The genomic and cellular basis of biosynthetic innovation in rove beetles.

How evolution at the cellular level potentiates change at the macroevolutionary level is a major question in evolutionary biology. With >66,000 described species, rove beetles (Staphylinidae) comprise the largest metazoan family. Their exceptional radiation has been coupled to pervasive biosynthetic innovation whereby numerous lineages bear defensive glands with diverse chemistries. Here, we combine comparative genomic and single-cell transcriptomic data from across the largest rove beetle clade, Aleocharinae. We retrace the functional evolution of two novel secretory cell types that together comprise the tergal gland-a putative catalyst behind Aleocharinae's megadiversity. We identify key genomic contingencies that were critical to the assembly of each cell type and their organ-level partnership in manufacturing the beetle's defensive secretion. This process hinged on evolving a mechanism for regulated production of noxious benzoquinones that appears convergent with plant toxin release systems, and synthesis of an effective benzoquinone solvent that weaponized the total secretion. We show that this cooperative biosynthetic system arose at the Jurassic-Cretaceous boundary, and that following its establishment, both cell types underwent ∼150 million years of stasis, their chemistry and core molecular architecture maintained almost clade-wide as Aleocharinae radiated globally into tens of thousands of lineages. Despite this deep conservation, we show that the two cell types have acted as substrates for the emergence of adaptive, biochemical novelties-most dramatically in symbiotic lineages that have infiltrated social insect colonies and produce host behavior-manipulating secretions. Our findings uncover genomic and cell type evolutionary processes underlying the origin, functional conservation and evolvability of a chemical innovation in beetles.

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.

Fine scale population structure of Acropora palmata and Acropora cervicornis in the Colombian Caribbean.

Using a standardized SNP array, we identified two populations of Acropora cervicornis and one population of A. palmata in the Caribbean coast of Colombia. San Andrés was the most genetically differentiated location for both species. An average pairwise FST value of 0.131 and 0.050 between San Andrés and neighboring collection sites was estimated, for A. cervicornis and A. palmata, respectively. Based on population patterns of both acroporid species, we inferred that Magdalena River is not a barrier of genetic connectivity among Colombian populations. Genetic comparisons between the Colombian coast of Caribbean with other Caribbean locations agree with previous studies for both species, where four populations were identified in A. cervicornis and three in A. palmata. Our results support published bio-physical model predictions and highlight the Panama-Colombia gyre as a possible isolating mechanism within the western Caribbean. However, the genetic diversity in both species was about half (mean HE per site = 0.321 in A. palmata and 0.369 in A. cervicornis) than previous estimates in acroporid populations in the Caribbean. The lower genetic diversity as well their relative isolation and high levels of reef degradation may be of particular conservation concern that may require species-specific management coupled with science-based restoration efforts.

Coral larvae suppress heat stress response during the onset of symbiosis decreasing their odds of survival.

The endosymbiosis between most corals and their photosynthetic dinoflagellate partners begins early in the host life history, when corals are larvae or juvenile polyps. The capacity of coral larvae to buffer climate-induced stress while in the process of symbiont acquisition could come with physiological trade-offs that alter behaviour, development, settlement and survivorship. Here we examined the joint effects of thermal stress and symbiosis onset on colonization dynamics, survival, metamorphosis and host gene expression of Acropora digitifera larvae. We found that thermal stress decreased symbiont colonization of hosts by 50% and symbiont density by 98.5% over 2 weeks. Temperature and colonization also influenced larval survival and metamorphosis in an additive manner, where colonized larvae fared worse or prematurely metamorphosed more often than noncolonized larvae under thermal stress. Transcriptomic responses to colonization and thermal stress treatments were largely independent, while the interaction of these treatments revealed contrasting expression profiles of genes that function in the stress response, immunity, inflammation and cell cycle regulation. The combined treatment either cancelled or lowered the magnitude of expression of heat-stress responsive genes in the presence of symbionts, revealing a physiological cost to acquiring symbionts at the larval stage with elevated temperatures. In addition, host immune suppression, a hallmark of symbiosis onset under ambient temperature, turned to immune activation under heat stress. Thus, by integrating the physical environment and biotic pressures that mediate presettlement event in corals, our results suggest that colonization may hinder larval survival and recruitment under projected climate scenarios.

