Multiple Stressors Drive Changes in Coral Microbiome Diversity and Composition Demonstrating the Role of a Bacterial Symbiont in Microbial Community Sensitivity and Resilience

On coral reefs, disturbances rarely occur in isolation. Global stressors such as increasing seawater temperature often coincide with local stressors like nutrient pollution. In the face of increasing anthropogenic stress, corals can function as environmental sentinels, although little is known about how multiple stressors interact to disrupt their associated bacterial communities and how symbiotic bacteria contribute to coral resistance and resilience to stress. Previous studies have shown that coral microbiomes under stress demonstrate shifts to more disease-associated states, increases in community variability, compromised function of beneficial microbiota, and selection for potentially pathogenic bacteria. Given that coral microbiota are thought to play an important role in host nutrient cycling and antimicrobial protection, it is important to understand how their response to individual and combined stressors can mitigate or exacerbate host susceptibility. My dissertation work coupled ecological experiments with DNA sequencing, bioinformatics, and multivariate statistics to describe compositional changes in coral microbiomes under various combinations of stressors and to infer the role of symbiotic bacteria in holobiont sensitivity and resilience to environmental disturbance. Ecological literature classifies interactions between combinations of individual stressors as synergistic, antagonistic, or having no interaction. We utilized common multivariate analyses to apply this multiple stressor framework to host-associated microbial communities. We conducted multiple stressor experiments both in mesocosm tanks and in the field on corals in Mo’orea, French Polynesia to evaluate how their microbiomes change compositionally with increasing and interacting levels of perturbation. We applied either no stress or single, double, or triple stressors of nutrient enrichment, simulated predation, and increased seawater temperature in mesocosm tanks to the coral Pocillopora meandrina for 21 days. We predicted that when sequentially adding stressors to the experimental system, we would observe a compounding increase in changes to the microbiome. In contrast, we found that microbiome disruption or variation from healthy controls does not scale positively with increasing number of stressors. In contrast to more heterogeneous communities that may be more robust to changes due to high diversity and functional redundancy, healthy P. meandrina controls were so dominated by Endozoicomonas, a proposed beneficial symbiont, such that any amount or type of stress was sufficient to increase community evenness. Single stressors of high temperature and scarring produced the largest shifts in community structure and additional stressors acted antagonistically to produce a less-than-additive response. We found that microbiome variability or dispersion increased with any number of stressors and community dysbiosis is characterized by a proliferation of opportunistic bacteria such as Rhodobacteraceae and Desulfovibrionaceae rather than a depletion of the dominant taxon, Endozoicomonas. To study this phenomenon in situ, we enriched corals on the reef before, during, and after a natural thermal stress event in 2016 to observe changes in the microbiome of Acropora, Pocillopora, and Porites corals undergoing concurrent temperature and nutrient stress. Concurrent work showed that corals exposed to nitrate exhibited more frequent bleaching, bleaching for longer duration, and were more likely to die. However, as observed in the tank experiment, we found that nutrients are less important for shaping the microbial community than temperature. The addition of nitrate and urea to the water column had no effect on the coral microbiome and did not interact with temperature. The three coral hosts displayed varying degrees of sensitivity to warm temperatures suggesting alternate strategies for coping with stress. Overall, post-stress microbiomes did not return to pre-stress community composition, but rather were less diverse and increasingly dominated by Endozoicomonas which could suggest its ability to utilize host metabolic products of thermal stress for a sustained competitive advantage against other microbial taxa. While we are only beginning to uncover the functional role of Endozoicomonas, these results suggest it may contribute to microbiome resilience to thermal stress. Endozoicomonas species associate with a wide variety of marine hosts, and within corals, are hypothesized to breakdown dimethylsulfoniopropionate (DMSP) which the coral host produces during thermal stress. To investigate this role and other potential roles in nutrition and defense, I used culture-independent methods of genome sequencing, metagenomics, to assemble the Endozoicomonas symbiont residing in Pocillopora meandrina corals in Mo’orea. Comparative genomics was subsequently conducted on all available Endozoicomonas genomes to better understand their shared and distinct functional characteristics. Despite their large genomes, Endozoicomonas species do not appear to contribute significantly to the cycling of carbon, nitrogen, or sulfur within the holobiont. Instead, they have many genomic features that could facilitate the maintenance of a stable symbiosis with their marine eukaryotic hosts. Only two Endozoicomonas species, not including our MAG (metagenome-assembled genome), encode genes involved in the breakdown of DMSP. Thus, we found no obvious genomic features that would suggest susceptibility or tolerance of this symbiont to thermal stress. In this dissertation, I demonstrate how a dominant member of the coral host-associated microbial community can drive patterns in community diversity and variability, ultimately influencing community resistance and resilience. While I investigate the genetic potential of this dominant taxon, genomic features that contribute to community sensitivity or resilience remain elusive. Overall, this work highlights the complexity of possible interaction outcomes for environmental stressors to coral reefs when assessed at the microbial scale and presents foundational patterns in microbiome dysbiosis and recovery that can inform reef persistence into the future

