real-time reef management and decision-making. By reviewing the current state of knowledge, we provide a comprehensive framework (defined as five phases of research and innovation) to catalyse a shift from fundamental to applied research, allowing us to move from descriptive to predictive reef monitoring, and from reactive to proactive reef management.

Microbes guide corals looking to find a home.

Coral reefs are facing unprecedented anthropogenic pressures impacting critical processes such as recruitment of juvenile corals. Through larval choice assays and co-occurrence network analyses, a recent study by Turnlund et al. identified microbial taxa within reef biofilms that positively correlate and therefore have potential key roles in inducing coral settlement.

Horizon scanning the application of probiotics for wildlife.

The provision of probiotics benefits the health of a wide range of organisms, from humans to animals and plants. Probiotics can enhance stress resilience of endangered organisms, many of which are critically threatened by anthropogenic impacts. The use of so-called 'probiotics for wildlife' is a nascent application, and the field needs to reflect on standards for its development, testing, validation, risk assessment, and deployment. Here, we identify the main challenges of this emerging intervention and provide a roadmap to validate the effectiveness of wildlife probiotics. We cover the essential use of inert negative controls in trials and the investigation of the probiotic mechanisms of action. We also suggest alternative microbial therapies that could be tested in parallel with the probiotic application. Our recommendations align approaches used for humans, aquaculture, and plants to the emerging concept and use of probiotics for wildlife.

A coral-associated actinobacterium mitigates coral bleaching under heat stress.

BACKGROUND: The positive effects of exposing corals to microorganisms have been reported though how the benefits are conferred are poorly understood. Here, we isolated an actinobacterial strain (SCSIO 13291) from Pocillopora damicornis with capabilities to synthesize antioxidants, vitamins, and antibacterial and antiviral compounds supported with phenotypic and/or genomic evidence. Strain SCSIO 13291 was labeled with 5 (and - 6)-carboxytetramethylrhodamine, succinimidyl ester and the labeled cell suspension directly inoculated onto the coral polyp tissues when nubbins were under thermal stress in a mesocosm experiment. We then visualized the labelled bacterial cells and analyzed the coral physiological, transcriptome and microbiome to elucidate the effect this strain conferred on the coral holobiont under thermal stress. RESULTS: Subsequent microscopic observations confirmed the presence of the bacterium attached to the coral polyps. Addition of the SCSIO 13291 strain reduced signs of bleaching in the corals subjected to heat stress. At the same time, alterations in gene expression, which were involved in reactive oxygen species and light damage mitigation, attenuated apoptosis and exocytosis in addition to metabolite utilization, were observed in the coral host and Symbiodiniaceae populations. In addition, the coral associated bacterial community altered with a more stable ecological network for samples inoculated with the bacterial strain. CONCLUSIONS: Our results provide insights into the benefits of a putative actinobacterial probiotic strain that mitigate coral bleaching signs. This study suggests that the inoculation of bacteria can potentially directly benefit the coral holobiont through conferring metabolic activities or through indirect mechanisms of suppling additional nutrient sources.

Validation of key sponge symbiont pathways using genome-centric metatranscriptomics.

The sponge microbiome underpins host function through provision and recycling of essential nutrients in a nutrient poor environment. Genomic data suggest that carbohydrate degradation, carbon fixation, nitrogen metabolism, sulphur metabolism and supplementation of B-vitamins are central microbial functions. However, validation beyond the genomic potential of sponge symbiont pathways is rarely explored. To evaluate metagenomic predictions, we sequenced the metagenomes and metatranscriptomes of three common coral reef sponges: Ircinia ramosa, Ircinia microconulosa and Phyllospongia foliascens. Multiple carbohydrate active enzymes were expressed by Poribacteria, Bacteroidota and Cyanobacteria symbionts, suggesting these lineages have a central role in assimilating dissolved organic matter. Expression of entire pathways for carbon fixation and multiple sulphur compound transformations were observed in all sponges. Gene expression for anaerobic nitrogen metabolism (denitrification and nitrate reduction) were more common than aerobic metabolism (nitrification), where only the I. ramosa microbiome expressed the nitrification pathway. Finally, while expression of the biosynthetic pathways for B-vitamins was common, the expression of additional transporter genes was far more limited. Overall, we highlight consistencies and disparities between metagenomic and metatranscriptomic results when inferring microbial activity, while uncovering new microbial taxa that contribute to the health of their sponge host via nutrient exchange.