Inheritance of somatic mutations by animal offspring.

Since 1892, it has been widely assumed that somatic mutations are evolutionarily irrelevant in animals because they cannot be inherited by offspring. However, some nonbilaterians segregate the soma and germline late in development or never, leaving the evolutionary fate of their somatic mutations unknown. By investigating uni- and biparental reproduction in the coral Acropora palmata (Cnidaria, Anthozoa), we found that uniparental, meiotic offspring harbored 50% of the 268 somatic mutations present in their parent. Thus, somatic mutations accumulated in adult coral animals, entered the germline, and were passed on to swimming larvae that grew into healthy juvenile corals. In this way, somatic mutations can increase allelic diversity and facilitate adaptation across habitats and generations in animals.

The coral symbiont Candidatus Aquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally.

The symbiont "Candidatus Aquarickettsia rohweri" infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid ("Ac. prolifera"). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral's algal symbiont (Symbiodinium "fitti"). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.

Genomic conservation and putative downstream functionality of the phosphatidylinositol signalling pathway in the cnidarian-dinoflagellate symbiosis.

The mutualistic cnidarian-dinoflagellate symbiosis underpins the evolutionary success of stony corals and the persistence of coral reefs. However, a molecular understanding of the signalling events that lead to the successful establishment and maintenance of this symbiosis remains unresolved. For example, the phosphatidylinositol (PI) signalling pathway has been implicated during the establishment of multiple mutualistic and parasitic interactions across the kingdoms of life, yet its role within the cnidarian-dinoflagellate symbiosis remains unexplored. Here, we aimed to confirm the presence and assess the specific enzymatic composition of the PI signalling pathway across cnidaria and dinoflagellates by compiling 21 symbiotic anthozoan (corals and sea anemones) and 28 symbiotic dinoflagellate (Symbiodiniaceae) transcriptomic and genomic datasets and querying genes related to this pathway. Presence or absence of PI-kinase and PI-phosphatase orthologs were also compared between a broad sampling of taxonomically related symbiotic and non-symbiotic species. Across the symbiotic anthozoans analysed, there was a complete and highly conserved PI pathway, analogous to the pathway found in model eukaryotes. The Symbiodiniaceae pathway showed similarities to its sister taxon, the Apicomplexa, with the absence of PI 4-phosphatases. However, conversely to Apicomplexa, there was also an expansion of homologs present in the PI5-phosphatase and PI5-kinase groups, with unique Symbiodiniaceae proteins identified that are unknown from non-symbiotic unicellular organisms. Additionally, we aimed to unravel the putative functionalities of the PI signalling pathway in this symbiosis by analysing phosphoinositide (PIP)-binding proteins. Analysis of phosphoinositide (PIP)-binding proteins showed that, on average, 2.23 and 1.29% of the total assemblies of anthozoan and Symbiodiniaceae, respectively, have the potential to bind to PIPs. Enrichment of Gene Ontology (GO) terms associated with predicted PIP-binding proteins within each taxon revealed a broad range of functions, including compelling links to processes putatively involved in symbiosis regulation. This analysis establishes a baseline for current understanding of the PI pathway across anthozoans and Symbiodiniaceae, and thus a framework to target future research.

Evolutionary assembly of cooperating cell types in an animal chemical defense system.