Macroborer Presence on Corals Increases with Nutrient Input and Promotes Parrotfish Bioerosion

Bioerosion by reef-dwelling organisms influences net carbonate budgets on reefs worldwide. External bioeroders, such as parrotfish and sea urchins, and internal bioeroders, including sponges and lithophagid bivalves, are major contributors to bioerosion on reefs. Despite their importance, few studies have examined how environmental (e.g., nutrients) or biological drivers (e.g., the actions of other bioeroders) may influence bioeroder dynamics on reefs. For example, internal bioeroders could promote external bioerosion by weakening the coral skeletal matrix. Our study investigated: ( 1) whether nutrient supply influences the dynamics between internal and external bioeroders and ( 2) how the presence of a boring bivalve, Lithophaga spp., influences parrotfish bioerosion on massive Porites corals. We hypothesized that nutrient supply would be positively correlated with Lithophaga densities on massive Porites colonies, and that as bivalve density increased, the frequency and intensity of parrotfish bioerosion would increase. To test these hypotheses, we analyzed six time points over a 10-yr period from a time series of benthic images and nitrogen content of a dominant macroalga from the fringing reefs around Moorea, French Polynesia. We found Lithophaga densities were positively correlated with nitrogen availability. Further, massive Porites that are more infested with Lithophaga had both a higher probability of being bitten by parrotfish and a higher density of bite scars from parrotfishes. Our findings indicate that increasing nutrient availability may strengthen the relationship between internal and external bioeroders, suggesting that colonies at more eutrophic sites may experience higher bioerosion rates

DNM1 encephalopathy: A new disease of vesicle fission.

ObjectiveTo evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling.MethodsWe reviewed phenotypic data of 21 patients (7 previously published) with DNM1 mutations. We compared mutation data to known functional data and undertook biomolecular modeling to assess the effect of the mutations on protein function.ResultsWe identified 19 patients with de novo mutations in DNM1 and a sibling pair who had an inherited mutation from a mosaic parent. Seven patients (33.3%) carried the recurrent p.Arg237Trp mutation. A common phenotype emerged that included severe to profound intellectual disability and muscular hypotonia in all patients and an epilepsy characterized by infantile spasms in 16 of 21 patients, frequently evolving into Lennox-Gastaut syndrome. Two patients had profound global developmental delay without seizures. In addition, we describe a single patient with normal development before the onset of a catastrophic epilepsy, consistent with febrile infection-related epilepsy syndrome at 4 years. All mutations cluster within the GTPase or middle domains, and structural modeling and existing functional data suggest a dominant-negative effect on DMN1 function.ConclusionsThe phenotypic spectrum of DNM1-related encephalopathy is relatively homogeneous, in contrast to many other genetic epilepsies. Up to one-third of patients carry the recurrent p.Arg237Trp variant, which is now one of the most common recurrent variants in epileptic encephalopathies identified to date. Given the predicted dominant-negative mechanism of this mutation, this variant presents a prime target for therapeutic intervention

