UNASSIGNED: Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis.
UNASSIGNED: Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones.
UNASSIGNED: Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments.
UNASSIGNED: Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.

The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK\'s functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth\'s biosphere.

Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) - the most common receptors of bilaterian neuropeptides - but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by 1 to 3 of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively connected by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.

Studies of the starlet sea anemone provide important insights into the early evolution of the circadian clock in animals.

In nearly all animals, light-sensing mediated by opsin visual pigments is important for survival and reproduction. Eyeless light-sensing systems, though vital for many animals, have received relatively less attention than forms with charismatic or complex eyes. Despite no single light-sensing organ, the sea anemone Nematostella vectensis has 29 opsin genes and multiple light-mediated behaviors throughout development and reproduction, suggesting a deceptively complex light-sensing system. To characterize one aspect of this light-sensing system, we analyzed larval swimming behavior at high wavelength resolution across the ultraviolet and visual spectrum. N. vectensis larvae respond to light at least from 315 to 650 nm, which is a broad sensitivity range even compared to many animals with complex eyes. Planktonic swimming is induced by ultraviolet (UV) and violet wavelengths until 420 nm. Between 420 and 430 nm a behavioral switch occurs where at wavelengths longer than 430 nm, larvae respond to light by swimming down. Swimming down toward the substrate is distinct from light avoidance, as animals do not exhibit positive or negative phototaxis at any wavelength tested. At wavelengths longer than 575 nm, animals in the water column take increasingly longer to respond and this behavior is more variable until 650 nm where larval response is no different from the dark, suggesting these longer wavelengths lie outside of their sensitivity range. Larval swimming is the only motile stage in the life history of N. vectensis, and increased planktonic swimming could lead to greater dispersal range in potentially damaging shallow environments with short-wavelength light exposure. Longer wavelength environments may indicate more suitable substrates for metamorphosis into the polyp stage, where the individual will remain for the rest of its life. Future work will test whether this robust behavior is mediated by multiple opsins.

Bilaterian animals have evolved complex sensory organs comprised of distinct cell types that function coordinately to sense the environment. Each sensory unit has a defined architecture built from component cell types, including sensory cells, non-sensory support cells, and dedicated sensory neurons. Whether this characteristic cellular composition is present in the sensory organs of non-bilaterian animals is unknown. Here, we interrogate the cell type composition and gene regulatory networks controlling development of the larval apical sensory organ in the sea anemone Nematostella vectensis. Using single cell RNA sequencing and imaging approaches, we reveal two unique cell types in the Nematostella apical sensory organ, GABAergic sensory cells and a putative non-sensory support cell population. Further, we identify the paired-like (PRD) homeodomain gene prd146 as a specific sensory cell marker and show that Prd146+ sensory cells become post-mitotic after gastrulation. Genetic loss of function approaches show that Prd146 is essential for apical sensory organ development. Using a candidate gene knockdown approach, we place prd146 downstream of FGF signaling in the apical sensory organ gene regulatory network. Further, we demonstrate that an aboral FGF activity gradient coordinately regulates the specification of both sensory and support cells. Collectively, these experiments define the genetic basis for apical sensory organ development in a non-bilaterian animal and reveal an unanticipated degree of complexity in a prototypic sensory structure.

BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.

Across diverse taxa, sublethal exposure to abiotic stressors early in life can lead to benefits such as increased stress tolerance upon repeat exposure. This phenomenon, known as hormetic priming, is largely unexplored in early life stages of marine invertebrates, which are increasingly threatened by anthropogenic climate change. To investigate this phenomenon, larvae of the sea anemone and model marine invertebrate Nematostella vectensis were exposed to control (18 °C) or elevated (24 °C, 30 °C, 35 °C, or 39 °C) temperatures for 1 h at 3 days post-fertilization (DPF), followed by return to control temperatures (18 °C). The animals were then assessed for growth, development, metabolic rates, and heat tolerance at 4, 7, and 11 DPF. Priming at intermediately elevated temperatures (24 °C, 30 °C, or 35 °C) augmented growth and development compared to controls or priming at 39 °C. Indeed, priming at 39 °C hampered developmental progression, with around 40% of larvae still in the planula stage at 11 DPF, in contrast to 0% for all other groups. Total protein content, a proxy for biomass, and respiration rates were not significantly affected by priming, suggesting metabolic resilience. Heat tolerance was quantified with acute heat stress exposures, and was significantly higher for animals primed at intermediate temperatures (24 °C, 30 °C, or 35 °C) compared to controls or those primed at 39 °C at all time points. To investigate a possible molecular mechanism for the observed changes in heat tolerance, the expression of heat shock protein 70 (HSP70) was quantified at 11 DPF. Expression of HSP70 significantly increased with increasing priming temperature, with the presence of a doublet band for larvae primed at 39 °C, suggesting persistent negative effects of priming on protein homeostasis. Interestingly, primed larvae in a second cohort cultured to 6 weeks post-fertilization continued to display hormetic growth responses, whereas benefits for heat tolerance were lost; in contrast, negative effects of short-term exposure to extreme heat stress (39 °C) persisted. These results demonstrate that some dose-dependent effects of priming waned over time while others persisted, resulting in heterogeneity in organismal performance across ontogeny following priming. Overall, these findings suggest that heat priming may augment the climate resilience of marine invertebrate early life stages via the modulation of key developmental and physiological phenotypes, while also affirming the need to limit further anthropogenic ocean warming.

UNASSIGNED: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human LRRK2 gene (nvLRRK2). The leucine rich repeat kinase 2 (LRRK2) gene, when mutated, is the most common cause of inherited Parkinson\'s Disease (PD). Its protein product (LRRK2) has implications in a variety of cellular processes, however, the full function of LRRK2 is not well established. Current research is focusing on understanding the function of LRRK2, including both its physiological role as well as its pathobiological underpinnings.
UNASSIGNED: We used bioinformatics to determine the cross-species conservation of LRRK2, then applied drugs targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis.
UNASSIGNED: An in-silico characterization and phylogenetic analysis of nvLRRK2 comparing it to human LRRK2 highlighted key conserved motifs and residues. In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function.
UNASSIGNED: Our work introduces a new model organism with which to study LRRK biology. We show a necessity for LRRK2 in development and regeneration. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson\'s.

Jellyfish and sea anemones fire single-use, venom-covered barbs to immobilize prey or predators. We previously showed that the anemone Nematostella vectensis uses a specialized voltage-gated calcium (CaV) channel to trigger stinging in response to synergistic prey-derived chemicals and touch (Weir et al., 2020). Here, we use experiments and theory to find that stinging behavior is suited to distinct ecological niches. We find that the burrowing anemone Nematostella uses uniquely strong CaV inactivation for precise control of predatory stinging. In contrast, the related anemone Exaiptasia diaphana inhabits exposed environments to support photosynthetic endosymbionts. Consistent with its niche, Exaiptasia indiscriminately stings for defense and expresses a CaV splice variant that confers weak inactivation. Chimeric analyses reveal that CaVβ subunit adaptations regulate inactivation, suggesting an evolutionary tuning mechanism for stinging behavior. These findings demonstrate how functional specialization of ion channel structure contributes to distinct organismal behavior.