How the functions of multicellular organs emerge from the underlying evolution of cell types is poorly understood. We deconstructed evolution of an organ novelty: a rove beetle gland that secretes a defensive cocktail. We show how gland function arose via assembly of two cell types that manufacture distinct compounds. One cell type, comprising a chemical reservoir within the abdomen, produces alkane and ester compounds. We demonstrate that this cell type is a hybrid of cuticle cells and ancient pheromone and adipocyte-like cells, executing its function via a mosaic of enzymes from each parental cell type. The second cell type synthesizes benzoquinones using a chimera of conserved cellular energy and cuticle formation pathways. We show that evolution of each cell type was shaped by coevolution between the two cell types, yielding a potent secretion that confers adaptive value. Our findings illustrate how cooperation between cell types arises, generating new, organ-level behaviors.

Genomic variation of an endosymbiotic dinoflagellate (Symbiodinium 'fitti') among closely related coral hosts.

Mutualisms where hosts are coupled metabolically to their symbionts often exhibit high partner fidelity. Most reef-building coral species form obligate symbioses with a specific species of photosymbionts, dinoflagellates in the family Symbiodiniaceae, despite needing to acquire symbionts early in their development from environmental sources. Three Caribbean acroporids (Acropora palmata, A. cervicornis and their F1 hybrid) are sympatric across much of their range, but often occupy different depth and light habitats. Throughout this range, both species and their hybrid associate with the endosymbiotic dinoflagellate Symbiodinium 'fitti'. Because light (and therefore depth) influences the physiology of dinoflagellates, we investigated whether S. 'fitti' populations from each host taxon were differentiated genetically. Single nucleotide polymorphisms (SNPs) among S. 'fitti' strains were identified by aligning shallow metagenomic sequences of acroporid colonies sampled from across the Caribbean to a ~600-Mb draft assembly of the S. 'fitti' genome (from the CFL14120 A. cervicornis metagenome). Phylogenomic and multivariate analyses revealed that genomic variation among S. 'fitti' strains partitioned to each host taxon rather than by biogeographical origin. This is particularly noteworthy because the hybrid has a sparse fossil record and may be of relatively recent origin. A subset (37.6%) of the SNPs putatively under selection were nonsynonymous mutations predicted to alter protein efficiency. Differences in genomic variation of S. 'fitti' strains from each host taxon may reflect the unique selection pressures created by the microenvironments associated with each host. The nonrandom sorting among S. 'fitti' strains to different hosts could be the basis for lineage diversification via disruptive selection, leading to ecological specialization and ultimately speciation.

Phylogenetic analysis of cell-cycle regulatory proteins within the Symbiodiniaceae.

In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arrest of the symbiont cell cycle in the G1 phase, though the cellular mechanisms involved remain unknown. Cell-cycle progression in eukaryotes is controlled by the conserved family of cyclin-dependent kinases (CDKs) and their partner cyclins. We identified CDKs and cyclins in different Symbiodiniaceae species and examined their relationship to homologs in other eukaryotes. Cyclin proteins related to eumetazoan cell-cycle-related cyclins A, B, D, G/I and Y, and transcriptional cyclin L, were identified in the Symbiodiniaceae, alongside several alveolate-specific cyclin A/B proteins, and proteins related to protist P/U-type cyclins and apicomplexan cyclins. The largest expansion of Symbiodiniaceae cyclins was in the P/U-type cyclin groups. Proteins related to eumetazoan cell-cycle-related CDKs (CDK1) were identified as well as transcription-related CDKs. The largest expansion of CDK groups was, however, in alveolate-specific groups which comprised 11 distinct CDK groups (CDKA-J) with CDKB being the most widely distributed CDK protein. As a result of its phylogenetic position, conservation across Symbiodiniaceae species, and the presence of the canonical CDK motif, CDKB emerged as a likely candidate for a Saccharomyces cerevisiae Cdc28/Pho85-like homolog in Symbiodiniaceae. Similar to cyclins, two CDK-groups found in Symbiodiniaceae species were solely associated with apicomplexan taxa. A comparison of Breviolum minutum CDK and cyclin gene expression between free-living and symbiotic states showed that several alveolate-specific CDKs and two P/U-type cyclins exhibited altered expression in hospite, suggesting that symbiosis influences the cell cycle of symbionts on a molecular level. These results highlight the divergence of Symbiodiniaceae cell-cycle proteins across species. These results have important implications for host control of the symbiont cell cycle in novel cnidarian-dinoflagellate symbioses.

