Photosymbiont Density Is Correlated with Constitutive And Induced Immunity in The Facultatively Symbiotic Coral, Astrangia Poculata.

Scleractinian corals, essential ecosystem engineers that form the base of coral reef ecosystems, have faced unprecedented mortality in recent decades due to climate-change related stressors, including disease outbreaks. Despite this emergent threat to corals, many questions still remain regarding mechanisms underlying observed variation in disease susceptibility. Recent data suggests at least some degree of variation in disease response may be linked to variability in the relationship between host corals and their algal photosymbionts (Family Symbiodiniaceae). Still, the nuances of connections between symbiosis and immunity in cnidarians, including scleractinian corals, remain poorly understood. Here we leveraged an emergent model species, the facultatively symbiotic, temperate, scleractinian coral Astrangia poculata, to investigate associations between symbiont density and both constitutive and induced immunity. We used a combination of controlled immune challenges with heat-inactivated pathogens and transcriptomic analyses. Our results demonstrate that A. poculata mounts a robust initial response to pathogenic stimuli that is highly similar to responses documented in tropical corals. We document positive associations between symbiont density and both constitutive and induced immune responses, in agreement with recent preliminary studies in A. poculata. A suite of immune genes, including those coding for antioxidant peroxiredoxin biosynthesis, are positively associated with symbiont density in A. poculata under constitutive conditions. Furthermore, variation in symbiont density is associated with distinct patterns of immune response; low symbiont density corals induce preventative immune mechanisms whereas high symbiont density corals mobilize energetic resources to fuel humoral immune responses. In summary, our study reveals the need for more nuanced study of symbiosis-immune interplay across diverse scleractinian corals, preferably including quantitative energy budget analysis for full disentanglement of these complex associations and their effects on pathogen susceptibility.

A spectral framework to map QTLs affecting joint differential networks of gene co-expression.

Studying the mechanisms underlying the genotype-phenotype association is crucial in genetics. Gene expression studies have deepened our understanding of the genotype → expression → phenotype mechanisms. However, traditional expression quantitative trait loci (eQTL) methods often overlook the critical role of gene co-expression networks in translating genotype into phenotype. This gap highlights the need for more powerful statistical methods to analyze genotype → network → phenotype mechanism. Here, we develop a network-based method, called snQTL, to map quantitative trait loci affecting gene co-expression networks. Our approach tests the association between genotypes and joint differential networks of gene co-expression via a tensor-based spectral statistics, thereby overcoming the ubiquitous multiple testing challenges in existing methods. We demonstrate the effectiveness of snQTL in the analysis of three-spined stickleback (Gasterosteus aculeatus) data. Compared to conventional methods, our method snQTL uncovers chromosomal regions affecting gene co-expression networks, including one strong candidate gene that would have been missed by traditional eQTL analyses. Our framework suggests the limitation of current approaches and offers a powerful network-based tool for functional loci discoveries.

Characterization of trade-offs between immunity and reproduction in the coral species Astrangia poculata.

Background: Living organisms face ubiquitous pathogenic threats and have consequently evolved immune systems to protect against potential invaders. However, many components of the immune system are physiologically costly to maintain and engage, often drawing resources away from other organismal processes such as growth and reproduction. Evidence from a diversity of systems has demonstrated that organisms use complex resource allocation mechanisms to manage competing needs and optimize fitness. However, understanding of resource allocation patterns is limited across taxa. Cnidarians, which include ecologically important organisms like hard corals, have been historically understudied in the context of resource allocations. Improving understanding of resource allocation-associated trade-offs in cnidarians is critical for understanding future ecological dynamics in the face of rapid environmental change. Methods: Here, we characterize trade-offs between constitutive immunity and reproduction in the facultatively symbiotic coral Astrangia poculata. Male colonies underwent ex situ spawning and sperm density was quantified. We then examined the effects of variable symbiont density and energetic budget on physiological traits, including immune activity and reproductive investment. Furthermore, we tested for potential trade-offs between immune activity and reproductive investment. Results: We found limited associations between energetic budget and immune metrics; melanin production was significantly positively associated with carbohydrate concentration. However, we failed to document any associations between immunity and reproductive output which would be indicative of trade-offs, possibly due to experimental limitations. Our results provide a preliminary framework for future studies investigating immune trade-offs in cnidarians.

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Single-Cell RNA Sequencing Reveals Microevolution of the Stickleback Immune System.

