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Anthropogenic climate change has significantly altered terrestrial and marine ecosystems globally, often in the form of climate-related events such as thermal anomalies and disease outbreaks. Although the isolated effects of these stressors have been well documented, a growing body of literature suggests that stressors often interact, resulting in complex effects on ecosystems. This includes coral reefs where sequential associations between heat stress and disease have had profound impacts. Here we used the model cnidarian Exaiptasia diaphana to investigate mechanisms linking prior heat stress to increased disease susceptibility. We examined anemone pathogen susceptibility and physiology (symbiosis, immunity, and energetics) following recovery from heat stress. We observed significantly increased pathogen susceptibility in anemones previously exposed to heat stress. Notably, prior heat stress reduced anemone energetic reserves (carbohydrate concentration), and activity of multiple immune components. Minimal effects of prior heat stress on symbiont density were observed. Together, results suggest changes in energetic availability might have the strongest effect on pathogen susceptibility and immunity following heat stress. The results presented here provide critical insight regarding the interplay between heat stress recovery and pathogen susceptibility in cnidarians and are an important first step towards understanding temporal associations between these stressors., (© 2024. The Author(s).)

Many animals share a lifelong capacity to adapt their growth rates and body sizes to changing environmental food supplies. However, the cellular and molecular basis underlying this plasticity remains only poorly understood. We therefore studied how the sea anemones Nematostella vectensis and Aiptasia (Exaiptasia pallida) respond to feeding and starvation. Combining quantifications of body size and cell numbers with mathematical modelling, we observed that growth and shrinkage rates in Nematostella are exponential, stereotypic and accompanied by dramatic changes in cell numbers. Notably, shrinkage rates, but not growth rates, are independent of body size. In the facultatively symbiotic Aiptasia, we show that growth and cell proliferation rates are dependent on the symbiotic state. On a cellular level, we found that >7% of all cells in Nematostella juveniles reversibly shift between S/G2/M and G1/G0 cell cycle phases when fed or starved, respectively. Furthermore, we demonstrate that polyp growth and cell proliferation are dependent on TOR signalling during feeding. Altogether, we provide a benchmark and resource for further investigating the nutritional regulation of body plasticity on multiple scales using the genetic toolkit available for Nematostella., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Reef-building corals depend on symbiosis with photosynthetic algae that reside within their cells. As important as this relationship is for maintaining healthy reefs, it is strikingly delicate. When ocean temperatures briefly exceed the average summer maximum, corals can bleach, losing their endosymbionts. Although the mechanisms governing bleaching are unknown, studies implicate uncoupling of coral and algal cell divisions at high temperatures. Still, little is known regarding the coordination of host and algal cell divisions. Control of nutrient exchange is one likely mechanism. Both nitrogen and phosphate are necessary for dividing cells, and although nitrogen enrichment is known to increase symbiont density in the host, the consequences of phosphate enrichment are poorly understood. Here, we examined the effects of phosphate depletion on symbiont growth in culture and compared the physiology of phosphate-starved symbionts in culture to symbionts that were freshly isolated from a host. We found that available phosphate is as low in freshly isolated symbionts as it is in phosphate-starved cultures. Furthermore, RNAseq revealed that phosphate-limited and freshly isolated symbionts have similar patterns of gene expression for phosphate-dependent genes, most notably upregulation of phosphatases, which is consistent with phosphate recycling. Similarly, lipid profiling revealed a substantial decrease in phospholipid abundance in both phosphate-starved cultures and freshly isolated symbionts. These findings are important because they suggest that limited access to phosphate controls algal cell divisions within a host., Importance: The corals responsible for building tropical reefs are disappearing at an alarming rate as elevated sea temperatures cause them to bleach and lose the algal symbionts they rely on. Without these symbionts, corals are unable to harvest energy from sunlight and, therefore, struggle to thrive or even survive in the nutrient-poor waters of the tropics. To devise solutions to address the threat to coral reefs, it is necessary to understand the cellular events underpinning the bleaching process. One model for bleaching proposes that heat stress impairs algal photosynthesis and transfer of sugar to the host. Consequently, the host's demands for nitrogen decrease, increasing nitrogen availability to the symbionts, which leads to an increase in algal proliferation that overwhelms the host. Our work suggests that phosphate may play a similar role to nitrogen in this feedback loop., Competing Interests: The authors declare no conflict of interest.

Contemporary symbioses in extreme environments can give an insight into mechanisms that stabilize species interactions during environmental change. The intertidal sea anemone, Anthopleura elegantissima, engages in a nutritional symbiosis with microalgae similar to tropical coral, but withstands more intense environmental fluctuations during tidal inundations. In this study, we compare baseline symbiotic traits and their sensitivity to thermal stress within and among anemone aggregations across the intertidal using a laboratory-based tank experiment to better understand how fixed genotypic and plastic environmental effects contribute to the successful maintenance of this symbiosis in extreme habitats. High intertidal anemones had lower baseline symbiont-to-host cell ratios under control conditions, but their symbionts had higher baseline photosynthetic efficiency compared to low intertidal anemone symbionts. Symbiont communities were identical across all samples, suggesting that shifts in symbiont density and photosynthetic performance could be an acclimatory mechanism to maintain symbiosis in different environments. Despite lower baseline symbiont-to-host cell ratios, high intertidal anemones maintained greater symbiont-to-host cell ratios under heat stress compared with low intertidal anemones, suggesting greater thermal tolerance of high intertidal holobionts. However, the thermal tolerance of clonal anemones acclimatized to different zones was not explained by tidal height alone, indicating additional environmental variables contribute to physiological differences. Host genotype significantly influenced anemone weight, but only explained a minor proportion of variation among symbiotic traits and their response to thermal stress, further implicating environmental history as the primary driver of holobiont tolerance. These results indicate that this symbiosis is highly plastic and may be able to acclimatize to climate change over ecological timescales, defying the convention that symbiotic organisms are more susceptible to environmental stress., (© 2024 The Author(s). Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

Fish species of the genus Amphiprion (Perciformes: Pomacentridae) seek protection from predators among the tentacles of sea anemones as their natural habitat, where they live essentially unharmed from stinging by the host's nematocysts. The skin mucus of these anemonefish has been suggested as a protective mechanism that prevents the discharge of the nematocysts upon contact. Whereas some anemonefish species seem to produce their own protective mucous coating, others may acquire mucus (or biomolecules within) from the sea anemone during an acclimation period. In controlled experiments, we show that Amphiprion ocellaris acclimated successfully to their natural host anemone species Stichodactyla gigantea, and also to Stichodactyla haddoni, and in some cases Heteractis crispa, neither of which are natural host species. No symbiosis was observed for three other anemone species tested, Entacmaea quadricolor, Macrodactyla doreensis, and Heteractis malu. We explored the skin mucous protein profile from naive and experienced A. ocellaris during their acclimation to natural and unnatural host anemones. We confidently report the presence of metabolic and structural proteins in the skin mucus of all samples, likely involved in immunological defense, molecular transport, stress response, and signal transduction. For those anemonefish that established symbiosis, there was a clear increase in ribosomal-type proteins. We additionally provide evidence for the presence of anemone proteins only in the skin mucus of individuals that established symbiosis. Our results support previous speculation of the role of skin mucous-associated proteins in anemonefish-anemone symbiosis. Further exploration of these mucosal proteins could reveal the mechanism of anemonefish acclimation to host anemones., (© 2024 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