Life-stage specificity and cross-generational climate effects on the microbiome of a tropical sea urchin (Echinodermata: Echinoidea).

Microbes play a critical role in the development and health of marine invertebrates, though microbial dynamics across life stages and host generations remain poorly understood in most reef species, especially in the context of climate change. Here, we use a 4-year multigenerational experiment to explore microbe-host interactions under the Intergovernmental Panel on Climate Change (IPCC)-forecast climate scenarios in the rock-boring tropical urchin Echinometra sp. A. Adult urchins (F0 ) were exposed for 18 months to increased temperature and pCO2 levels predicted for years 2050 and 2100 under RCP 8.5, a period which encompassed spawning. After rearing F1 offspring for a further 2 years, spawning was induced, and F2 larvae were raised under current day and 2100 conditions. Cross-generational climate effects were also explored in the microbiome of F1 offspring through a transplant experiment. Using 16S rRNA gene sequence analysis, we determined that each life stage and generation was associated with a distinct microbiome, with higher microbial diversity observed in juveniles compared to larval stages. Although life-stage specificity was conserved under climate conditions projected for 2050 and 2100, we observed changes in the urchin microbial community structure within life stages. Furthermore, we detected a climate-mediated parental effect when juveniles were transplanted among climate treatments, with the parental climate treatment influencing the offspring microbiome. Our findings reveal a potential for cross-generational impacts of climate change on the microbiome of a tropical invertebrate species.

The effects of marine heatwaves on acute heat tolerance in corals.

Scleractinian coral populations are increasingly exposed to conditions above their upper thermal limits due to marine heatwaves, contributing to global declines of coral reef ecosystem health. However, historic mass bleaching events indicate there is considerable inter- and intra-specific variation in thermal tolerance whereby species, individual coral colonies and populations show differential susceptibility to exposure to elevated temperatures. Despite this, we lack a clear understanding of how heat tolerance varies across large contemporary and historical environmental gradients, or the selective pressures that underpin this variation. Here we conducted standardised acute heat stress experiments to identify variation in heat tolerance among species and isolated reefs spanning a large environmental gradient across the Coral Sea Marine Park. We quantified the photochemical yield (Fv /Fm ) of coral samples in three coral species, Acropora cf humilis, Pocillopora meandrina, and Pocillopora verrucosa, following exposure to four temperature treatments (local ambient temperatures, and + 3°C, +6°C and + 9°C above local maximum monthly mean). We quantified the temperature at which Fv /Fm decreased by 50% (termed ED50) and used derived values to directly compare acute heat tolerance across reefs and species. The ED50 for Acropora was 0.4-0.7°C lower than either Pocillopora species, with a 0.3°C difference between the two Pocillopora species. We also recorded 0.9°C to 1.9°C phenotypic variation in heat tolerance among reefs within species, indicating spatial heterogeneity in heat tolerance across broad environmental gradients. Acute heat tolerance had a strong positive relationship to mild heatwave exposure over the past 35 years (since 1986) but was negatively related to recent severe heatwaves (2016-2020). Phenotypic variation associated with mild thermal history in local environments provides supportive evidence that marine heatwaves are selecting for tolerant individuals and populations; however, this adaptive potential may be compromised by the exposure to recent severe heatwaves.

Coral restoration and adaptation in Australia: The first five years.

