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

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

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.

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis.

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48-72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system.

The scavenger receptor repertoire in six cnidarian species and its putative role in cnidarian-dinoflagellate symbiosis.

Many cnidarians engage in a mutualism with endosymbiotic photosynthetic dinoflagellates that forms the basis of the coral reef ecosystem. Interpartner interaction and regulation includes involvement of the host innate immune system. Basal metazoans, including cnidarians have diverse and complex innate immune repertoires that are just beginning to be described. Scavenger receptors (SR) are a diverse superfamily of innate immunity genes that recognize a broad array of microbial ligands and participate in phagocytosis of invading microbes. The superfamily includes subclades named SR-A through SR-I that are categorized based on the arrangement of sequence domains including the scavenger receptor cysteine rich (SRCR), the C-type lectin (CTLD) and the CD36 domains. Previous functional and gene expression studies on cnidarian-dinoflagellate symbiosis have implicated SR-like proteins in interpartner communication and regulation. In this study, we characterized the SR repertoire from a combination of genomic and transcriptomic resources from six cnidarian species in the Class Anthozoa. We combined these bioinformatic analyses with functional experiments using the SR inhibitor fucoidan to explore a role for SRs in cnidarian symbiosis and immunity. Bioinformatic searches revealed a large diversity of SR-like genes that resembled SR-As, SR-Bs, SR-Es and SR-Is. SRCRs, CTLDs and CD36 domains were identified in multiple sequences in combinations that were highly homologous to vertebrate SRs as well as in proteins with novel domain combinations. Phylogenetic analyses of CD36 domains of the SR-B-like sequences from a diversity of metazoans grouped cnidarian with bilaterian sequences separate from other basal metazoans. All cnidarian sequences grouped together with moderate support in a subclade separately from bilaterian sequences. Functional experiments were carried out on the sea anemone Aiptasia pallida that engages in a symbiosis with Symbiodinium minutum (clade B1). Experimental blocking of the SR ligand binding site with the inhibitor fucoidan reduced the ability of S. minutum to colonize A. pallida suggesting that host SRs play a role in host-symbiont recognition. In addition, incubation of symbiotic anemones with fucoidan elicited an immune response, indicating that host SRs function in immune modulation that results in host tolerance of the symbionts.

Uptake and partitioning of copper and cadmium in the coral Pocillopora damicornis.

Coral-reef ecosystems are increasingly being impacted by a wide variety of anthropogenic inputs, including heavy metals, which could be contributing to coral reef stress and bleaching episodes. Fragments of Pocillopora damicornis were exposed in the laboratory to cadmium (Cd) or copper (Cu) chlorides (0, 5, 50 microg l(-1)) for 14 days and analyzed for metal content in the whole association, algal or animal fractions. Various physiological and biochemical parameters were also measured, such as, algal cell counts, mitotic index, chlorophyll content and levels of the antioxidant glutathione (GSH). Cd and Cu accumulation were observed at all time points and doses; there was no evidence of differential metal partitioning between the algal or animal fractions. No changes in algal cell density, mitotic index or chlorophyll content from the controls were observed in any of the metal treatments. GSH levels were significantly higher in the 5 microg l(-1) Cd (Day 4) and Cu (Days 4 and 14) treatments compared with controls at the same time point. Although no evidence of a bleaching response occurred, corals in both 50 microg l(-1) metal exposures sloughed off tissues and did not survive the duration of the exposure period. Our results demonstrate the accumulation of Cd and Cu in P. damicornis and mortality in the absence of a bleaching response.

Knockdown of actin and caspase gene expression by RNA interference in the symbiotic anemone Aiptasia pallida.

Since the discovery of the ancient eukaryotic process of RNA-mediated gene silencing, the reverse-genetics technique RNA interference (RNAi) has increasingly been used to examine gene function in vertebrate and invertebrate systems. In this study, we report on the use of RNAi, adapted from studies on animal model systems, to manipulate gene expression in a symbiotic marine cnidarian. We describe gene knockdown of actin and of acasp--a cysteine protease, or caspase--in the symbiotic sea anemone Aiptasia pallida. Knockdown was assessed qualitatively with in situ hybridizations for both genes. Quantitative PCR and caspase activity assays were used as a quantitative measure of knockdown for acasp.

Highly conserved caspase and Bcl-2 homologues from the sea anemone Aiptasia pallida: lower metazoans as models for the study of apoptosis evolution.

Key insight into the complexities of apoptosis may be gained from the study of its evolution in lower metazoans. In this study we describe two genes from a cnidarian, Aiptasia pallida, that are homologous to key genes in the apoptotic pathway from vertebrates. The first is a novel ancient caspase, acasp, that displays attributes of both initiator and executioner caspases and includes a caspase recruitment domain (CARD). The second, a Bcl-2 family member, abhp, contains a BH1 and BH2 domain and shares structural characteristics and phylogenetic affinity with a group of antiapoptotic Bcl-2s including A1 and Bcl-2L10. The breadth of occurrence of other invertebrate homologues across the phylogenetic trees of both genes suggests that the complexity of apoptotic pathways is an ancient trait that predates the evolution of vertebrates and higher invertebrates such as nematodes and flies. This paves the way for establishing new lower metazoan model systems for the study of apoptosis.