Behavioural syndromes are suites of behaviours that corelate between-individuals but the same behaviours may also show within-individual correlations owing to state dependency or trade-offs. Therefore, overall phenotypic behavioural correlations must be separated into their between- and within-individual components. We investigate how startle response duration (an index of boldness) and time taken to reject an inert item (an index of investigation thoroughness) covary in beadlet sea anemones, Actinia equina . Anemones took longer to reject a more complex item compared to a simpler one, validating this measure of investigation thoroughness. We then quantified between- and within-individual correlations using a Bayesian analysis and an alternative frequentist analysis, which returned the same results. Startle responses decreased with anemone size while thoroughness decreased across repeated observations, indicative of simple learning. For each behaviour, repeatability was significant but relatively low and there was no behavioural syndrome. Rather, the two behaviours showed a negative within-individual correlation in most individuals. Thus, boldness and thoroughness are unlikely to be under correlative selection, and they may instead be expressed independently, in line with the general pattern that cross-contextual behavioural syndromes are comparatively rare. It now appears that this pattern may extend broadly across animal diversity.

Kashimoto et al. introduce the giant sea anemones, which form mutualistic relationships with anemonefish., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 ( NvClk Δ ), 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, NvClk Δ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClk Δ/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., Competing Interests: RA, MR, NS, TD, LA, OL No competing interests declared, (© 2023, Aguillon et al.)

Studies of the starlet sea anemone provide important insights into the early evolution of the circadian clock in animals., Competing Interests: EK, PE No competing interests declared, (© 2024, Kwiatkowski and Emery.)

Artificial light at night (ALAN) is becoming a widespread stressor in coastal ecosystems, affecting species that rely on natural day/night cycles. Yet, studies examining ALAN effects remain limited, particularly in the case of sessile species. This study assessed the effects of ALAN upon the feeding activity and two molecular indicators in the widespread plumose sea anemone Metridium senile. Anemones were exposed to either natural day/night or ALAN conditions to monitor feeding activity, and tissue samples were collected to quantify proteins and superoxide dismutase (SOD) enzyme concentrations. In day/night conditions, sea anemones showed a circadian rhythm of activity in which feeding occurs primarily at night. This rhythm was altered by ALAN, which turned it into a reduced and more uniform pattern of feeding. Consistently, proteins and SOD concentrations were significantly lower in anemones exposed to ALAN, suggesting that ALAN can be harmful to sea anemones and potentially other marine sessile species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Specimens of the Mediterranean sea anemone Anemonia viridis were exposed to methylmercury (MeHg) and bacterial infection to study their immune responses to a well-known toxic pollutant. Anemones were housed in laboratory conditions and divided into five experimental groups: 1. control (no microinjection); 2. filtered seawater + buffer injection; 3. filtered seawater + Escherichia coli injection; 4. MeHg + buffer injection; 5. MeHg + E. coli injection. Data showed an increase in antioxidant enzyme production compared to the constitutive condition, while methylmercury inhibited lysozyme production. The buffer inoculation had no statistically significant effects on the animals. In addition, electrophoretic and protease analyses revealed differences in the type of proteins produced, as well as a modulation of proteases depending on the treatment. The study demonstrated the immunomodulatory effect of the organic pollutant on A. viridis, validating its use as a model organism for marine coastal biomonitoring programmes and multiple stress studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mutualistic symbioses between cnidarians and photosynthetic algae are modulated by complex interactions between host immunity and environmental conditions. Here, we investigate how symbiosis interacts with food limitation to influence gene expression and stress response programming in the sea anemone Exaiptasia pallida (Aiptasia). Transcriptomic responses to starvation were similar between symbiotic and aposymbiotic Aiptasia; however, aposymbiotic anemone responses were stronger. Starved Aiptasia of both symbiotic states exhibited increased protein levels of immune-related transcription factor NF-κB, its associated gene pathways, and putative target genes. However, this starvation-induced increase in NF-κB correlated with increased immunity only in symbiotic anemones. Furthermore, starvation had opposite effects on Aiptasia susceptibility to pathogen and oxidative stress challenges, suggesting distinct energetic priorities under food scarce conditions. Finally, when we compared starvation responses in Aiptasia to those of a facultative coral and non-symbiotic anemone, 'defence' responses were similarly regulated in Aiptasia and the facultative coral, but not in the non-symbiotic anemone. This pattern suggests that capacity for symbiosis influences immune responses in cnidarians. In summary, expression of certain immune pathways-including NF-κB-does not necessarily predict susceptibility to pathogens, highlighting the complexities of cnidarian immunity and the influence of symbiosis under varying energetic demands.

Climate change threatens the survival of symbiotic cnidarians by causing photosymbiosis breakdown in a process known as bleaching. Direct effects of temperature on cnidarian host physiology remain difficult to describe because heatwaves depress symbiont performance, leading to host stress and starvation. The symbiotic sea anemone Exaiptasia diaphana provides an opportune system to disentangle direct versus indirect heat effects on the host, as it can survive indefinitely without symbionts. We tested the hypothesis that heat directly impairs cnidarian physiology by comparing symbiotic and aposymbiotic individuals of two laboratory subpopulations of a commonly used clonal strain of E. diaphana, CC7. We exposed anemones to a range of temperatures (ambient, +2°C, +4°C and +6°C) for 15-18 days, then measured their symbiont population densities, autotrophic carbon assimilation and translocation, photosynthesis, respiration and host intracellular pH (pHi). Symbiotic anemones from the two subpopulations differed in size and symbiont density and exhibited distinct heat stress responses, highlighting the importance of acclimation to different laboratory conditions. Specifically, the cohort with higher initial symbiont densities experienced dose-dependent symbiont loss with increasing temperature and a corresponding decline in host photosynthate accumulation. In contrast, the cohort with lower initial symbiont densities did not lose symbionts or assimilate less photosynthate when heated, similar to the response of aposymbiotic anemones. However, anemone pHi decreased at higher temperatures regardless of cohort, symbiont presence or photosynthate translocation, indicating that heat consistently disrupts cnidarian acid-base homeostasis independent of symbiotic status or mutualism breakdown. Thus, pH regulation may be a critical vulnerability for cnidarians in a changing climate., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Cellular mechanisms responsible for the regulation of nutrient exchange, immune responses, and symbiont population growth in the cnidarian-dinoflagellate symbiosis are poorly resolved, particularly with respect to the dinoflagellate symbiont. Here, we characterized proteomic changes in the native symbiont Breviolum minutum during colonization of its host sea anemone Exaiptasia diaphana ("Aiptasia"). We also compared the proteome of this native symbiont in the established symbiotic state with that of a non-native symbiont, Durusdinium trenchii. The onset of symbiosis between Aiptasia and Breviolum minutum increased the accumulation of symbiont proteins associated with the acquisition of inorganic carbon and photosynthesis, nitrogen metabolism, micro- and macronutrient starvation, suppression of host immune responses, tolerance to low pH, and management of oxidative stress. Such responses are consistent with a functional, persistent symbiosis. In contrast, D. trenchii predominantly showed elevated levels of immunosuppressive proteins, consistent with the view that this symbiont is an opportunist that forms a less beneficial, less well-integrated symbiosis with this model anemone. By adding symbiont analysis to the already known responses of the host proteome, our results provide a more holistic view of cellular processes that determine host-symbiont specificity and how differences in symbiont partners (i.e. native versus non-native symbionts) may impact the fitness of the cnidarian-dinoflagellate symbiosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Cnidarians (corals, hydras, jellyfish, sea anemones) are prey-devouring creatures with a simple nervous system, the function of which is largely unknown. A new study on the freshwater polyp Hydra has now uncovered the neuronal circuits that control its feeding behavior., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Photosymbiotic cnidarians generally seek bright environments so that their symbionts can be photosynthetically active. However, excess light may result in a breakdown of symbiosis due to the accumulation of photodamage in symbionts causing symbiont loss (bleaching). It is currently unknown if photosymbiotic cnidarians sense light only to regulate spawning time and to facilitate predation, or whether they also use their light-sensing capacities to protect their symbionts from photodamage. In this study, we examined how the sea anemone Aiptasia changes its behaviour when exposed to excess light. We reveal that Aiptasia polyps, when carrying symbionts, contract their bodies when exposed to high light intensities and subsequently migrate away in a direction perpendicular to the light source. Interestingly, this negative phototaxis was only evident under blue light and absent upon UV, green and red light exposure. Non-symbiotic Aiptasia did not exhibit this light response. Our study demonstrates that photosymbiotic Aiptasia polyps display negative phototactic behaviour in response to blue light, and that they also can perceive its direction, despite lacking specialized eye structures. We postulate that Aiptasia uses blue light, which penetrates seawater efficiently, as a general proxy for sunlight exposure to protect its symbionts from photodamage., (© 2023. Springer Nature Limited.)

