Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq’s reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.

Understanding the response of the coral holobiont to environmental change is crucial to inform conservation efforts. The most pressing problem is “coral bleaching,” usually precipitated by prolonged thermal stress. We used untargeted, polar metabolite profiling to investigate the physiological response of the coral species Montipora capitata and Pocillopora acuta to heat stress. Our goal was to identify diagnostic markers present early in the bleaching response. From the untargeted UHPLC-MS data, a variety of co-regulated dipeptides were found that have the highest differential accumulation in both species. The structures of four dipeptides were determined and showed differential accumulation in symbiotic and aposymbiotic (alga-free) populations of the sea anemone Aiptasia (Exaiptasia pallida), suggesting the deep evolutionary origins of these dipeptides and their involvement in symbiosis. These and other metabolites may be used as diagnostic markers for thermal stress in wild coral.

While SARS-CoV-2 has sporadically infected a wide range of animal species worldwide1, the virus has been repeatedly and frequently detected in white-tailed deer in North America2–7. The zoonotic origins of this pandemic virus highlight the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, which disseminated across hundreds of kilometers. We discovered that alpha and delta variants evolved in white-tailed deer at three-times the rate observed in humans. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only faster in white-tailed deer but driven by different mutational biases and selection pressures. White-tailed deer are not just short-term recipients of human viral diversity but serve as reservoirs for alpha and other variants to evolve in new directions after going extinct in humans. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal model experiments using viruses isolated from white-tailed deer. Still, SARS-CoV-2 viruses have transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Background Corals, which form the foundation of biodiverse reef ecosystems, are under threat from warming oceans. Reefs provide essential ecological services, including food, income from tourism, nutrient cycling, waste removal, and the absorption of wave energy to mitigate erosion. Here, we studied the coral thermal stress response using network methods to analyze transcriptomic and polar metabolomic data generated from the Hawaiian rice coral Montipora capitata. Coral nubbins were exposed to ambient or thermal stress conditions over a 5-week period, coinciding with a mass spawning event of this species. The major goal of our study was to expand the inventory of thermal stress-related genes and metabolites present in M. capitata and to study gene-metabolite interactions. These interactions provide the foundation for functional or genetic analysis of key coral genes as well as provide potentially diagnostic markers of pre-bleaching stress. A secondary goal of our study was to analyze the accumulation of sex hormones prior to and during mass spawning to understand how thermal stress may impact reproductive success in M. capitata. Methods M. capitata was exposed to thermal stress during its spawning cycle over the course of 5 weeks, during which time transcriptomic and polar metabolomic data were collected. We analyzed these data streams individually, and then integrated both data sets using MAGI (Metabolite Annotation and Gene Integration) to investigate molecular transitions and biochemical reactions. Results Our results reveal the complexity of the thermal stress phenome in M. capitata, which includes many genes involved in redox regulation, biomineralization, and reproduction. The size and number of modules in the gene co-expression networks expanded from the initial stress response to the onset of bleaching. The later stages involved the suppression of metabolite transport by the coral host, including a variety of sodium-coupled transporters and a putative ammonium transporter, possibly as a response to reduction in algal productivity. The gene-metabolite integration data suggest that thermal treatment results in the activation of animal redox stress pathways involved in quenching molecular oxygen to prevent an overabundance of reactive oxygen species. Lastly, evidence that thermal stress affects reproductive activity was provided by the downregulation of CYP-like genes and the irregular production of sex hormones during the mass spawning cycle. Overall, redox regulation and metabolite transport are key components of the coral animal thermal stress phenome. Mass spawning was highly attenuated under thermal stress, suggesting that global climate change may negatively impact reproductive behavior in this species.