While coral reefs in Australia have historically been a showcase of conventional management informed by research, recent declines in coral cover have triggered efforts to innovate and integrate intervention and restoration actions into management frameworks. Here we outline the multi-faceted intervention approaches that have developed in Australia since 2017, from newly implemented in-water programs, research to enhance coral resilience and investigations into socio-economic perspectives on restoration goals. We describe in-water projects using coral gardening, substrate stabilisation, coral repositioning, macro-algae removal, and larval-based restoration techniques. Three areas of research focus are also presented to illustrate the breadth of Australian research on coral restoration, (1) the transdisciplinary Reef Restoration and Adaptation Program (RRAP), one of the world's largest research and development programs focused on coral reefs, (2) interventions to enhance coral performance under climate change, and (3) research into socio-cultural perspectives. Together, these projects and the recent research focus reflect an increasing urgency for action to confront the coral reef crisis, develop new and additional tools to manage coral reefs, and the consequent increase in funding opportunities and management appetite for implementation. The rapid progress in trialling and deploying coral restoration in Australia builds on decades of overseas experience, and advances in research and development are showing positive signs that coral restoration can be a valuable tool to improve resilience at local scales (i.e., high early survival rates across a variety of methods and coral species, strong community engagement with local stakeholders). RRAP is focused on creating interventions to help coral reefs at multiple scales, from micro scales (i.e., interventions targeting small areas within a specific reef site) to large scales (i.e., interventions targeting core ecosystem function and social-economic values at multiple select sites across the Great Barrier Reef) to resist, adapt to and recover from the impacts of climate change. None of these interventions aim to single-handedly restore the entirety of the Great Barrier Reef, nor do they negate the importance of urgent climate change mitigation action.

The pulsating soft coral Xenia umbellata shows high resistance to warming when nitrate concentrations are low.

The resistance of hard corals to warming can be negatively affected by nitrate eutrophication, but related knowledge for soft corals is scarce. We thus investigated the ecophysiological response of the pulsating soft coral Xenia umbellata to different levels of nitrate eutrophication (control = 0.6, medium = 6, high = 37 µM nitrate) in a laboratory experiment, with additional warming (27.7 to 32.8 °C) from days 17 to 37. High nitrate eutrophication enhanced cellular chlorophyll a content of Symbiodiniaceae by 168%, while it reduced gross photosynthesis by 56%. After additional warming, polyp pulsation rate was reduced by 100% in both nitrate eutrophication treatments, and additional polyp loss of 7% d-1 and total fragment mortality of 26% was observed in the high nitrate eutrophication treatment. Warming alone did not affect any of the investigated response parameters. These results suggest that X. umbellata exhibits resistance to warming, which may facilitate ecological dominance over some hard corals as ocean temperatures warm, though a clear negative physiological response occurs when combined with nitrate eutrophication. This study thus confirms the importance of investigating combinations of global and local factors to understand and manage changing coral reefs.

Algal turf structure and composition vary with particulate loads on coral reefs.

Algal turfs trap and retain particulates, however, little is known about the relationship between particulate accumulation and taxonomic composition of algal turfs. We investigated how particulate mass related to algal turf structure (length and density) and community composition (taxonomic and functional) on two disparate reefs. Particulate mass was positively related to algal turf length. By contrast, the relationship between particulate mass and turf density was more complex and followed a negative parabolic shape; density increased with particulate mass before stabilising and then declining. Community analyses showed taxonomic, but not functional group compositions differed significantly between reefs and with increasing particulate mass. Our results suggest high loads of particulates accumulated in algal turfs are related to a longer, lower density turf structure, typified by filamentous forms such as Cladophora. Changes in algal turf structure and composition could have a variety of bottom-up influences on coral reef ecosystems.

A High-Throughput LC-MS/MS Method for the Simultaneous Quantification of Twenty-Seven Drug Molecules in Ocular Tissues.