AbstractIntegrated chemo- and mechanosensory pathways, along with activated calcium influxes, regulate nematocyst discharge from sea anemone tentacles. Discharge from vibration-sensitive Type A cnidocyte supporting cell complexes use calcium-conducting transient receptor potential V4-like channels. Because calcium influxes often couple with calcium-activated, large-conductance potassium (BK) channels, we hypothesized that BK channels function in nematocyst discharge. To verify this hypothesis, we first tested five selective BK channel blockers on nematocyst-mediated prey killing in Diadumene lineata (aka Haliplanella luciae ). All tested BK channel blockers inhibited prey killing at concentrations comparable to their inhibition of vertebrate BK channels. In addition, the BK channel blocker paxilline selectively inhibited prey killing mediated by vibration-sensitive Type A cnidocyte supporting cell complexes. We queried a mammalian BK α amino acid sequence to the Exaiptasia diaphena database, from which we identified a putative anemone, pore-forming BK α subunit sequence. Using the E. diaphena BK α sequence as a template, we assembled a BK α transcript from our assembled D. lineata transcriptome. In addition, the hydra homolog of D. lineata BK α localizes to nematocytes on the hydra single-cell RNA sequencing map. Our findings suggest that D. lineata expresses BK channels that play a role in vibration-sensitive nematocyst discharge from Type A cnidocyte supporting cell complexes. We believe this is the first functional demonstration of BK channels in nonbilaterians. Because stimulated chemoreceptors frequency tune Type A cnidocyte supporting cell complexes to frequencies matching swimming movements of prey via a protein kinase A signaling pathway and protein kinase A generally activates BK channels, we suggest that D. lineata BK channels may participate in protein kinase A-mediated frequency tuning.

To survive in the nutrient-poor waters of the tropics, reef-building corals rely on intracellular, photosynthetic dinoflagellate symbionts. Photosynthates produced by the symbiont are translocated to the host, and this enables corals to form the structural foundation of the most biodiverse of all marine ecosystems. Although the regulation of nutrient exchange between partners is critical for ecosystem stability and health, the mechanisms governing how nutrients are sensed, transferred, and integrated into host cell processes are largely unknown. Ubiquitous among eukaryotes, the mechanistic target of the rapamycin (mTOR) signaling pathway integrates intracellular and extracellular stimuli to influence cell growth and cell-cycle progression and to balance metabolic processes. A functional role of mTOR in the integration of host and symbiont was demonstrated in various nutritional symbioses, and a similar role of mTOR was proposed for coral-algal symbioses. Using the endosymbiosis model Aiptasia, we examined the role of mTOR signaling in both larvae and adult polyps across various stages of symbiosis. We found that symbiosis enhances cell proliferation, and using an Aiptasia-specific antibody, we localized mTOR to symbiosome membranes. We found that mTOR signaling is activated by symbiosis, while inhibition of mTOR signaling disrupts intracellular niche establishment and symbiosis altogether. Additionally, we observed that dysbiosis was a conserved response to mTOR inhibition in the larvae of a reef-building coral species. Our data confim that mTOR signaling plays a pivotal role in integrating symbiont-derived nutrients into host metabolism and symbiosis stability, ultimately allowing symbiotic cnidarians to thrive in challenging environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

AbstractExtracellular calcium has been known to be required for in situ nematocyst discharge for more than 60 years, yet calcium's role in nematocyst discharge is poorly understood. Currently, we know that extracellular calcium plays at least two distinct roles in in situ nematocyst discharge. First, calcium plays a role in the triggering of discharge by physical contact, most likely involving transient receptor potential channels. Second, activated L-type calcium channels desensitize nematocyst discharge predisposed to discharge by stimulated chemoreceptors for N -acetylated sugars, such as N -acetylneuraminic acid (NANA). It is not known whether the stimulated NANA signaling pathway activates L-type channels electrogenically through membrane depolarization or directly by phosphorylation of the channel. We hypothesize that the activated NANA signaling pathway initiates desensitization by depolarizing cell membrane potentials to activate voltage-gated L-type calcium channels. Consistent with our hypothesis, we show that depolarization induced by blocking voltage-gated potassium channels with 4-aminopyridine selectively activates Ca 2+ influx into tentacle ectodermal cells via L-type channels and inhibits in situ nematocyst discharge from chemosensitized anemones. Furthermore, preventing membrane depolarization with valinomycin or hyperpolarizing resting membrane potentials with low-potassium seawater suppresses NANA-induced Ca 2+ influx, prevents desensitization of in situ nematocyst discharge, and enhances NANA sensitivity. Thus, changing resting membrane potentials modulates NANA sensitivity, and NANA-induced depolarization drives desensitization. We suggest that desensitization of the NANA signaling pathway occurs by a feedback pathway involving calcium channels that are activated by NANA-induced depolarization. Elucidating the desensitization pathway may suggest methods to protect or prevent public health cases of nematocyst stinging.