The risk and severity of pathogen infections in humans, livestock, or wild organisms depend on host immune function, which can vary between closely related host populations or even among individuals. This immune variation can entail between-population differences in immune gene coding sequences, copy number, or expression. In recent years, many studies have focused on population divergence in immunity using whole-tissue transcriptomics. But, whole-tissue transcriptomics cannot distinguish between evolved differences in gene regulation within cells, versus changes in cell composition within the focal tissue. Here, we leverage single-cell transcriptomic approaches to document signatures of microevolution of immune system structure in a natural system, the three-spined stickleback (Gasterosteus aculeatus). We sampled nine adult fish from three populations with variability in resistance to a cestode parasite, Schistocephalus solidus, to create the first comprehensive immune cell atlas for G. aculeatus. Eight broad immune cell types, corresponding to major vertebrate immune cells, were identified. We were also able to document significant variation in both abundance and expression profiles of the individual immune cell types among the three populations of fish. Furthermore, we demonstrate that identified cell type markers can be used to reinterpret traditional transcriptomic data: we reevaluate previously published whole-tissue transcriptome data from a quantitative genetic experimental infection study to gain better resolution relating infection outcomes to inferred cell type variation. Our combined study demonstrates the power of single-cell sequencing to not only document evolutionary phenomena (i.e., microevolution of immune cells) but also increase the power of traditional transcriptomic data sets.

Variation in symbiont density is linked to changes in constitutive immunity in the facultatively symbiotic coral, Astrangia poculata.

Scleractinian corals are essential ecosystem engineers, forming the basis of coral reef ecosystems. However, these organisms are in decline globally, in part due to rising disease prevalence. Most corals are dependent on symbiotic interactions with single-celled algae from the family Symbiodiniaceae to meet their nutritional needs, however, suppression of host immunity may be essential to this relationship. To explore immunological consequences of algal symbioses in scleractinian corals, we investigated constitutive immune activity in the facultatively symbiotic coral, Astrangia poculata. We compared immune metrics (melanin synthesis, antioxidant production and antibacterial activity) between coral colonies of varying symbiont density. Symbiont density was positively correlated to both antioxidant activity and melanin concentration, likely as a result of the dual roles of these pathways in immunity and symbiosis regulation. Our results confirm the complex nature of relationships between algal symbiosis and host immunity and highlight the need for nuanced approaches when considering these relationships.

Evolutionary gain and loss of a pathological immune response to parasitism.

Parasites impose fitness costs on their hosts. Biologists often assume that natural selection favors infection-resistant hosts. Yet, when the immune response itself is costly, theory suggests that selection may sometimes favor loss of resistance, which may result in alternative stable states where some populations are resistant and others are tolerant. Intraspecific variation in immune costs is rarely surveyed in a manner that tests evolutionary patterns, and there are few examples of adaptive loss of resistance. Here, we show that when marine threespine stickleback colonized freshwater lakes, they gained resistance to the freshwater-associated cestode Schistocephalus solidus. Extensive peritoneal fibrosis and inflammation are a commonly observed phenotype that contributes to suppression of cestode growth and viability but also imposes a substantial cost on fecundity. Combining genetic mapping and population genomics, we find that opposing selection generates immune system differences between tolerant and resistant populations, consistent with divergent optimization.

Population-level variation in parasite resistance due to differences in immune initiation and rate of response.

Closely related populations often differ in resistance to a given parasite, as measured by infection success or failure. Yet, the immunological mechanisms of these evolved differences are rarely specified. Does resistance evolve via changes to the host's ability to recognize that an infection exists, actuate an effective immune response, or attenuate that response? We tested whether each of these phases of the host response contributed to threespine sticklebacks' recently evolved resistance to their tapeworm Schistocephalus solidus. Although marine stickleback and some susceptible lake fish permit fast-growing tapeworms, other lake populations are resistant and suppress tapeworm growth via a fibrosis response. We subjected lab-raised fish from three populations (susceptible marine "ancestors," a susceptible lake population, and a resistant lake population) to a novel immune challenge using an injection of (1) a saline control, (2) alum, a generalized pro-inflammatory adjuvant that causes fibrosis, (3) a tapeworm protein extract, or (4) a combination of alum and tapeworm protein. With enough time, all three populations generated a robust fibrosis response to the alum treatments. Yet, only the resistant population exhibited a fibrosis response to the tapeworm protein alone. Thus, these populations differed in their ability to respond to the tapeworm protein but shared an intact fibrosis pathway. The resistant population also initiated fibrosis faster in response to alum, and was able to attenuate fibrosis, unlike the susceptible populations' slow but longer lasting response to alum. As fibrosis has pathological side effects that reduce fecundity, the faster recovery by the resistant population may reflect an adaptation to mitigate the costs of immunity. Broadly, our results confirm that parasite detection and immune initiation, activation speed, and immune attenuation simultaneously contribute to the evolution of parasite resistance and adaptations to infection in natural populations.