The purpose of this study was to develop a validated LC-MS/MS analytical method for the simultaneous analysis of a large cassette containing a wide range of drug substances with positive, negative, or neutral charge and further apply the method to assess octanol partition coefficient and eye tissue recovery of the drug cassette. A twenty-seven-drug cassette (N-in-one) including beta blockers, NSAIDs, and corticosteroids that range from extremely hydrophilic (sotalol) to very hydrophobic (triamcinolone hexacetanide) was used to develop an LC-MS/MS assay using QTrap 4500. An LC-MS/MS method based on gradient elution, with an eighteen-minute run time including equilibration time, was developed and validated for the rapid and simultaneous quantitation of drugs with a wide range of lipophilicities. Scheduled multiple reaction monitoring was used to maximize the scan time for each peak, ensuring sufficient scans. Method validation included lower limit of quantitation (LLOQ) and intra- and inter-day reproducibility. The LLOQ ranged from 0.5 (sotalol) to 40 fmols (dexamethasone) on column with a %RSD < 20%. The method was tested by measuring octanol:water and octanol:buffer (PBS, pH 7.4) partition coefficients and by quantitation of the drug cassette extracted from rabbit aqueous humor and cornea. Measured partition coefficients correlated positively with predicted values (r2=0.5-0.7). Drug recovery was ≥ 79% from aqueous humor and between 61 and 67% on average from cornea. A rapid, sensitive LC-MS/MS method suitable for N-in-one drug delivery screening was developed for simultaneous quantification of twenty-seven drugs in aqueous solutions and eye tissues.

High-resolution spatial and genomic characterization of coral-associated microbial aggregates in the coral Stylophora pistillata.

Bacteria commonly form aggregates in a range of coral species [termed coral-associated microbial aggregates (CAMAs)], although these structures remain poorly characterized despite extensive efforts studying the coral microbiome. Here, we comprehensively characterize CAMAs associated with Stylophora pistillata and quantify their cell abundance. Our analysis reveals that multiple Endozoicomonas phylotypes coexist inside a single CAMA. Nanoscale secondary ion mass spectrometry imaging revealed that the Endozoicomonas cells were enriched with phosphorus, with the elemental compositions of CAMAs different from coral tissues and endosymbiotic Symbiodiniaceae, highlighting a role in sequestering and cycling phosphate between coral holobiont partners. Consensus metagenome-assembled genomes of the two dominant Endozoicomonas phylotypes confirmed their metabolic potential for polyphosphate accumulation along with genomic signatures including type VI secretion systems allowing host association. Our findings provide unprecedented insights into Endozoicomonas-dominated CAMAs and the first direct physiological and genomic linked evidence of their biological role in the coral holobiont.

Cultured Bacteria Provide Insight into the Functional Potential of the Coral-Associated Microbiome.

Improving the availability of representative isolates from the coral microbiome is essential for investigating symbiotic mechanisms and applying beneficial microorganisms to improve coral health. However, few studies have explored the diversity of bacteria which can be isolated from a single species. Here, we isolated a total of 395 bacterial strains affiliated with 49 families across nine classes from the coral Pocillopora damicornis. Identification results showed that most of the strains represent potential novel bacterial species or genera. We also sequenced and assembled the genomes of 118 of these isolates, and then the putative functions of these isolates were identified based on genetic signatures derived from the genomes and this information was combined with isolate-specific phenotypic data. Genomic information derived from the isolates identified putative functions including nitrification and denitrification, dimethylsulfoniopropionate transformation, and supply of fixed carbon, amino acids, and B vitamins which may support their eukaryotic partners. Furthermore, the isolates contained genes associated with chemotaxis, biofilm formation, quorum sensing, membrane transport, signal transduction, and eukaryote-like repeat-containing and cell-cell attachment proteins, all of which potentially help the bacterium establish association with the coral host. Our work expands on the existing culture collection of coral-associated bacteria and provides important information on the metabolic potential of these isolates which can be used to refine understanding of the role of bacteria in coral health and are now available to be applied to novel strategies aimed at improving coral resilience through microbiome manipulation. IMPORTANCE Microbes underpin the health of corals which are the building blocks of diverse and productive reef ecosystems. Studying the culturable fraction of coral-associated bacteria has received less attention in recent times than using culture-independent molecular methods. However, the genomic and phenotypic characterization of isolated strains allows assessment of their functional role in underpinning coral health and identification of beneficial microbes for microbiome manipulation. Here, we isolated 395 bacterial strains from tissues of Pocillopora damicornis with many representing potentially novel taxa and therefore providing a significant contribution to coral microbiology through greatly enlarging the existing cultured coral-associated bacterial bank. Through analysis of the genomes obtained in this study for the coral-associated bacteria and coral host, we elucidate putative metabolic linkages and symbiotic establishment. The results of this study will help to elucidate the role of specific isolates in coral health and provide beneficial microbes for efforts aimed at improving coral health.