Frequent coral bleaching has drawn attention to the mechanisms of coral dinoflagellate endosymbiosis. Owing to the difficulty of rearing corals in the laboratory, model symbiosis systems are desired. The sea anemone Exaiptasia diaphana , hosting clade B1 of the genus Breviolum , has long been studied as a model system; however, a single species is insufficient for comparative studies and thus provides only limited resources for symbiosis research, especially regarding the specificity of host-symbiont associations. We established a clonal strain of the sea anemone Anthopleura atodai , whose symbiont was identified as a novel subclade of Symbiodinium (clade A) using a novel feeding method. We also developed a method to efficiently bleach various sea anemone species using a quinoclamine-based herbicide. Bleached A. atodai polyps were vital and able to reproduce asexually, exhibiting no signs of harmful effects of the drug treatment. Pilot studies have suggested that host-symbiont specificity is influenced by multiple steps differently in A. atodai and E. diaphana . RNAseq analyses of A. atodai showed that multiple NPC2 genes were expressed in the symbiotic state, which have been suggested to function in the transport of sterols from symbionts to host cells. These results reveal the usefulness of A. atodai in comparative studies of cnidarian-algal symbiosis.

The ability to learn and form memories allows animals to adapt their behavior based on previous experiences. Associative learning, the process through which organisms learn about the relationship between two distinct events, has been extensively studied in various animal taxa. However, the existence of associative learning, prior to the emergence of centralized nervous systems in bilaterian animals, remains unclear. Cnidarians such as sea anemones or jellyfish possess a nerve net, which lacks centralization. As the sister group to bilaterians, they are particularly well suited for studying the evolution of nervous system functions. Here, we probe the capacity of the starlet sea anemone Nematostella vectensis to form associative memories by using a classical conditioning approach. We developed a protocol combining light as the conditioned stimulus with an electric shock as the aversive unconditioned stimulus. After repetitive training, animals exhibited a conditioned response to light alone-indicating that they learned the association. In contrast, all control conditions did not form associative memories. Besides shedding light on an aspect of cnidarian behavior, these results root associative learning before the emergence of NS centralization in the metazoan lineage and raise fundamental questions about the origin and evolution of cognition in brainless animals.

Climate change induced heat stress has increased coral bleaching events worldwide. Differentially regulated immune genes are one of the primary responses to heat stress suggesting that immune activation is critical. However, the cellular immune mechanisms of coral bleaching is currently unknown, and it is still not known if the immune response documented during heat stress is a consequence of bleaching or is directly caused by the heat stress itself. To address this question, we have used two model system sea anemones (Order: Actiniaria): Exaiptasia diaphana and Nematostella vectensis . E. diaphana is an established sea anemone model for algal symbiont interaction, while N. vectensis is an established sea anemone model that lacks the algal symbiont. Here, we examined the effect of increased temperature on phagocytic activity, as an indication of immune function. Our data shows that immune cell activity increases during heat stress, while small molecule pinocytosis remains unaffected. We observed an increase in cellular production of reactive oxygen species with increasing temperatures. We also found that the cellular immune activity was not affected by the presence of the Symbiodiniaceae. Our results suggest that the immune activity observed in heat-stress induced bleaching in corals is a fundamental and basic response independent of the bleaching effect. These results establish a foundation for improving our understanding of hexacorallian immune cell biology, and its potential role in coral bleaching., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Eliachar, Snyder, Barkan, Talice, Otolenghi, Jaimes-Becerra, Sharoni, Sultan, Hadad, Levy, Moran, Gershoni-Yahalom, Traylor-Knowles and Rosental.)

The establishment and maintenance of the symbiosis between a cnidarian host and its dinoflagellate symbionts is central to the success of coral reefs. To explore the metabolite production underlying this symbiosis, we focused on a group of low molecular weight secondary metabolites, biogenic volatile organic compounds (BVOCs). BVOCs are released from an organism or environment, and can be collected in the gas phase, allowing non-invasive analysis of an organism's metabolism (i.e. 'volatilomics'). We characterised volatile profiles of the sea anemone Aiptasia (Exaiptasia diaphana), a model system for cnidarian-dinoflagellate symbiosis, using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. We compared volatile profiles between: (1) symbiotic anemones containing their native symbiont, Breviolum minutum; (2) aposymbiotic anemones; and (3) cultured isolates of B. minutum. Overall, 152 BVOCs were detected, and classified into 14 groups based on their chemical structure, the most numerous groups being alkanes and aromatic compounds. A total of 53 BVOCs were differentially abundant between aposymbiotic anemones and B. minutum cultures; 13 between aposymbiotic and symbiotic anemones; and 60 between symbiotic anemones and cultures of B. minutum. More BVOCs were differentially abundant between cultured and symbiotic dinoflagellates than between aposymbiotic and symbiotic anemones, suggesting that symbiosis may modify symbiont physiology more than host physiology. This is the first volatilome analysis of the Aiptasia model system and provides a foundation from which to explore how BVOC production is perturbed under environmental stress, and ultimately the role they play in this important symbiosis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Vertebrate growth can be phenotypically plastic in response to predator-prey and competitive interactions. It is unknown however, if it can be plastic in response to mutualistic interactions. Here we investigate plasticity of vertebrate growth in response to variation in mutualistic interactions, using clown anemonefish and their anemone hosts. In the wild, there is a positive correlation between the size of the fish and the size of the anemone, but the cause of this correlation is unknown. Plausible hypotheses are that fish exhibit growth plasticity in response to variation in food or space provided by the host. In the lab, we pair individuals with real anemones of various sizes and show that fish on larger anemones grow faster than fish on smaller anemones. By feeding the fish a constant food ration, we exclude variation in food availability as a cause. By pairing juveniles with artificial anemones of various sizes, we exclude variation in space availability as a single cause. We argue that variation in space availability in conjunction with host cues cause the variability in fish growth. By adjusting their growth, anemonefish likely maximize their reproductive value given their anemone context. More generally, we demonstrate vertebrate growth plasticity in response to variation in mutualistic interactions., (© 2022. The Author(s).)

Bidirectional nutrient flow between partners is integral to the cnidarian-dinoflagellate endosymbiosis. However, our current knowledge of the transporter proteins that regulate nutrient and metabolite trafficking is nascent. Four transmembrane transporters that likely play an important role in interpartner nitrogen and carbon exchange were investigated with immunocytochemistry in the model sea anemone Exaiptasia diaphana ("Aiptasia"; strain NZ1): ammonium transporter 1 (AMT1), V-type proton ATPase (VHA), facilitated glucose transporter member 8 (GLUT8), and aquaporin-3 (AQP3). Anemones lacking symbionts were compared with those in symbiosis with either their typical, homologous dinoflagellate symbiont, Breviolum minutum , or the heterologous species, Durusdinium trenchii and Symbiodinium microadriaticum . AMT1 and VHA were only detected in symbiotic Aiptasia, irrespective of symbiont type. However, GLUT8 and AQP3 were detected in both symbiotic and aposymbiotic states. All transporters were localized to both the epidermis and gastrodermis, though localization patterns in host tissues were heavily influenced by symbiont identity, with S. microadriaticum -colonized anemones showing the most distinct patterns. These patterns suggested disruption of fixed carbon and inorganic nitrogen fluxes when in symbiosis with heterologous versus homologous symbionts. This study enhances our understanding of nutrient transport and host-symbiont integration, while providing a platform for further investigation of nutrient transporters and the host-symbiont interface in the cnidarian-dinoflagellate symbiosis. IMPORTANCE Coral reefs are in serious decline, in particular due to the thermally induced dysfunction of the cnidarian-dinoflagellate symbiosis that underlies their success. Yet our ability to react to this crisis is hindered by limited knowledge of how this symbiosis functions. Indeed, we still have much to learn about the cellular integration that determines whether a particular host-symbiont combination can persist, and hence whether corals might be able to adapt by acquiring new, more thermally resistant symbionts. Here, we employed immunocytochemistry to localize and quantify key nutrient transporters in tissues of the sea anemone Aiptasia, a globally adopted model system for this symbiosis, and compared the expression of these transporters when the host is colonized by native versus nonnative symbionts. We showed a clear link between transporter expression and symbiont identity, elucidating the cellular events that dictate symbiosis success, and we provide a methodological platform for further examination of cellular integration in this ecologically important symbiosis.