Between-population differences in constitutive and infection-induced gene expression in threespine stickleback.

Vertebrate immunity is a complex system consisting of a mix of constitutive and inducible defences. Furthermore, host immunity is subject to selective pressure from a range of parasites and pathogens which can produce variation in these defences across populations. As populations evolve immune responses to parasites, they may adapt via a combination of (1) constitutive differences, (2) shared inducible responses, or (3) divergent inducible responses. Here, we leverage a powerful natural host-parasite model system (Gasterosteus aculeatus and Schistochephalus solidus) to tease apart the relative contributions of these three types of adaptations to among-population divergence in response to parasites. Gene expression analyses revealed limited evidence of significant divergence in constitutive expression of immune defence, and strong signatures of conserved inducible responses to the parasite. Furthermore, our results highlight a handful of immune-related genes which show divergent inducible responses which may contribute disproportionately to functional differences in infection success or failure. In addition to investigating variation in evolutionary adaptation to parasite selection, we also leverage this unique data set to improve understanding of cellular mechanisms underlying a putative resistance phenotype (fibrosis). Combined, our results provide a case study in evolutionary immunology showing that a very small number of genes may contribute to genotype differences in infection response.

Immune Gene Expression Covaries with Gut Microbiome Composition in Stickleback.

Commensal microbial communities have immense effects on their vertebrate hosts, contributing to a number of physiological functions, as well as host fitness. In particular, host immunity is strongly linked to microbiota composition through poorly understood bi-directional links. Gene expression may be a potential mediator of these links between microbial communities and host function. However, few studies have investigated connections between microbiota composition and expression of host immune genes in complex systems. Here, we leverage a large study of laboratory-raised fish from the species Gasterosteus aculeatus (three-spined stickleback) to document correlations between gene expression and microbiome composition. First, we examined correlations between microbiome alpha diversity and gene expression. Our results demonstrate robust positive associations between microbial alpha diversity and expression of host immune genes. Next, we examined correlations between host gene expression and abundance of microbial taxa. We identified 15 microbial families that were highly correlated with host gene expression. These families were all tightly correlated with host expression of immune genes and processes, falling into one of three categories-those positively correlated, negatively correlated, and neutrally related to immune processes. Furthermore, we highlight several important immune processes that are commonly associated with the abundance of these taxa, including both macrophage and B cell functions. Further functional characterization of microbial taxa will help disentangle the mechanisms of the correlations described here. In sum, our study supports prevailing hypotheses of intimate links between host immunity and gut microbiome composition.IMPORTANCE Here, we document associations between host gene expression and gut microbiome composition in a nonmammalian vertebrate species. We highlight associations between expression of immune genes and both microbiome diversity and abundance of specific microbial taxa. These findings support other findings from model systems which have suggested that gut microbiome composition and host immunity are intimately linked. Furthermore, we demonstrate that these correlations are truly systemic; the gene expression detailed here was collected from an important fish immune organ (the head kidney) that is anatomically distant from the gut. This emphasizes the systemic impact of connections between gut microbiota and host immune function. Our work is a significant advancement in the understanding of immune-microbiome links in nonmodel, natural systems.

Investigating the roles of transforming growth factor-beta in immune response of Orbicella faveolata, a scleractinian coral.

Symbiotic relationships range from parasitic to mutualistic, yet all endosymbionts face similar challenges, including evasion of host immunity. Many symbiotic organisms have evolved similar mechanisms to face these challenges, including manipulation of the host's transforming growth factor-beta (TGFß) pathway. Here we investigate the TGFß pathway in scelaractinian corals which are dependent on symbioses with dinoflagellates from the family Symbiodiniaceae. Using the Caribbean coral, Orbicella faveolata, we explore the effects of enhancement and inhibition of the TGFß pathway on host gene expression. Following transcriptomic analyses, we demonstrated limited effects of pathway manipulation in absence of immune stimulation. However, manipulation of the TGFß pathway significantly affects the subsequent ability of host corals to mount an immune response. Enhancement of the TGFß pathway eliminates transcriptomic signatures of host coral immune response, while inhibition of the pathway maintains the response. This is, to our knowledge, the first evidence of an immunomodulatory role for TGFß in a scelaractinian coral. These findings suggest variation in TGFß signaling may have implications in the face of increasing disease prevelance. Our results suggest that the TGFß pathway can modulate tradeoffs between symbiosis and immunity. Further study of links between symbiosis, TGFß, and immunity is needed to better understand the ecological implications of these findings.

