Tentacle patterning during Exaiptasia diaphana pedal lacerate development differs between symbiotic and aposymbiotic animals

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.

Trial Assay for Safe First-Aid Protocol for the Stinging Sea Anemone Anemonia viridis (Cnidaria: Anthozoa) and a Severe Toxic Reaction

Anemonia viridis is an abundant and widely distributed temperate sea anemone that can form dense congregations of individuals. Despite the potential severity of its sting, few detailed cases have been reported. We report a case of a severe toxic reaction following an A. viridis sting in a 35-year-old oceanographer. She developed severe pain, itching, redness, and burning sensation, which worsened one week after treatment with anti-inflammatories, antihistamines and corticosteroids. Prompted by this event, and due to the insufficient risk prevention, lack of training for marine-environment users, and lack of research into sting-specific first-aid protocols, we evaluated the cnidocyst response to five different compounds commonly recommended as rinse solutions in first-aid protocols (seawater, vinegar, ammonia, baking soda, and freshwater) by means of the Tentacle Solution Assay. Vinegar and ammonia triggered an immediate and massive cnidocyst discharge after their application and were classified as activator solutions. Baking soda and freshwater were also classified as activator solutions, although with a lower intensity of discharge. Only seawater was classified as a neutral solution and therefore recommended as a rinse solution after A. viridis sting, at least until an inhibitory solution is discovered.

Common Environmental Pollutants Negatively Affect Development and Regeneration in the Sea Anemone Nematostella vectensis Holobiont

The anthozoan sea anemone Nematostella vectensis belongs to the phylum of cnidarians which also includes jellyfish and corals. Nematostella are native to United States East Coast marsh lands, where they constantly adapt to changes in salinity, temperature, oxygen concentration and pH. Its natural ability to continually acclimate to changing environments coupled with its genetic tractability render Nematostella a powerful model organism in which to study the effects of common pollutants on the natural development of these animals. Potassium nitrate, commonly used in fertilizers, and Phthalates, a component of plastics are frequent environmental stressors found in coastal and marsh waters. Here we present data showing how early exposure to these pollutants lead to dramatic defects in development of the embryos and eventual mortality possibly due to defects in feeding ability. Additionally, we examined the microbiome of the animals and identified shifts in the microbial community that correlated with the type of water that was used to grow the animals, and with their exposure to pollutants.

The cyclic dinucleotide 2′3′-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2′3′ cyclic guanosine monophosphate-adenosine monophosphate (2′3′-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis. Using RNA sequencing, we found that 2′3′-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis. Many of the antiviral genes induced by 2′3′-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2′3′-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2′3′-cGAMP signaling in animals.

Trial Assay for Safe First-Aid Protocol for the Stinging Sea Anemone Anemonia viridis (Cnidaria: Anthozoa) and a Severe Toxic Reaction

Anemonia viridis is an abundant and widely-distributed temperate sea anemone that can form dense congregations of individuals. Despite the potential severity of its sting, few detailed cases have been reported. We report a case of a severe toxic reaction following an A. viridis sting in a 35-year-old oceanographer. She developed severe pain, itching, redness and burning sensation, which worsened one week after treatment with anti-inflammatories, antihistamines and corticosteroids. Prompted by this event, and due to the insufficient risk prevention, lack of training for marine-environment users and lack of research into sting-specific first-aid protocols, we evaluated the cnidocyst response to five different compounds commonly recommended as rinse solutions in first-aid protocols (seawater, vinegar, ammonia, baking soda and freshwater) by means of the Tentacle Solution Assay. Vinegar and ammonia triggered an immediate and massive cnidocyst discharge after their application and were classified as activator solutions. Baking soda and freshwater were also classified as activator solutions, although with a lower intensity of discharge. Only seawater was classified as a neutral solution and therefore recommended as a rinse solution after A. viridis sting, at least until an inhibitory solution is discovered.

Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone

The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against KV1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.

The Influence of Symbiont Diversity on the Functional Biology of a Model Sea Anemone