Here we describe Stylobates calcifer sp. nov. (Cnidaria, Actiniaria, Actiniidae), a new carcinoecium-forming sea anemone from the deep-sea floor of Japan. Stylobates produces a carcinoecium that thinly covers the snail shells inhabited by host hermit crabs Pagurodofleinia doederleini . The new species is distinct from other species by the shape of the marginal sphincter muscle, the distribution of cnidae, the direction of the oral disk, and host association. The species' novelty is supported by the data of its mitochondrial genes 12S , 16S , and COIII and nuclear genes 18S and 28S . Also, we conducted behavioral observation of this new species, focusing on the feeding behavior and interaction with the specific host hermit crab. Our observations suggest that this sea anemone potentially feeds on the suspended particulate organic matter from the water column or the food residuals of hermit crabs. When the host's shell changed, intensive manipulation for transference of S. calcifer sp. nov. was recorded. However, although the hermit crab detached and transferred the sea anemone to the new shell after shell change, the sea anemone did not exhibit active or cooperative participation. Our data suggest that the sea anemone may not produce a carcinoecium synchronously to its host's growth, contrary to the anecdotal assumption about carcinoecium-forming sea anemones. Conversely, the host hermit crab's growth may not depend entirely on the carcinoecium produced by the sea anemone. This study is perhaps the first observation of the behavioral interaction of the rarely studied carcinoecium-forming mutualism in the deep sea.

Toxin production in nematocysts by Cnidaria phylum represents an important source of bioactive compounds. Using electrophysiology and, heterologous expression of mammalian ion channels in the Xenopus oocyte membrane, we identified two main effects produced by the sea anemone Bartholomea annulata venom. Nematocysts isolation and controlled discharge of their content, revealed that venom had potent effects on both voltage-dependent Na + (Na v ) channels and GABA type A channel receptors (GABA A R), two essential proteins in central nervous system signaling. Unlike many others sea anemone toxins, which slow the inactivation rate of Na v channels, B. annulata venom potently inhibited the neuronal action potential and the Na + currents generated by distinct Na v channels opening, including human TTX-sensitive (hNa v 1.6) and TTX-insensitive Na v channels (hNa v 1.5). A second effect of B. annulata venom was an agonistic action on GABA A R that activated distinct receptors conformed by either α1β2γ2, α3β2γ1 or, ρ1 homomeric receptors. Since GABA was detected in venom samples by ELISA assay at low nanomolar range, it was excluded that GABA from nematocysts directly activated the GABA A Rs. This revealed that substances in B. annulata nematocysts generated at least two potent and novel effects on mammalian ion channels that are crucial for nervous system signaling., (© 2022. The Author(s).)

Crude oil released into the environment undergoes weathering processes that gradually change its composition and toxicity. Co-exposure to petroleum mixtures and other stressors, including ultraviolet (UV) radiation, may lead to synergistic effects and increased toxicity. Laboratory studies should consider these factors when testing the effects of oil exposure on aquatic organisms. Here, we study transcriptomic responses of the estuarine sea anemone Nematostella vectensis to naturally weathered oil, with or without co-exposure to environmental levels of UV radiation. We find that co-exposure greatly enhances the response. We use bioinformatic analyses to identify molecular pathways implicated in this response, which suggest phototoxicity and oxidative damage as mechanisms for the enhanced stress response. Nematostella's stress response shares similarities with the vertebrate oxidative stress response, implying deep conservation of certain stress pathways in animals. We show that exposure to weathered oil along with surface-level UV exposure has substantial physiological consequences in a model cnidarian., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Signaling pathways are fundamental for the establishment and maintenance of diverse symbioses. The symbiosis of cnidarians and dinoflagellate algae is the foundation for the ecological success of coral reefs, involving the transfer of photosynthetic products from the symbiont to host. However, signal transduction pathways for this symbiosis remain uncharacterized. Cultured and natural cnidarian symbionts can produce glycerol, one of the main translocated photosynthates. Here, we investigate whether a signal transduction pathway may be involved in inducing glycerol synthesis in cultured symbionts under an osmotic stress model. We evaluated the effect of specific inhibitors of the main transduction pathways, p38, JNK, and ERK 1/2 in Brevolium minutum, the symbiont of the Aiptasia model system. We found that glycerol production and the specific activity of the enzyme Gpdh were selectively inhibited by a p38 Mitogen-Activated Protein Kinase (MAPK) inhibitor. Additionally, the phosphorylation of a putative p38-like protein was rapidly detected. Finally, we studied the presence of each of the components of the p38 MAPK pathway in silico in genomes and transcriptomes reported up to date for different symbiont types. We propose a model for the arrangement of this pathway in the family of dinoflagellate symbionts known as Symbiodiniaceae., (© 2021 International Society of Protistologists.)

AbstractCnidarians require mechanical stimuli to trigger nematocyst discharge and initiate feeding behaviors. The interval from triggering stimulus to response is tens of microseconds, making it likely that mechanically gated ion channels trigger nematocyst discharge. Because many transient receptor potential channels are mechanically gated, we hypothesized that nematocyst discharge involves transient receptor potential channels. We therefore tested various transient receptor potential channel inhibitors to determine whether they inhibit nematocyst discharge and prey killing in the acontiate sea anemone (Actinaria) Diadumene lineata (a.k.a. Haliplanella luciae ). Three types of cnidocyte supporting cell complexes regulate nematocyst discharge in anemones: Types C, B, and A. Discharge from Type Cs is directly triggered by stimulation of contact-sensitive mechanoreceptors, while Type Bs require activation of chemoreceptors from prey-derived N -acetylated sugars to sensitize contact-sensitive mechanoreceptors. In Type As, activated chemoreceptors tune vibration-sensitive mechanoreceptors that predispose contact-sensitive mechanoreceptors for triggering. The non-selective transient receptor potential channel blockers lanthanum and gadolinium dose-dependently inhibited about 80% of prey killing and all nematocyst discharge from Type Bs and Type Cs, but not Type As. The selective transient receptor potential vanilloid 4 (TRPV4) blocker GSK2193874 inhibited Type As and Type Bs. However, the selective TRPV4 blockers HC-067047 and RN-1734 inhibited only Type As. Thus, three TRPV4-selective blockers implicate TRPV-like involvement in discharge from Type As, whereas GSK2193874 also affected Type Bs. Our results suggest that a TRPV-like homolog plays an essential role in nematocyst-mediated prey killing from Type As, whereas other transient receptor potential channels are likely involved in discharge from Type B and C cnidocyte supporting cell complexes.