Uncovering a mitochondrial unfolded protein response in corals and its role in adapting to a changing world.

The Anthropocene will be characterized by increased environmental disturbances, leading to the survival of stress-tolerant organisms, particularly in the oceans, where novel marine diseases and elevated temperatures are re-shaping ecosystems. These environmental changes underscore the importance of identifying mechanisms which promote stress tolerance in ecologically important non-model species such as reef-building corals. Mitochondria are central regulators of cellular stress and have dedicated recovery pathways including the mitochondrial unfolded protein response, which increases the transcription of protective genes promoting protein homeostasis, free radical detoxification and innate immunity. In this investigation, we identify a mitochondrial unfolded protein response in the endangered Caribbean coral Orbicella faveolata, by performing in vivo functional replacement using a transcription factor (Of-ATF5) originating from a coral in the model organism Caenorhabditis elegans. In addition, we use RNA-seq network analysis and transcription factor-binding predictions to identify a transcriptional network of genes likely to be regulated by Of-ATF5 which is induced during the immune challenge and temperature stress. Overall, our findings uncover a conserved cellular pathway which may promote the ability of reef-building corals to survive increasing levels of environmental stress.

Proteomic Investigation of a Diseased Gorgonian Coral Indicates Disruption of Essential Cell Function and Investment in Inflammatory and Other Immune Processes.

As scleractinian coral cover declines in the face of increased frequency in disease outbreaks, future reefs may become dominated by octocorals. Understanding octocoral disease responses and consequences is therefore necessary if we are to gain insight into the future of ecosystem services provided by coral reefs. In Florida, populations of the octocoral Eunicea calyculata infected with Eunicea black disease (EBD) were observed in the field in the fall of 2011. This disease was recognized by a stark, black pigmentation caused by heavy melanization. Histological preparations of E. calyculata infected with EBD demonstrated granular amoebocyte (GA) mobilization, melanin granules in much of the GA population, and the presence of fungal hyphae penetrating coral tissue. Previous transcriptomic analysis also identified immune trade-offs evidenced by increased immune investment at the expense of growth. Our investigation utilized proteogenomic techniques to reveal decreased investment in general cell signaling while increasing energy production for immune responses. Inflammation was also prominent in diseased E. calyculata and sheds light on factors driving the extreme phenotype observed with EBD. With disease outbreaks continuing to increase in frequency, our results highlight new targets within the cnidarian immune system and provide a framework for understanding transcriptomics in the context of an organismal disease phenotype and its protein expression.

Transcriptional analyses provide new insight into the late-stage immune response of a diseased Caribbean coral.

Increasing global temperatures due to climate change have resulted in respective increases in the severity and frequency of epizootics around the globe. Corals in particular have faced rapid declines due to disease outbreaks. Understanding immune responses and associated potential life-history trade-offs is therefore a priority. In the autumn of 2011, a novel disease of octocorals of the genus Eunicea was first documented in the Florida Keys. Termed Eunicea Black Disease (EBD), the disease is easily identified by the dark appearance of affected tissue, caused by a strong melanization response on the part of the host. In order to better understand the response of corals to EBD, we conducted full transcriptome analysis of 3 healthy and 3 diseased specimens of Eunicea calyculata collected from offshore southeast Florida. Differential expression and protein analyses revealed a strong, diverse immune response to EBD characterized by phagocytosis, adhesion and melanization on the part of the host. Furthermore, coexpression network analyses suggested this might come at the cost of reduced cell cycle progression and growth. This is in accordance with past histological studies of naturally infected hard corals, suggesting that potential trade-offs during infection may affect post-outbreak recovery of reef ecosystems by reducing both organismal growth and fecundity. Our findings highlight the importance of considering factors beyond mortality when estimating effects of disease outbreaks on ecosystems.

A conserved Toll-like receptor-to-NF-κB signaling pathway in the endangered coral Orbicella faveolata.