<p>Cnidarian–dinoflagellate symbioses, particularly those between anthozoans and dinoflagellates of the genus Symbiodinium (commonly referred to as zooxanthellae) are widespread in the marine environment. They are responsible for the formation of coral reefs and are thus of great ecological importance. In recent years there has been an increase in the frequency and severity of episodes of coral bleaching resulting in degradation and mortality of coral reefs on a global scale. In order to gain a deeper understanding of how corals can adapt to changing environmental conditions, the effect that symbiont type has on the persistence and physiology of an association needs to be ascertained. The aim of this research was to determine how different symbiont types affect the nutritional biology and intracellular physiology of the symbiosis when in association with the sea anemone Aiptasia pulchella. The specific objectives of the study were to; (1) determine whether different symbiont types are equally as adept at supporting the energetic demands of the same host; (2) determine if internal pH (pHi) is a reflection of symbiont type and whether the optimal pH for photosynthesis coincides with the host cell pHi; and (3) test the influence of Symbiodinium type on host tissue glycerol and glucose pools. In order to answer these questions, aposymbiotic (i.e. symbiont-free) sea anemones were infected with different Symbiodinium types and the relationship between symbiont type, photosynthetic performance and autotrophic potential was tested. A range of ‘normal’ and novel cnidarian–dinoflagellate symbioses was also used to measure host cell pHi and to determine the optimal pHi of isolated intact symbiosomes (i.e. the vacuoles that house the symbionts), as well as to compare the amounts of free glycerol and glucose (metabolites) present in the host tissues. Different host-symbiont combinations were found to have different photosynthetic and respiratory attributes. Earlier onset of full autotrophy (i.e. when all metabolic carbon demands of the symbiosis were met by photosynthesis) and higher CZAR values (i.e. the contribution of zooxanthellae to animal respiration) were demonstrated by symbioses hosting Symbiodinium B1 both from the original (homologous) and different (heterologous) host. The study showed that Symbiodinium types differ in their pH optima and that the optimal pHi for photosynthesis does not always match the actual measured pHi. Symbiont type was also shown to have an effect on host tissue glycerol and glucose pools, with the associations harbouring the homologous Symbiodinium B1 attaining the highest concentrations of both metabolites. Findings from this study suggest that corals may be able to maintain an association with a range of Symbiodinium types, and hence potentially switch as a consequence of bleaching. The new symbiont type may not be as nutritionally advantageous as the original type however, which could have implications for the growth and survivorship of the coral, unless it is able to supplement its carbon demands heterotrophically. The rapid proliferation of some of the heterologous Symbiodinium types (e.g. Symbiodinium E2) inside the host indicates that, after bleaching, there is potential for fast symbiont establishment. The reduced carbon contribution of these heterologous symbionts may not be a major concern should the coral be able to reinstate the more nutritionally advantageous symbiont as the dominant type during bleaching recovery. Finally, the rapid proliferation demonstrated by the heterologous Symbiodinium types and the associated metabolic cost to the host, could be an indication of the opportunistic nature of some of these types and may indicate a shift towards parasitism. It is imperative to extend this type of work to corals in the field to determine how these associations behave in nature. Also, in order to get a clearer picture of the diversity in symbiosis physiology, a wider range of Symbiodinium types needs to be investigated.</p>

The Influence of Symbiont Diversity on the Functional Biology of a Model Sea Anemone

<p>Cnidarian–dinoflagellate symbioses, particularly those between anthozoans and dinoflagellates of the genus Symbiodinium (commonly referred to as zooxanthellae) are widespread in the marine environment. They are responsible for the formation of coral reefs and are thus of great ecological importance. In recent years there has been an increase in the frequency and severity of episodes of coral bleaching resulting in degradation and mortality of coral reefs on a global scale. In order to gain a deeper understanding of how corals can adapt to changing environmental conditions, the effect that symbiont type has on the persistence and physiology of an association needs to be ascertained. The aim of this research was to determine how different symbiont types affect the nutritional biology and intracellular physiology of the symbiosis when in association with the sea anemone Aiptasia pulchella. The specific objectives of the study were to; (1) determine whether different symbiont types are equally as adept at supporting the energetic demands of the same host; (2) determine if internal pH (pHi) is a reflection of symbiont type and whether the optimal pH for photosynthesis coincides with the host cell pHi; and (3) test the influence of Symbiodinium type on host tissue glycerol and glucose pools. In order to answer these questions, aposymbiotic (i.e. symbiont-free) sea anemones were infected with different Symbiodinium types and the relationship between symbiont type, photosynthetic performance and autotrophic potential was tested. A range of ‘normal’ and novel cnidarian–dinoflagellate symbioses was also used to measure host cell pHi and to determine the optimal pHi of isolated intact symbiosomes (i.e. the vacuoles that house the symbionts), as well as to compare the amounts of free glycerol and glucose (metabolites) present in the host tissues. Different host-symbiont combinations were found to have different photosynthetic and respiratory attributes. Earlier onset of full autotrophy (i.e. when all metabolic carbon demands of the symbiosis were met by photosynthesis) and higher CZAR values (i.e. the contribution of zooxanthellae to animal respiration) were demonstrated by symbioses hosting Symbiodinium B1 both from the original (homologous) and different (heterologous) host. The study showed that Symbiodinium types differ in their pH optima and that the optimal pHi for photosynthesis does not always match the actual measured pHi. Symbiont type was also shown to have an effect on host tissue glycerol and glucose pools, with the associations harbouring the homologous Symbiodinium B1 attaining the highest concentrations of both metabolites. Findings from this study suggest that corals may be able to maintain an association with a range of Symbiodinium types, and hence potentially switch as a consequence of bleaching. The new symbiont type may not be as nutritionally advantageous as the original type however, which could have implications for the growth and survivorship of the coral, unless it is able to supplement its carbon demands heterotrophically. The rapid proliferation of some of the heterologous Symbiodinium types (e.g. Symbiodinium E2) inside the host indicates that, after bleaching, there is potential for fast symbiont establishment. The reduced carbon contribution of these heterologous symbionts may not be a major concern should the coral be able to reinstate the more nutritionally advantageous symbiont as the dominant type during bleaching recovery. Finally, the rapid proliferation demonstrated by the heterologous Symbiodinium types and the associated metabolic cost to the host, could be an indication of the opportunistic nature of some of these types and may indicate a shift towards parasitism. It is imperative to extend this type of work to corals in the field to determine how these associations behave in nature. Also, in order to get a clearer picture of the diversity in symbiosis physiology, a wider range of Symbiodinium types needs to be investigated.</p>