Exaiptasia diaphana , a tropical sea anemone known as Aiptasia, is a tractable model system for studying the cellular, physiological, and ecological characteristics of cnidarian-dinoflagellate symbiosis. Aiptasia is widely used as a proxy for coral-algal symbiosis, since both Aiptasia and corals form a symbiosis with members of the family Symbiodiniaceae. Laboratory strains of Aiptasia can be maintained in both the symbiotic (Sym) and aposymbiotic (Apo, without algae) states. Apo Aiptasia allow for the study of the influence of symbiosis on different biological processes and how different environmental conditions impact symbiosis. A key feature of Aiptasia is the ease of propagating both Sym and Apo individuals in the laboratory through a process called pedal laceration. In this form of asexual reproduction, small pieces of tissue rip away from the pedal disc of a polyp, then these lacerates eventually develop tentacles and grow into new polyps. While pedal laceration has been described in the past, details of how tentacles are formed or how symbiotic and nutritional state influence this process are lacking. Here we describe the stages of development in both Sym and Apo pedal lacerates. Our results show that Apo lacerates develop tentacles earlier than Sym lacerates, while over the course of 20 days, Sym lacerates end up with a greater number of tentacles. We describe both tentacle and mesentery patterning during lacerate development and show that they form through a single pattern in early stages regardless of symbiotic state. In later stages of development, Apo lacerate tentacles and mesenteries progress through a single pattern, while variable patterns were observed in Sym lacerates. We discuss how Aiptasia lacerate mesentery and tentacle patterning differs from oral disc regeneration and how these patterning events compare to postembryonic development in Nematostella vectensis , another widely-used sea anemone model. In addition, we demonstrate that Apo lacerates supplemented with a putative nutrient source developed an intermediate number of tentacles between un-fed Apo and Sym lacerates. Based on these observations, we hypothesize that pedal lacerates progress through two different, putatively nutrient-dependent phases of development. In the early phase, the lacerate, regardless of symbiotic state, preferentially uses or relies on nutrients carried over from the adult polyp. These resources are sufficient for lacerates to develop into a functional polyp. In the late phase of development, continued growth and tentacle formation is supported by nutrients obtained from either symbionts and/or the environment through heterotrophic feeding. Finally, we advocate for the implementation of pedal lacerates as an additional resource in the Aiptasia model system toolkit for studies of cnidarian-dinoflagellate symbiosis., Competing Interests: The authors declare there are no competing interests., (©2022 Presnell et al.)

The capacity to regenerate lost or injured body parts is a widespread feature within metazoans and has intrigued scientists for centuries. One of the most extreme types of regeneration is the so-called whole body regenerative capacity, which enables regeneration of fully functional organisms from isolated body parts. While not exclusive to this habitat, whole body regeneration is widespread in aquatic/marine invertebrates. Over the past decade, new whole-body research models have emerged that complement the historical models Hydra and planarians. Among these, the sea anemone Nematostella vectensis has attracted increasing interest in regard to deciphering the cellular and molecular mechanisms underlying the whole-body regeneration process. This manuscript will present an overview of the biological features of this anthozoan cnidarian as well as the available tools and resources that have been developed by the scientific community studying Nematostella . I will further review our current understanding of the cellular and molecular mechanisms underlying whole-body regeneration in this marine organism, with emphasis on how comparing embryonic development and regeneration in the same organism provides insight into regeneration specific elements.

Sea anemones vary immensely in life history strategies, environmental niches and their ability to regenerate. While the sea anemone Nematostella vectensis is the starlet of many key regeneration studies, recent work is emerging on the diverse regeneration strategies employed by other sea anemones. This manuscript will explore current molecular mechanisms of regeneration employed by non-model sea anemones Exaiptasia diaphana (an emerging model species for coral symbiosis studies) and Calliactis polypus (a less well-studied species) and examine how these species compare to the model sea anemone N. vectensis . We summarize the field of regeneration within sea anemones, within the greater context of phylum Cnidaria and in other invertebrate models of regeneration. We also address the current knowledge on two key systems that may be implemented in regeneration: the innate immune system and developmental pathways, including future aspects of work and current limitations.

Background: Coral reefs are among the most diverse and productive ecosystems on Earth. This success relies on the coral's association with a wide range of microorganisms, including dinoflagellates of the family Symbiodiniaceae that provide coral hosts with most of their organic carbon requirements. While bacterial associates have long been overlooked, research on these microorganisms is gaining traction, and deciphering bacterial identity and function is greatly enhancing our understanding of cnidarian biology. Here, we investigated bacterial communities in defensive tissues (acontia) of the coral model, the sea anemone Exaiptasia diaphana. Acontia are internal filaments that are ejected upon detection of an external threat and release toxins to repel predators., Results: Using culturing techniques and 16S rRNA gene metabarcoding we identified bacterial communities associated with acontia of four Great Barrier Reef-sourced E. diaphana genotypes. We show that bacterial communities are similar across genotypes, and dominated by Alteromonadaceae, Vibrionaceae, Rhodobacteraceae, and Saprospiraceae. By analyzing abundant amplicon sequence variants (ASVs) from metabarcoding data from acontia and comparing these to data from whole anemones, we identified five potentially important bacterial genera of the acontia microbiome: Vibrio, Sulfitobacter, Marivita, Alteromonas, and Lewinella. The role of these bacteria within the acontia remains uninvestigated but could entail assistance in defense processes such as toxin production., Conclusions: This study provides insight into potential bacterial involvement in cnidarian defense tissues and highlights the need to study bacterial communities in individual compartments within a holobiont.

Nematostella has fascinating features such as whole-body regeneration, the absence of signs of aging and importantly, the absence of age-related diseases. Easy to culture and spawn, this little sea anemone in spite of its "simple" aspect, displays interesting morphological characteristics similar to vertebrates and an unexpected similarity in gene content/genome organization. Importantly, the scientific community working on Nematostella is developing a variety of functional genomics tools that enable scientists to use this anemone in the field of regenerative medicine, longevity and mecano-sensory diseases. As a complementary research model to vertebrates, this marine invertebrate is emerging and promising to dig deeper into those fields of research in an integrative manner (entire organism) and provides new opportunities for scientists to lift specific barriers that can be encountered with other commonly used animal models., (© 2021 médecine/sciences – Inserm.)

Animal regeneration is a biological process leading to the reformation of injured or lost tissues/body parts. One of the most fascinating regenerative phenomena is the so-called whole-body regeneration, leading to the reformation of fully functional organisms within days after bisection. The sea anemone Nematostella vectensis is currently emerging as novel whole-body regeneration model. Here we describe the methods of inducing the regenerative process in this cnidarian as well as the fixation and staining protocols for morphological, molecular, and cellular analysis.