Management and Associated Toxicokinetics of Massive Valproic Acid Ingestion with High Flow Continuous Venovenous Hemodiafiltration.

INTRODUCTION: Valproic acid (VPA) toxicity commonly results in a self-limited state of CNS depression that is managed with supportive care and levocarnitine. In massive overdose, patients can develop toxic encephalopathy, shock, multisystem organ failure, and death. We present a case with relevant toxicokinetics of a patient presenting with a profoundly elevated VPA concentration resulting in survival, treated with supportive care including high-dose continuous venovenous hemodiafiltration (CVVHDF). CASE REPORT: A 17-year-old female presented to an emergency department after being found unresponsive at home with concern for massive VPA ingestion. She arrived obtunded and hypotensive with initial VPA concentration of 2226 mg/L, estimated 9 h post-ingestion. Her early hospital course was marked by hypotension requiring multiple vasopressors, and her workup was notable for multiple severe metabolic derangements. High-dose CVVHDF was initiated upon transfer to a tertiary children's hospital with the aim to enhance VPA removal and normalize metabolic derangements. At that time, her VPA concentration was 1071 mg/L. Apparent half-life of VPA improved modestly with extracorporeal treatment, but her metabolic derangements and hemodynamic instability corrected rapidly. Her clinical course was complicated by necrotizing pancreatitis, pancytopenia requiring transfusions of multiple cell lines, coma, and seizures. She ultimately recovered with normal neurological function.

Probiotics for coral aquaculture: challenges and considerations.

Globally, coral reefs are under pressure from climate change, with concerning declines in coral abundance observed due to increasing cumulative impacts. Active intervention measures that mitigate the declines are increasingly being applied to buy time for coral reefs as the world transitions to a low-carbon economy. One such mitigation strategy is coral restoration based on large-scale coral aquaculture to provide stock for reseeding reefs, with the added potential of selecting corals that better tolerate environmental stress. Application of probiotics during production and deployment, to modulate the naturally occurring bacteria associated with corals, may confer health benefits such as disease resistance, increased environmental tolerance or improved coral nutrition. Here, we briefly describe coral associated bacteria and their role in the coral holobiont, identify probiotics traits potentially beneficial to coral, and discuss current research directions required to develop, test and verify the feasibility for probiotics to improve coral aquaculture at industrial scales.

Surface Modification of Erythrocytes with Lipid Anchors: Structure-Activity Relationship for Optimal Membrane Incorporation, in vivo Retention, and Immunocompatibility.

Red blood cells (RBCs) are natural carriers for sustained drug delivery, imaging, and in vivo sensing. One of the popular approaches to functionalize RBCs is through lipophilic anchors, but the structural requirements for anchor stability and in vivo longevity remain to be investigated. Using fluorescent lipids with the same cyanine 3 (Cy3) headgroup but different lipid chain and linker, the labeling efficiency of RBCs and in vivo stability are investigated. Short-chain derivatives exhibited better insertion efficiency, and mouse RBCs are better labeled than human RBCs. Short-chain derivatives demonstrate low retention in vivo. Derivatives with ester bonds are especially unstable, due to removal and degradation. On the other hand, long-chain, covalently linked derivatives show remarkably long retention and stability (over 80 days half life in the membrane). The clearance organs are liver and spleen with evidence of lipid transfer to the liver sinusoidal endothelium. Notably, RBCs modified with PEGylated lipid show decreased macrophage uptake. Some of the derivatives promote binding of antibodies in human plasma and mouse sera and modest increase in complement deposition and hemolysis, but these do not correlate with in vivo stability of RBCs. Ultra-stable anchors can enable functionalization of RBCs for drug delivery, imaging, and sensing.