Considerations for maximizing the adaptive potential of restored coral populations in the western Atlantic.

Active coral restoration typically involves two interventions: crossing gametes to facilitate sexual larval propagation; and fragmenting, growing, and outplanting adult colonies to enhance asexual propagation. From an evolutionary perspective, the goal of these efforts is to establish self-sustaining, sexually reproducing coral populations that have sufficient genetic and phenotypic variation to adapt to changing environments. Here, we provide concrete guidelines to help restoration practitioners meet this goal for most Caribbean species of interest. To enable the persistence of coral populations exposed to severe selection pressure from many stressors, a mixed provenance strategy is suggested: genetically unique colonies (genets) should be sourced both locally as well as from more distant, environmentally distinct sites. Sourcing three to four genets per reef along environmental gradients should be sufficient to capture a majority of intraspecies genetic diversity. It is best for practitioners to propagate genets with one or more phenotypic traits that are predicted to be valuable in the future, such as low partial mortality, high wound healing rate, high skeletal growth rate, bleaching resilience, infectious disease resilience, and high sexual reproductive output. Some effort should also be reserved for underperforming genets because colonies that grow poorly in nurseries sometimes thrive once returned to the reef and may harbor genetic variants with as yet unrecognized value. Outplants should be clustered in groups of four to six genets to enable successful fertilization upon maturation. Current evidence indicates that translocating genets among distant reefs is unlikely to be problematic from a population genetic perspective but will likely provide substantial adaptive benefits. Similarly, inbreeding depression is not a concern given that current practices only raise first-generation offspring. Thus, proceeding with the proposed management strategies even in the absence of a detailed population genetic analysis of the focal species at sites targeted for restoration is the best course of action. These basic guidelines should help maximize the adaptive potential of reef-building corals facing a rapidly changing environment.

Genomic Variants Among Threatened Acropora Corals.

Genomic sequence data for non-model organisms are increasingly available requiring the development of efficient and reproducible workflows. Here, we develop the first genomic resources and reproducible workflows for two threatened members of the reef-building coral genus Acropora We generated genomic sequence data from multiple samples of the Caribbean A. cervicornis (staghorn coral) and A. palmata (elkhorn coral), and predicted millions of nucleotide variants among these two species and the Pacific A. digitifera A subset of predicted nucleotide variants were verified using restriction length polymorphism assays and proved useful in distinguishing the two Caribbean acroporids and the hybrid they form ("A. prolifera"). Nucleotide variants are freely available from the Galaxy server (usegalaxy.org), and can be analyzed there with computational tools and stored workflows that require only an internet browser. We describe these data and some of the analysis tools, concentrating on fixed differences between A. cervicornis and A. palmata In particular, we found that fixed amino acid differences between these two species were enriched in proteins associated with development, cellular stress response, and the host's interactions with associated microbes, for instance in the ABC transporters and superoxide dismutase. Identified candidate genes may underlie functional differences in how these threatened species respond to changing environments. Users can expand the presented analyses easily by adding genomic data from additional species, as they become available.

Uptake and localization of fluorescently-labeled Karenia brevis metabolites in non-toxic marine microbial taxa.