Mutations in NLRP5 are associated with reproductive wastage and multilocus imprinting disorders in humans

This is the final version. It first appeared at http://www.nature.com/ncomms/2015/150901/ncomms9086/full/ncomms9086.html.Human-imprinting disorders are congenital disorders of growth, development and metabolism, associated with disturbance of parent of origin-specific DNA methylation at imprinted loci across the genome. Some imprinting disorders have higher than expected prevalence of monozygotic twinning, of assisted reproductive technology among parents, and of disturbance of multiple imprinted loci, for which few causative trans-acting mutations have been found. Here we report mutations in NLRP5 in five mothers of individuals affected by multilocus imprinting disturbance. Maternal-effect mutations of other human NLRP genes, NLRP7 and NLRP2, cause familial biparental hydatidiform mole and multilocus imprinting disturbance, respectively. Offspring of mothers with NLRP5 mutations have heterogenous clinical and epigenetic features, but cases include a discordant monozygotic twin pair, individuals with idiopathic developmental delay and autism, and families affected by infertility and reproductive wastage. NLRP5 mutations suggest connections between maternal reproductive fitness, early zygotic development and genomic imprinting.L.E.D. and F.I.R. were supported by the Medical Research Council (MR/J000329/1). J.B., K.B., B.H., L.S. M.B. and T.E. were supported by Bundesministerium fu?r Bildung und Forschung (grant number 01GM1513A and 01GM1513C) and C.T. was supported by an Ipsen Fellowship Grant. The cohort ?Imprinting Disorders-Finding out Why? was accrued through the support of the Newlife Foundation for Disabled Children and through support from the Wessex NIHR clinical research network and NIHR Wellcome Southampton clinical research facility. Funding for DNA collection and methylation analysis of normal control samples was provided in part by the National Institutes of Health R01 AI091905-01, R01 AI061471 and R01 HL082925. ERM thanks Action Medical Research for support

Alzheimer\u27s Disease cerebrospinal fluid biomarkers are not influenced by gravity drip or aspiration extraction methodology

Introduction Cerebrospinal fluid (CSF) biomarkers, although of established utility in the diagnostic evaluation of Alzheimer’s disease (AD), are known to be sensitive to variation based on pre-analytical sample processing. We assessed whether gravity droplet collection versus syringe aspiration was another factor influencing CSF biomarker analyte concentrations and reproducibility. Methods Standardized lumbar puncture using small calibre atraumatic spinal needles and CSF collection using gravity fed collection followed by syringe aspirated extraction was performed in a sample of elderly individuals participating in a large long-term observational research trial. Analyte assay concentrations were compared. Results For the 44 total paired samples of gravity collection and aspiration, reproducibility was high for biomarker CSF analyte assay concentrations (concordance correlation [95%CI]: beta-amyloid1-42 (Aβ42) 0.83 [0.71 - 0.90]), t-tau 0.99 [0.98 - 0.99], and phosphorylated tau (p-tau) 0.82 [95 % CI 0.71 - 0.89]) and Bonferroni corrected paired sample t-tests showed no significant differences (group means (SD): Aβ42 366.5 (86.8) vs 354.3 (82.6), p = 0.10; t-tau 83.9 (46.6) vs 84.7 (47.4) p = 0.49; p-tau 43.5 (22.8) vs 40.0 (17.7), p = 0.05). The mean duration of collection was 10.9 minutes for gravity collection andaspiration. Conclusions Our results demonstrate that aspiration of CSF is comparable to gravity droplet collection for AD biomarker analyses but could considerably accelerate throughput and improve the procedural tolerability for assessment of CSF biomarkers. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated