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.

The sea anemone Nematostella vectensis (Anthozoa, Cnidaria) is a powerful model for characterizing the evolution of genes functioning in venom and nervous systems. Although venom has evolved independently numerous times in animals, the evolutionary origin of many toxins remains unknown. In this work, we pinpoint an ancestral gene giving rise to a new toxin and functionally characterize both genes in the same species. Thus, we report a case of protein recruitment from the cnidarian nervous to venom system. The ShK-like1 peptide has a ShKT cysteine motif, is lethal for fish larvae and packaged into nematocysts, the cnidarian venom-producing stinging capsules. Thus, ShK-like1 is a toxic venom component. Its paralog, ShK-like2, is a neuropeptide localized to neurons and is involved in development. Both peptides exhibit similarities in their functional activities: They provoke contraction in Nematostella polyps and are toxic to fish. Because ShK-like2 but not ShK-like1 is conserved throughout sea anemone phylogeny, we conclude that the two paralogs originated due to a Nematostella -specific duplication of a ShK-like2 ancestor, a neuropeptide-encoding gene, followed by diversification and partial functional specialization. ShK-like2 is represented by two gene isoforms controlled by alternative promoters conferring regulatory flexibility throughout development. Additionally, we characterized the expression patterns of four other peptides with structural similarities to studied venom components and revealed their unexpected neuronal localization. Thus, we employed genomics, transcriptomics, and functional approaches to reveal one venom component, five neuropeptides with two different cysteine motifs, and an evolutionary pathway from nervous to venom system in Cnidaria., Competing Interests: The authors declare no competing interest.

Ocean warming is causing the symbioses between cnidarians and their algal symbionts to breakdown more frequently, resulting in bleaching. For sea anemones, nutritional benefits derived from hosting anemonefishes increase their algal symbiont density. The sea anemone-anemonefish relationship could, therefore, facilitate bleaching recovery. To test this, bleached and unbleached sea anemones, both with and without anemonefish, were monitored in the laboratory. At the start of our experiment, algal symbiont density and colour score were lower in the bleached than unbleached sea anemones, whereas total chlorophyll remained similar. After 106 days, bleached sea anemones with anemonefish had an algal symbiont density and colour score equal to the controls (unbleached sea anemones and without anemonefish), indicating recovery had occurred. Furthermore, total chlorophyll was 66% higher in the bleached sea anemones with anemonefish than the controls. In contrast, recovery did not occur for the bleached sea anemones without anemonefish as they had 78% fewer algal symbionts than the controls, and colour score remained lower. Unbleached sea anemones with anemonefish also showed positive changes in algal symbiont density and total chlorophyll, which increased by 103% and 264%, respectively. Consequently, anemonefishes give their host sea anemones a distinct ecological advantage by enhancing resilience to bleaching, highlighting the benefits of symbioses in a changing climate.

Exaiptasia pallida has been applied as a cnidarian model to assess the toxicity of various contaminants using endpoints related to growth, reproduction and mortality. However, increasingly accepted behavioural and biochemical endpoints are underrepresented in ecotoxicity testing with cnidarian species. The aim of this study was to assess the suitability of tentacle retraction and superoxide dismutase activity as behavioural and biochemical endpoints for ecotoxicity testing with E. pallida. A concentration-dependent, tentacle retraction response was found in sub-lethal toxicity testing for anemones exposed to 1-65 μg L -1 Cu and 2-630 μg L -1 Zn for 24 and 96 h. Semi-quantitative and quantitative approaches to tentacle retraction analysis showed a difference in response sensitivity, however, both methods resulted in similar 24- and 96-h EC50 values for Cu and Zn. Additionally, tentacle retraction analysis provided the benefit of identifying recovery in anemones previously exposed to 359 μg L -1 Zn following a 96-h recovery period. Conversely, no significant difference in superoxide dismutase activity was detected in anemones exposed to the Cu and Zn solutions compared with controls, after either 24- or 96-h exposures. These findings support the ease of application and sensitivity of tentacle retraction as an endpoint in ecotoxicity testing with E. pallida and recommend its suitability for use in acute, sub-lethal toxicity testing. Moreover, evidence of recovery in E. pallida following exposure suggests that recovery should be incorporated into future toxicity assessments., Competing Interests: Declaration of competing interest We declare that there are no known conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

The San Francisco Bay outflow creates a tidally influenced low-salinity plume that affects adjacent coastal sites. In the study region, Anthopleura elegantissima (Cnidaria; Anthozoa) hosts a single symbiont, the dinoflagellate Breviolum muscatinei. Salinity, temperature, and aerial stress induce a bleaching response similar to corals where symbionts are expelled, causing further energetic stress. Using field observations of environmental conditions and symbiont abundance at sites on a gradient of exposure to estuarine outflow, along with a fully crossed multifactorial lab experiment, we tested for changes in symbiont abundance in response to various combinations of three stressors. Lab experiments were designed to mimic short term outflow events with low salinity, high temperature, and aerial exposure treatments. The lab aerial exposure treatment was a statistically significant factor in suppressing symbiont repopulation (ANOVA, p = .017). In the field, symbiont density decreased with increasing tidal height at the site closest to freshwater outflow (ANOVA, p = .007), suggesting that aerial exposure may affect symbiont density more than sea surface temperature and salinity. Unanticipated documentation of survival in 9 months of sand burial and subsequent repopulation of symbionts is reported as a six-month extension to past observations, exemplifying strong tolerance to environmental insult in this Cnidarian mutualism. The study of this symbiosis is useful in examining predicted changes in ocean conditions in tidepool communities and considering relative sources of stress., Competing Interests: The authors have declared that no competing interests exist.

The anemone-crab mutualism is ubiquitous in temperate and tropical marine environments. In this symbiosis, one or more anemones live on a shell inhabited by a hermit crab and reciprocal phoretic, trophic, and defensive benefits are exchanged between the partners. Sea anemone-hermit crab symbionts belong to three families: Hormathiidae (Calliactis and Paracalliactis), Sagartiidae (Carcinactis and Verrillactis), and Actiniidae (Stylobates). Hermit crabs establish most partnerships by detaching anemones and placing them on their shell; sea anemones can also mount shells unaided, triggered by a mollusc-derived substance in the periostracum of the shell. At least partial cooperation by the anemones is necessary for successful establishment of the symbiosis. Here, we expand the evolutionary framework for hormathiid symbionts by generating a phylogeny with at least one member of each actiniarian symbiotic genus with hermit crabs using five molecular markers (16S, 12S, 18S, 28S, CO3). We not only corroborated the results from a previous study by finding two origins of hermit crab symbiosis within Hormathiidae, but also found additional origins for hermit crab symbiosis within Actiniaria. We provide for the first time evidence of a close relationship between symbionts Carcinactis dolosa and V. paguri. The ability to secrete chitin by the ectoderm of the column is inferred to be broadly convergent within Actiniaria whereas the secretion of a chitinous carcinoecium by the pedal disc is a distinct but convergent morphological adaptation of several lineages within Actiniaria. Our finding of multiple origins for both the hermit crab and gastropod symbioses suggests that the shell-mounting behavior might only have been the precursor of the hermit crab association among Calliactis spp., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