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY-labeled conjugate of brevetoxin congener PbTx-2 (B-PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid-rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20-200 µm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B-PbTx. Uptake and intracellular fluorescence of B-PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY-conjugated K. brevis associated secondary metabolites, brevenal (B-Bn) and brevisin (B-Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently-tagged brevetoxin conjugates in non-toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B-PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

BACKGROUND: The potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na+) and calcium (Ca2+) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na+ VGCs. METHODS: We examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na+/Ca2+ VGC of the non-toxic diatom Odontella sinensis using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin's effect on voltage gated Na+/Ca2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na+/Ca2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na+ VGCs, previously identified as the PbTx binding site in animals. RESULTS: Up to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na+/Ca2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na+/Ca2+ VGCs than the 4-domain eukaryote channels. DISCUSSION: Although membrane excitability and the kinetics of action potential currents were unaffected, the permeation of the channels underlying the diatom action potential was significantly altered in the presence of PbTx-3. However, at environmentally relevant concentrations the effects of PbTx- on diatom voltage activated currents and interference of cell signaling through this pathway may be limited. The relative insensitivity of phytoplankton VGCs may be due to divergence of site-5 (the putative PbTx binding site), and in some cases, such as O. sinensis, resistance to toxin effects may be because of evolutionary loss of the 4-domain eukaryote channel, while retaining a single domain bacterial-like VGC that can substitute in the generation of fast action potentials.

Phylogenetic characterization of transporter proteins in the cnidarian-dinoflagellate symbiosis.

Metabolic exchange between cnidarians and their symbiotic dinoflagellates is central to maintaining their mutualistic relationship. Sugars are translocated to the host, while ammonium and nitrate are utilized by the dinoflagellates (Symbiodinium spp.). We investigated membrane protein sequences of each partner to identify potential transporter proteins that move sugars into cnidarian cells and nitrogen products into Symbiodinium cells. We examined the facilitated glucose transporters (GLUT), sodium/glucose cotransporters (SGLT), and aquaporin (AQP) channels in the cnidarian host as mechanisms for sugar uptake, and the ammonium and high-affinity nitrate transporters (AMT and NRT2, respectively) in the algal symbiont as mechanisms for nitrogen uptake. Homologous protein sequences were used for phylogenetic analysis and tertiary structure deductions. In cnidarians, we identified putative glucose transporters of the GLUT family and glycerol transporting AQP proteins, as well as sodium monocarboxylate transporters and sodium myo-inositol cotransporters homologous to SGLT proteins. We hypothesize that cnidarians use GLUT proteins as the primary mechanism for glucose uptake, while glycerol moves into cells by passive diffusion. We also identified putative AMT proteins in several Symbiodinium clades and putative NRT2 proteins only in a single clade. We further observed an upregulation of expressed putative AMT proteins in Symbiodinium, which may have emerged as an adaptation to conditions experienced inside the host cell. This study is the first to identify transporter sequences from a diversity of cnidarian species and Symbiodinium clades, which will be useful for future experimental analyses of the host-symbiont proteome and the nutritional exchange of Symbiodinium cells in hospite.

The sphingosine rheostat is involved in the cnidarian heat stress response but not necessarily in bleaching.

Sphingolipids play important roles in mitigating cellular heat and oxidative stress by altering membrane fluidity, receptor clustering and gene expression. Accumulation of signaling sphingolipids that comprise the sphingosine rheostat, pro-apoptotic sphingosine (Sph) and pro-survival sphingosine-1-phosphate (S1P) is key to determining cell fate. Reef-building corals and other symbiotic cnidarians living in shallow tropical waters can experience elevated seawater temperature and high UV irradiance, two stressors that are increasing in frequency and severity with climate change. In symbiotic cnidarians, these stressors disrupt the photosynthetic machinery of the endosymbiont and ultimately result in the collapse of the partnership (dysbiosis), known as cnidarian bleaching. In a previous study, exogenously applied sphingolipids altered heat-induced bleaching in the symbiotic anemone Aiptasia pallida, but endogenous regulation of these lipids is unknown. Here, we characterized the role of the rheostat in the cnidarian heat stress response (HSR) and in dysbiosis. Gene expression of rheostat enzymes sphingosine kinase (AP-SPHK) and S1P phosphatase (AP-SGPP), and concentrations of sphingolipids were quantified from anemones incubated at elevated temperatures. We observed a biphasic HSR in A. pallida. At early exposure, rheostat gene expression and lipid levels were suppressed while gene expression of a heat stress biomarker increased and 40% of symbionts were lost. After longer incubations at the highest temperature, AP-SGPP and then Sph levels both increased. These results indicate that the sphingosine rheostat in A. pallida does not participate in initiation of dysbiosis, but instead functions in the chronic response to prolonged heat stress that promotes host survival.