Herein, we characterize the Toll-like receptor (TLR)-to-NF-κB innate immune pathway of Orbicella faveolata (Of), which is an ecologically important, disease-susceptible, reef-building coral. As compared to human TLRs, the intracellular TIR domain of Of-TLR is most similar to TLR4, and it can interact in vitro with the human TLR4 adapter MYD88. Treatment of O. faveolata tissue with lipopolysaccharide, a ligand for mammalian TLR4, resulted in gene expression changes consistent with NF-κB pathway mobilization. Biochemical and cell-based assays revealed that Of-NF-κB resembles the mammalian non-canonical NF-κB protein p100 in that C-terminal truncation results in translocation of Of-NF-κB to the nucleus and increases its DNA-binding and transcriptional activation activities. Moreover, human IκB kinase (IKK) and Of-IKK can both phosphorylate conserved residues in Of-NF-κB in vitro and induce C-terminal processing of Of-NF-κB in vivo. These results are the first characterization of TLR-to-NF-κB signaling proteins in an endangered coral, and suggest that these corals have conserved innate immune pathways.

Life or death: disease-tolerant coral species activate autophagy following immune challenge.

Global climate change has increased the number and severity of stressors affecting species, yet not all species respond equally to these stressors. Organisms may employ cellular mechanisms such as apoptosis and autophagy in responding to stressful events. These two pathways are often mutually exclusive, dictating whether a cell adapts or dies. In order to examine differences in cellular response to stress, we compared the immune response of four coral species with a range of disease susceptibility. Using RNA-seq and novel pathway analysis, we were able to identify differences in response to immune stimulation between these species. Disease-susceptible species Orbicella faveolata activated pathways associated with apoptosis. By contrast, disease-tolerant species Porites porites and Porites astreoides activated autophagic pathways. Moderately susceptible species Pseudodiploria strigosa activated a mixture of these pathways. These findings were corroborated by apoptotic caspase protein assays, which indicated increased caspase activity following immune stimulation in susceptible species. Our results indicate that in response to immune stress, disease-tolerant species activate cellular adaptive mechanisms such as autophagy, while susceptible species turn on cell death pathways. Differences in these cellular maintenance pathways may therefore influence the organismal stress response. Further study of these pathways will increase understanding of differential stress response and species survival in the face of changing environments.

Associations between transcriptional changes and protein phenotypes provide insights into immune regulation in corals.

Disease outbreaks in marine ecosystems have driven worldwide declines of numerous taxa, including corals. Some corals, such as Orbicella faveolata, are particularly susceptible to disease. To explore the mechanisms contributing to susceptibility, colonies of O. faveolata were exposed to immune challenge with lipopolysaccharides. RNA sequencing and protein activity assays were used to characterize the response of corals to immune challenge. Differential expression analyses identified 17 immune-related transcripts that varied in expression post-immune challenge. Network analyses revealed several groups of transcripts correlated to immune protein activity. Several transcripts, which were annotated as positive regulators of immunity were included in these groups, and some were downregulated following immune challenge. Correlations between expression of these transcripts and protein activity results further supported the role of these transcripts in positive regulation of immunity. The observed pattern of gene expression and protein activity may elucidate the processes contributing to the disease susceptibility of species like O. faveolata.

Up in Arms: Immune and Nervous System Response to Sea Star Wasting Disease.

Echinoderms, positioned taxonomically at the base of deuterostomes, provide an important system for the study of the evolution of the immune system. However, there is little known about the cellular components and genes associated with echinoderm immunity. The 2013-2014 sea star wasting disease outbreak is an emergent, rapidly spreading disease, which has led to large population declines of asteroids in the North American Pacific. While evidence suggests that the signs of this disease, twisting arms and lesions, may be attributed to a viral infection, the host response to infection is still poorly understood. In order to examine transcriptional responses of the sea star Pycnopodia helianthoides to sea star wasting disease, we injected a viral sized fraction (0.2 µm) homogenate prepared from symptomatic P. helianthoides into apparently healthy stars. Nine days following injection, when all stars were displaying signs of the disease, specimens were sacrificed and coelomocytes were extracted for RNA-seq analyses. A number of immune genes, including those involved in Toll signaling pathways, complement cascade, melanization response, and arachidonic acid metabolism, were differentially expressed. Furthermore, genes involved in nervous system processes and tissue remodeling were also differentially expressed, pointing to transcriptional changes underlying the signs of sea star wasting disease. The genomic resources presented here not only increase understanding of host response to sea star wasting disease, but also provide greater insight into the mechanisms underlying immune function in echinoderms.