Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis.

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here, we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis, which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFß, Notch, and FGF signalling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed, and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella, with implications for understanding the reproductive and regenerative biology of other cnidarian species.

Staphylococcus aureus Infection Induces the Production of the Neutrophil Chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by Murine Osteoblasts.

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

Environmental and molecular regulation of asexual reproduction in the sea anemone Nematostella vectensis.

Cnidarians exhibit incredible reproductive diversity, with most capable of sexual and asexual reproduction. Here we investigate factors that influence asexual reproduction in the burrowing sea anemone Nematostella vectensis , which can propagate asexually by transverse fission of the body column. By altering culture conditions, we demonstrate that the presence of a burrowing substrate strongly promotes transverse fission. In addition, we show that animal size does not affect fission rates, and that the plane of fission is fixed along the oral-aboral axis of the polyp. Homeobox transcription factors and components of the TGFß, Notch, and FGF signaling pathways are differentially expressed in polyps undergoing physal pinching suggesting they are important regulators of transverse fission. Gene ontology analyses further suggest that during transverse fission the cell cycle is suppressed and that cell adhesion and patterning mechanisms are downregulated to promote separation of the body column. Finally, we demonstrate that the rate of asexual reproduction is sensitive to population density. Collectively, these experiments provide a foundation for mechanistic studies of asexual reproduction in Nematostella , with implications for understanding the reproductive and regenerative biology of other cnidarian species.

Induction of protective interferon-ß responses in murine osteoblasts following Staphylococcus aureus infection.

Introduction: The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus. Methods: Here, we have assessed the local expression of IFN-ß by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-ß on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria. Results: We report the presence of markedly elevated IFN-ß levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-ß and ISG products. IFN-ß production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-ß by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-ß markedly reduces the number of viable bacteria harbored by these cells. Discussion: Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-ß that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.

Some like it hot: population-specific adaptations in venom production to abiotic stressors in a widely distributed cnidarian.

BACKGROUND: In cnidarians, antagonistic interactions with predators and prey are mediated by their venom, whose synthesis may be metabolically expensive. The potentially high cost of venom production has been hypothesized to drive population-specific variation in venom expression due to differences in abiotic conditions. However, the effects of environmental factors on venom production have been rarely demonstrated in animals. Here, we explore the impact of specific abiotic stresses on venom production of distinct populations of the sea anemone Nematostella vectensis (Actiniaria, Cnidaria) inhabiting estuaries over a broad geographic range where environmental conditions such as temperatures and salinity vary widely. RESULTS: We challenged Nematostella polyps with heat, salinity, UV light stressors, and a combination of all three factors to determine how abiotic stressors impact toxin expression for individuals collected across this species' range. Transcriptomics and proteomics revealed that the highly abundant toxin Nv1 was the most downregulated gene under heat stress conditions in multiple populations. Physiological measurements demonstrated that venom is metabolically costly to produce. Strikingly, under a range of abiotic stressors, individuals from different geographic locations along this latitudinal cline modulate differently their venom production levels. CONCLUSIONS: We demonstrate that abiotic stress results in venom regulation in Nematostella. Together with anecdotal observations from other cnidarian species, our results suggest this might be a universal phenomenon in Cnidaria. The decrease in venom production under stress conditions across species coupled with the evidence for its high metabolic cost in Nematostella suggests downregulation of venom production under certain conditions may be highly advantageous and adaptive. Furthermore, our results point towards local adaptation of this mechanism in Nematostella populations along a latitudinal cline, possibly resulting from distinct genetics and significant environmental differences between their habitats.

Population Genomic Analyses of the Sea Urchin Echinometra sp. EZ across an Extreme Environmental Gradient.

Extreme environmental gradients represent excellent study systems to better understand the variables that mediate patterns of genomic variation between populations. They also allow for more accurate predictions of how future environmental change might affect marine species. The Persian/Arabian Gulf is extreme in both temperature and salinity, whereas the adjacent Gulf of Oman has conditions more typical of tropical oceans. The sea urchin Echinometra sp. EZ inhabits both of these seas and plays a critical role in coral reef health as a grazer and bioeroder, but, to date, there have been no population genomic studies on this or any urchin species in this unique region. E sp. EZ's life history traits (e.g., large population sizes, large reproductive clutches, and long life spans), in theory, should homogenize populations unless nonneutral processes are occurring. Here, we generated a draft genome and a restriction site-associated DNA sequencing data set from seven populations along an environmental gradient across the Persian/Arabian Gulf and the Gulf of Oman. The estimated genome size of E. sp. EZ was 609 Mb and the heterozygosity was among the highest recorded for an echinoderm at 4.5%. We recovered 918 high-quality SNPs from 85 individuals which we then used in downstream analyses. Population structure analyses revealed a high degree of admixture between all sites, although there was population differentiation and significant pairwise FST values between the two seas. Preliminary results suggest migration is bidirectional between the seas and nine candidate loci were identified as being under putative natural selection, including one collagen gene. This study is the first to investigate the population genomics of a sea urchin from this extreme environmental gradient and is an important contribution to our understanding of the complex spatial patterns that drive genomic divergence.