All animals detect and integrate diverse environmental signals to mediate behavior. Cnidarians, including jellyfish and sea anemones, both detect and capture prey using stinging cells called nematocytes which fire a venom-covered barb via an unknown triggering mechanism. Here, we show that nematocytes from Nematostella vectensis use a specialized voltage-gated calcium channel (nCa V ) to distinguish salient sensory cues and control the explosive discharge response. Adaptations in nCa V confer unusually sensitive, voltage-dependent inactivation to inhibit responses to non-prey signals, such as mechanical water turbulence. Prey-derived chemosensory signals are synaptically transmitted to acutely relieve nCa V inactivation, enabling mechanosensitive-triggered predatory attack. These findings reveal a molecular basis for the cnidarian stinging response and highlight general principles by which single proteins integrate diverse signals to elicit discrete animal behaviors., Competing Interests: KW, CD, Lv, AL, NB No competing interests declared, (© 2020, Weir et al.)

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.

Immersing anemones in calcium-free seawater disorganizes hair bundle mechanoreceptors on tentacles of sea anemones while causing a loss of vibration sensitivity. Remarkably, anemone hair bundles recover after being returned to calcium-containing seawater. Reorganization of actin in stereocilia likely follows during the recovery of normal morphology of hair bundles after such immersion. Previous studies have reported that Rho G-proteins are located in the stereocilia of hair bundles in sea anemones where they participate in polymerizing actin in stereocilia upon activation of specific chemoreceptors. We here find that immersing anemones in calcium-free seawater significantly reduces the abundance of hair bundles. A partial recovery of abundance of hair bundles occurs within 3 h post-immersion, but a full recovery of abundance does not occur even 6 h after specimens are returned to calcium-containing seawater. Anemones recovering from immersion in calcium-free seawater feature hair bundles that are significantly wider at their tips than in controls. The hair bundles subsequently narrow at their tips, becoming comparable to those of untreated controls within 6 h. Stereocilia of hair bundles are significantly longer in experimental animals than in controls at 2 h of recovery before shortening to lengths comparable to untreated controls at 6 h. In the presence of Rho inhibitors, the recovery in abundance of hair bundles through 6 h is delayed or inhibited. Likewise, in the presence of Rho inhibitors, stereocilia fail to significantly elongate within 2 h of recovery. These data suggest that Rho G-proteins participate in the normal recovery of abundance and recovery of normal morphology of experimentally damaged hair bundle mechanoreceptors., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia ( Exaiptasia pallida ) and its native microalgal photosymbionts ( Breviolum minutum and Breviolum psygmophilum ). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G 2 /M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate. IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues., (Copyright © 2020 Tivey et al.)

Neuropeptides are ancient neuronal signaling molecules that have diversified across Cnidaria (e.g., jellyfish, corals, and sea anemones) and its sister group Bilateria (e.g., vertebrates, insects, and worms). Over the course of neuropeptide evolution emerged lineage-specific neuropeptides, but their roles in the evolution and diversification of nervous system function remain enigmatic. As a step toward filling in this knowledge gap, we investigated the expression pattern of a cnidarian-specific neuropeptide-RPamide-during the development of the starlet sea anemone Nematostella vectensis , using in situ hybridization and immunohistochemistry. We show that RPamide precursor transcripts first occur during gastrulation in scattered epithelial cells of the aboral ectoderm. These RPamide-positive epithelial cells exhibit a spindle-shaped, sensory-cell-like morphology, and extend basal neuronal processes that form a nerve net in the aboral ectoderm of the free-swimming planula larva. At the aboral end, RPamide-positive sensory cells become integrated into the developing apical organ that forms a bundle of long cilia referred to as the apical tuft. Later during planula development, RPamide expression becomes evident in sensory cells in the oral ectoderm of the body column and pharynx, and in the developing endodermal nervous system. At metamorphosis into a polyp, the RPamide-positive sensory nerve net in the aboral ectoderm degenerates by apoptosis, and RPamide expression begins in ectodermal sensory cells of growing oral tentacles. In addition, we find that the expression pattern of RPamide in planulae differs from that of conserved neuropeptides that are shared across Cnidaria and Bilateria, indicative of distinct functions. Our results not only provide the anatomical framework necessary to analyze the function of the cnidarian-specific neuropeptides in future studies, but also reveal previously unrecognized features of the sea anemone nervous system-the apical organ neurons of the planula larva, and metamorphosis-associated reorganization of the ectodermal nervous system., (Copyright © 2020 Zang and Nakanishi.)

Considerable advances in chronobiology have been made through controlled laboratory studies, but distinct temporal rhythms can emerge under natural environmental conditions. Lab-reared Nematostella vectensis sea anemones exhibit circadian behavioral and physiological rhythms . Given that these anemones inhabit shallow estuarine environments subject to tidal inputs, it was unclear whether circadian rhythmicity would persist following entrainment in natural conditions, or whether circatidal periodicity would predominate. Nematostella were conditioned within a marsh environment, where they experienced strong daily temperature cycles as well as brief tidal flooding around the full and new moons. Upon retrieval, anemones exhibited strong circadian (∼24 h) activity rhythms under a light-dark cycle or continuous darkness, but reduced circadian rhythmicity under continuous light. However, some individuals in each light condition showed circadian rhythmicity, and a few individuals showed circatidal rhythmicity. Consistent with the behavioral studies, a large number of transcripts (1640) exhibited diurnal rhythmicity compared with very few (64) with semidiurnal rhythmicity. Diurnal transcripts included core circadian regulators, and 101 of 434 (23%) genes that were previously found to be upregulated by exposure to ultraviolet radiation. Together, these behavioral and transcriptional studies show that circadian rhythmicity predominates and suggest that solar radiation drives physiological cycles in this sediment-dwelling subtidal animal., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Vision plays a major role in the life of most teleosts, and is assumingly well adapted to each species ecology and behaviour. Using a multidisciplinary approach, we scrutinised several aspects of the visual system and ecology of the Great Barrier Reef anemonefish, Amphiprion akindynos, including its orange with white patterning, retinal anatomy and molecular biology, its symbiosis with anemones and sequential hermaphroditism. Amphiprion akindynos possesses spectrally distinct visual pigments and opsins: one rod opsin, RH1 (498 nm), and five cone opsins, SWS1 (370 nm), SWS2B (408 nm), RH2B (498 nm), RH2A (520 nm), and LWS (554 nm). Cones were arranged in a regular mosaic with each single cone surrounded by four double cones. Double cones mainly expressed RH2B (53%) in one member and RH2A (46%) in the other, matching the prevailing light. Single cones expressed SWS1 (89%), which may serve to detect zooplankton, conspecifics and the host anemone. Moreover, a segregated small fraction of single cones coexpressed SWS1 with SWS2B (11%). This novel visual specialisation falls within the region of highest acuity and is suggested to increase the chromatic contrast of Amphiprion akindynos colour patterns, which might improve detection of conspecifics.