23Coral reef systems are under global threat due to warming and acidifying oceans 1 . 24Understanding the response of the coral holobiont to environmental change is crucial to aid 25 conservation efforts. The most pressing problem is "coral bleaching", usually precipitated 26 by prolonged thermal stress that disrupts the algal symbiosis sustaining the holobiont 2,3 . We 27 used metabolomics to understand how the coral holobiont metabolome responds to heat 28 stress with the goal of identifying diagnostic markers prior to bleaching onset. We studied 29 the heat tolerant Montipora capitata and heat sensitive Pocillopora acuta coral species from 30 the Hawaiian reef system in Kāne'ohe Bay, O'ahu. Untargeted LC-MS analysis uncovered 31 both known and novel metabolites that accumulate during heat stress. Among those showing 32 the highest differential accumulation were a variety of co-regulated dipeptides present in 33 both species. The structures of four of these compounds were determined (Arginine-34 Glutamine, Lysine-Glutamine, Arginine-Valine, and Arginine-Alanine). These dipeptides 35 also showed differential accumulation in symbiotic and aposymbiotic (alga free) individuals 36 of the sea anemone model Aiptasia 4 , suggesting their animal provenance and algal symbiont 37 related function. Our results identify a suite of metabolites associated with thermal stress 38 that can be used to diagnose coral health in wild samples. 39 40 The exchange of metabolites, either between organism-environment or organism-organism, gave 41 rise not only to early life, but complex life systems on Earth. Examples include bacterial deep vent 42communities 5 , the plant rhizosphere 6 , the human microbiome 7 , and the coral holobiont 8 . Exchange 43 of metabolites between stony corals (Scleractinia), their dinoflagellate algal photosymbionts 44 (Symbiodiniaceae), and associated microbes is the foundation for modern coral reef ecosystems 1,9 45 that cover ca. 255,000 km 2 of the planet surface 10 . Under ambient conditions, the algal cells 46 provide 90-95% of host energy needs in the form of lipids, carbohydrates, amino acids, and O2 11 . 47

The response of coral reef ecosystems to anthropogenic climate change is driven by a complex interaction between location, stress history, species composition, and genetic background of the reef system. The latter two factors are particularly relevant when considering the different reproductive strategies used by coral species. We studied the stress resistant coral Montipora capitata and the more stress sensitive Pocillopora acuta from Kāneʻohe Bay, Oʻahu, Hawaiʻi. High-quality genome assemblies were generated for both species with the M. capitata assembly at chromosome-level resolution and the P. acuta assembly derived from a triploid colony, representing the first non-diploid genome generated from a coral. We report significant differences in the reproductive strategies of these coral species that not only affect the genetic structure of their populations in Kāneʻohe Bay, but also impact algal symbiont composition. Single-nucleotide polymorphism analysis shows that P. acuta comprises at least nine distinct genotypes in the bay with ancestral diploid and derived triploid lineages. In contrast, M. capitata colonies are diploids with nearly all being genotypically distinct. Genotype has a strong effect on gene expression profiles in these species, largely outweighing the effects of environmental stress. Our insights advance understanding of how reproductive strategy and ploidy can vary between different coral species and among local populations, how these factors constrain coral holobiont genetic diversity, and how genotype constrains genome-wide gene expression.

Coral bleaching, precipitated by the expulsion of the algal symbionts that provide colonies with fixed carbon is a global threat to reef survival. To protect corals from anthropogenic stress, portable tools are needed to detect and diagnose stress syndromes and assess population health prior to extensive bleaching. Here, medical grade Urinalysis strips, used to detect an array of disease markers in humans, were tested on the lab stressed Hawaiian coral species, Montipora capitata (stress resistant) and Pocillopora acuta (stress sensitive), as well as samples from nature that also included Porites compressa. Of the 10 diagnostic reagent tests on these strips, two appear most applicable to corals: ketone and leukocytes. The test strip results from M. capitata were explored using existing transcriptomic data from the same samples and provided evidence of the stress syndromes detected by the strips. We designed a 3D printed smartphone holder and image processing software for field analysis of test strips (TestStripDX) and devised a simple strategy to generate color scores for corals (reflecting extent of bleaching) using a smartphone camera (CoralDX). Our approaches provide field deployable methods, that can be improved in the future (e.g., coral-specific stress test strips) to assess reef health using inexpensive tools and freely available software.

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