Sphingolipid Metabolism of a Sea Anemone Is Altered by the Presence of Dinoflagellate Symbionts.

In host-microbe interactions, signaling lipids function in interpartner communication during both the establishment and maintenance of associations. Previous evidence suggests that sphingolipids play a role in the mutualistic cnidarian-Symbiodinium symbiosis. Exogenously applied sphingolipids have been shown to alter this partnership, though endogenous host regulation of sphingolipids by the sphingosine rheostat under different symbiotic conditions has not been characterized. The rheostat regulates levels of pro-survival sphingosine-1-phosphate (S1P) and pro-apoptotic sphingosine (Sph) through catalytic activities of sphingosine kinase (SPHK) and S1P phosphatase (SGPP). The role of the rheostat in recognition and establishment of cnidarian-Symbiodinium symbiosis was investigated in the sea anemone Aiptasia pallida by measuring gene expression, protein levels, and sphingolipid metabolites in symbiotic, aposymbiotic, and newly recolonized anemones. Comparison of two host populations showed that symbiotic animals from one population had lower SGPP gene expression and Sph lipid concentrations compared to aposymbiotic animals, while the other population had higher S1P concentrations than their aposymbiotic counterparts. In both populations, the host rheostat trended toward host cell survival in the presence of symbionts. Furthermore, upregulation of both rheostat enzymes on the first day of host recolonization by symbionts suggests a role for the rheostat in host-symbiont recognition during symbiosis onset. Collectively, these data suggest a regulatory role of sphingolipid signaling in cnidarian-Symbiodinium symbiosis and symbiont uptake.

The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida.

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions.

De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes.

Many nonmodel species exemplify important biological questions but lack the sequence resources required to study the genes and genomic regions underlying traits of interest. Reef-building corals are famously sensitive to rising seawater temperatures, motivating ongoing research into their stress responses and long-term prospects in a changing climate. A comprehensive understanding of these processes will require extending beyond the sequenced coral genome (Acropora digitifera) to encompass diverse coral species and related anthozoans. Toward that end, we have assembled and annotated reference transcriptomes to develop catalogs of gene sequences for three scleractinian corals (Fungia scutaria, Montastraea cavernosa, Seriatopora hystrix) and a temperate anemone (Anthopleura elegantissima). High-throughput sequencing of cDNA libraries produced ~20-30 million reads per sample, and de novo assembly of these reads produced ~75,000-110,000 transcripts from each sample with size distributions (mean ~1.4 kb, N50 ~2 kb), comparable to the distribution of gene models from the coral genome (mean ~1.7 kb, N50 ~2.2 kb). Each assembly includes matches for more than half the gene models from A. digitifera (54-67%) and many reasonably complete transcripts (~5300-6700) spanning nearly the entire gene (ortholog hit ratios ≥0.75). The catalogs of gene sequences developed in this study made it possible to identify hundreds to thousands of orthologs across diverse scleractinian species and related taxa. We used these sequences for phylogenetic inference, recovering known relationships and demonstrating superior performance over phylogenetic trees constructed using single mitochondrial loci. The resources developed in this study provide gene sequences and genetic markers for several anthozoan species. To enhance the utility of these resources for the research community, we developed searchable databases enabling researchers to rapidly recover sequences for genes of interest. Our analysis of de novo assembly quality highlights metrics that we expect will be useful for evaluating the relative quality of other de novo transcriptome assemblies. The identification of orthologous sequences and phylogenetic reconstruction demonstrates the feasibility of these methods for clarifying the substantial uncertainties in the existing scleractinian phylogeny.