A model for presenting accelerometer paradata in large studies : ISCOLE

Background: We present a model for reporting accelerometer paradata (process-related data produced from survey administration) collected in the International Study of Childhood Obesity Lifestyle and the Environment (ISCOLE), a multi-national investigation of >7000 children (averaging 10.5 years of age) sampled from 12 different developed and developing countries and five continents. Methods: ISCOLE employed a 24-hr waist worn 7-day protocol using the ActiGraph GT3X+. Checklists, flow charts, and systematic data queries documented accelerometer paradata from enrollment to data collection and treatment. Paradata included counts of consented and eligible participants, accelerometers distributed for initial and additional monitoring (site specific decisions in the face of initial monitoring failure), inadequate data (e.g., lost/malfunction, insufficient wear time), and averages for waking wear time, valid days of data, participants with valid data (>= 4 valid days of data, including 1 weekend day), and minutes with implausibly high values (>= 20,000 activity counts/min). Results: Of 7806 consented participants, 7372 were deemed eligible to participate, 7314 accelerometers were distributed for initial monitoring and another 106 for additional monitoring. 414 accelerometer data files were inadequate (primarily due to insufficient wear time). Only 29 accelerometers were lost during the implementation of ISCOLE worldwide. The final locked data file consisted of 6553 participant files (90.0% relative to number of participants who completed monitoring) with valid waking wear time, averaging 6.5 valid days and 888.4 minutes/day (14.8 hours). We documented 4762 minutes with implausibly high activity count values from 695 unique participants (9.4% of eligible participants and Conclusions: Detailed accelerometer paradata is useful for standardizing communication, facilitating study management, improving the representative qualities of surveys, tracking study endpoint attainment, comparing studies, and ultimately anticipating and controlling costs.Peer reviewe

Improving wear time compliance with a 24-hour waist-worn accelerometer protocol in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE)

Background: We compared 24-hour waist-worn accelerometer wear time characteristics of 9-11 year old children in the International Study of Childhood Obesity, Lifestyle and the Environment (ISCOLE) to similarly aged U.S. children providing waking-hours waist-worn accelerometer data in the 2003-2006 National Health and Nutrition Examination Survey (NHANES). Methods: Valid cases were defined as having >= 4 days with >= 10 hours of waking wear time in a 24-hour period, including one weekend day. Previously published algorithms for extracting total sleep episode time from 24-hour accelerometer data and for identifying wear time (in both the 24-hour and waking-hours protocols) were applied. The number of valid days obtained and a ratio (percent) of valid cases to the number of participants originally wearing an accelerometer were computed for both ISCOLE and NHANES. Given the two surveys' discrepant sampling designs, wear time (minutes/day, hours/day) from U.S. ISCOLE was compared to NHANES using a meta-analytic approach. Wear time for the 11 additional countries participating in ISCOLE were graphically compared with NHANES. Results: 491 U.S. ISCOLE children (9.92 +/- 0.03 years of age [M +/- SE]) and 586 NHANES children (10.43 +/- 0.04 years of age) were deemed valid cases. The ratio of valid cases to the number of participants originally wearing an accelerometer was 76.7% in U.S. ISCOLE and 62.6% in NHANES. Wear time averaged 1357.0 +/- 4.2 minutes per 24-hour day in ISCOLE. Waking wear time was 884.4 +/- 2.2 minutes/day for U.S. ISCOLE children and 822.6 +/- 4.3 minutes/day in NHANES children (difference = 61.8 minutes/day, p <0.001). Wear time characteristics were consistently higher in all ISCOLE study sites compared to the NHANES protocol. Conclusions: A 24-hour waist-worn accelerometry protocol implemented in U.S. children produced 22.6 out of 24 hours of possible wear time, and 61.8 more minutes/day of waking wear time than a similarly implemented and processed waking wear time waist-worn accelerometry protocol. Consistent results were obtained internationally. The 24-hour protocol may produce an important increase in wear time compliance that also provides an opportunity to study the total sleep episode time separate and distinct from physical activity and sedentary time detected during waking-hours.Peer reviewe