Decoupling behavioral and transcriptional responses to color in an eyeless cnidarian.

BACKGROUND: Animals have specific molecular, physiological, and behavioral responses to light that are influenced by wavelength and intensity. Predictable environmental changes - predominantly solar and lunar cycles - drive endogenous daily oscillations by setting internal pacemakers, otherwise known as the circadian clock. Cnidarians have been a focal group to discern the evolution of light responsiveness due to their phylogenetic position as a sister phylum to bilaterians and broad range of light-responsive behaviors and physiology. Marine species that occupy a range of depths will experience different ranges of wavelengths and light intensities, which may result in variable phenotypic responses. Here, we utilize the eyeless sea anemone Nematostella vectensis, an estuarine anemone that typically resides in shallow water habitats, to compare behavioral and molecular responses when exposed to different light conditions. RESULTS: Quantitative measures of locomotion clearly showed that this species responds to light in the blue and green spectral range with a circadian activity profile, in contrast to a circatidal activity profile in the red spectral range and in constant darkness. Differences in average day/night locomotion was significant in each condition, with overall peak activity during the dark period. Comparative analyses of 96 transcriptomes from individuals sampled every 4 h in each lighting treatment revealed complex differences in gene expression between colors, including in many of the genes likely involved in the cnidarian circadian clock. Transcriptional profiling showed the majority of genes are differentially expressed when comparing mid-day with mid-night, and mostly in red light. Gene expression profiles were largely unique in each color, although animals in blue and green were overall more similar to each other than to red light. CONCLUSIONS: Together, these analyses support the hypothesis that cnidarians are sensitive to red light, and this perception results in a rich transcriptional and divergent behavioral response. Future work determining the specific molecular mechanisms driving the circadian and potential circatidal rhythms measured here would be impactful to connect gene expression variation with behavioral variation in this eyeless species.

Diel patterning in the bacterial community associated with the sea anemone Nematostella vectensis.

Microbes can play an important role in the physiology of animals by providing essential nutrients, inducing immune pathways, and influencing the specific species that compose the microbiome through competitive or facilitatory interactions. The community of microbes associated with animals can be dynamic depending on the local environment, and factors that influence the composition of the microbiome are essential to our understanding of how microbes may influence the biology of their animal hosts. Regularly repeated changes in the environment, such as diel lighting, can result in two different organismal responses: a direct response to the presence and absence of exogenous light and endogenous rhythms resulting from a molecular circadian clock, both of which can influence the associated microbiota. Here, we report how diel lighting and a potential circadian clock impacts the diversity and relative abundance of bacteria in the model cnidarian Nematostella vectensis using an amplicon-based sequencing approach. Comparisons of bacterial communities associated with anemones cultured in constant darkness and in light:dark conditions revealed that individuals entrained in the dark had a more diverse microbiota. Overall community composition showed little variation over a 24-hr period in either treatment; however, abundances of individual bacterial OTUs showed significant cycling in each treatment. A comparative analysis of genes involved in the innate immune system of cnidarians showed differential expression between lighting conditions in N. vectensis, with significant up-regulation during long-term darkness for a subset of genes. Together, our studies support a hypothesis that the bacterial community associated with this species is relatively stable under diel light conditions when compared with static conditions and that particular bacterial members may have time-dependent abundance that coincides with the diel photoperiod in an otherwise stable community.

Transcriptional remodelling upon light removal in a model cnidarian: Losses and gains in gene expression.

Organismal responses to light:dark cycles can result from two general processes: (a) direct response to light or (b) a free-running rhythm (i.e., a circadian clock). Previous research in cnidarians has shown that candidate circadian clock genes have rhythmic expression in the presence of diel lighting, but these oscillations appear to be lost quickly after removal of the light cue. Here, we measure whole-organism gene expression changes in 136 transcriptomes of the sea anemone Nematostella vectensis, entrained to a light:dark environment and immediately following light cue removal to distinguish two broadly defined responses in cnidarians: light entrainment and circadian regulation. Direct light exposure resulted in significant differences in expression for hundreds of genes, including more than 200 genes with rhythmic, 24-hr periodicity. Removal of the lighting cue resulted in the loss of significant expression for 80% of these genes after 1 day, including most of the hypothesized cnidarian circadian genes. Further, 70% of these candidate genes were phase-shifted. Most surprisingly, thousands of genes, some of which are involved in oxidative stress, DNA damage response and chromatin modification, had significant differences in expression in the 24 hr following light removal, suggesting that loss of the entraining cue may induce a cellular stress response. Together, our findings suggest that a majority of genes with significant differences in expression for anemones cultured under diel lighting are largely driven by the primary photoresponse rather than a circadian clock when measured at the whole animal level. These results provide context for the evolution of cnidarian circadian biology and help to disassociate two commonly confounded factors driving oscillating phenotypes.