Ocean Acidification: Comparative Impacts on the Photophysiology of a Temperate Symbiotic Sea Anemone and a Tropical Coral

<p>Ocean acidification has the potential to drastically alter the coral reef ecosystem by reducing the calcification rate of corals and other reef-builders, and hence a considerable amount of research is now focused on this issue. It also is conceivable that acidification may affect other physiological processes of corals. In particular, acidification may alter photosynthetic physiology and hence the productivity of the coraldinoflagellate symbiosis that is pivotal to the reef's survival and growth. However, very little is known about the impacts of acidification on the photophysiology of corals or, indeed, other invertebrate-algal symbioses. This gap in our knowledge was addressed here by measuring the impacts of acidification (pH 7.6 versus pH 8.1) on the photophysiology and health of the tropical coral Stylophora pistillata and its isolated dinoflagellate symbionts ('zooxanthellae'), and the temperate sea anemone Anthopleura aureoradiata. The comparative nature of this study allowed for any differences between tropical and temperate symbioses, and zooxanthellae in a symbiotic or free-living state, to be assessed. Corals, anemones and cultured zooxanthellae were maintained in flowthrough seawater systems, and treated either with non-acidified (control) seawater at pH 8.1, or seawater acidified with CO2 or HCl to pH 7.6. A variety of parameters, including zooxanthellar density, chlorophyll content, photosynthetic health (Yi), and the ratio of gross photosynthetic production to respiration (P:R) were measured via cell counts, spectrophotometry, respirometry and PAM fluorometry, at a series of time-points up to a maximum of 42 days. Acidification generated by the addition of CO2 had no discernible effect on Yi of either the corals or anemones. However, in the coral, chlorophyll content per zooxanthella cell increased by 25%, which was countered by a near-significant decline (22%) in the rate of gross photosynthesis per unit chlorophyll; as zooxanthellar density remained unchanged, this led to a constant P:R ratio. When acidified via CO2, the isolated zooxanthellae exhibited no impacts in recorded Yi or chlorophyll levels. The response of the anemone to acidification via CO2 was different to that observed in the coral, as the density of zooxanthellae increased, rather than the chlorophyll content per cell, leading to an increased rate of gross photosynthesis. However P:R again remained constant as the increased photosynthesis was matched by an increased rate of respiration. In contrast to the impacts of CO2, HCl adversely impacted the chlorophyll content per cell in both the isolated zooxanthellae and sea anemone, and Yi, gross photosynthesis per cell, and overall gross photosynthesis in the sea anemone; however, despite the decline in gross photosynthesis, P:R remained constant due to the concurrent decline in respiration. Unfortunately, the corals in the HCl experiment died due to technical issues. There are two plausible reasons for this difference between CO2 and HCl. Firstly, HCl may have caused intracellular acidosis which damaged chloroplast structure and photosynthetic function. Secondly, the increased levels of aqueous CO2 stimulated photosynthetic function and hence mitigated for the effects of lowered pH. In addition, evidence is presented for a pH threshold for A. aureoradiata of between pH 6 and pH 6.75 (acidified with HCl), at which point photosynthesis 'shuts-down'. This suggests that, even without the potentially beneficial effects from increased CO2 levels, it is likely that oceanic pH would need to decrease to less than pH 6.75 for any acidosis effects to compromise the productivity of this particular symbiosis. Since acidification will have the benefits of increased CO2 and will reach nowhere near such low pH levels as those extremes tested here, it is proposed that ocean acidification via increased dissolution of CO2 into our oceans will have no impact on the photosynthetic production of symbiotic cnidarians. Indeed, it is entirely likely that increased CO2 will add some benefit to the usually carbon-limited symbiotic zooxanthellae. Ocean acidification is not likely to benefit corals however, with compromised calcification rates likely to undermine the viability of the coral. Symbiotic sea anemones, which do not bio-mineralise CaCO3, are better placed to take advantage of the increased CO2 as we move toward more acidic oceans.</p>