Simon R. Dunn

Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose.

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.

Apoptosis as a post-phagocytic winnowing mechanism in a coral–dinoflagellate mutualism

This study was aimed at detecting apoptosis as a post-phagocytic mechanism of symbiont selection during the onset of symbiosis in larvae of the scleractinian coral Fungia scutaria. Larvae were infected with one of three Symbiodinium types: freshly isolated homologous ITS-type C1f from adult F. scutaria, heterologous C31 from adult Montipora capitata, known to be unable to successfully colonize F. scutaria larvae, and type B1 from the symbiotic sea anemone Aiptasia spp. Apoptosis was detected by the activation of caspases, enzymes specific to apoptosis. Caspase activity was measured in situ by cleavage of a specific fluorophore and detection with confocal microscopy. At 6 h post infection, there was a significant increase in caspase activation in gastrodermal cells in C31-infected larvae, compared with larvae infected with C1f or B1 types. Compared with control larvae infected with C31, which had decreased infection rates present by 24 h post infection, when C31-infected larvae were incubated with a broad-scale caspase inhibitor, the per cent of larvae infected with C31 did not significantly decrease over time. This indicates that the reduction in infection success observed in untreated C31-infected larvae can be rescued with inhibition of caspases and apoptosis. This suggests the presence of a post-phagocytic recognition mechanism. Larvae infected with freshly isolated B1 retained infection success over time compared with C31-infected larvae, suggesting that there is host discrimination between heterologous algae. Initiation of this post-phagocytic response may occur more readily with a highly specific heterologous symbiont type such as C31, compared with a generalist heterologous type such as clade B1.

Regulation of Apoptotic Mediators Reveals Dynamic Responses to Thermal Stress in the Reef Building Coral Acropora millepora

Background Mass coral bleaching is increasing in scale and frequency across the worlds coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts. Methodology/Principal Findings The present study reports the impacts of different stressors (colchicine and heat stress) on three phases of apoptosis: (i) the potential initiation by differential expression of Bcl-2 members, (ii) the execution of apoptotic events by activation of caspase 3-like proteases and (iii) and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity. Conclusions/Significance In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes.

Thermal stress promotes host mitochondrial degradation in symbiotic cnidarians: are the batteries of the reef going to run out?

The symbiotic relationship between cnidarians and their dinoflagellate symbionts, Symbiodinium spp, which underpins the formation of tropical coral reefs, can be destabilized by rapid changes to environmental conditions. Although some studies have concluded that a breakdown in the symbiosis begins with increased reactive oxygen species (ROS) generation within the symbiont due to a decoupling of photosynthesis, others have reported the release of viable symbionts via a variety of host cell derived mechanisms. We explored an alternative model focused upon changes in host cnidarian mitochondrial integrity in response to thermal stress. Mitochondria are often likened to being batteries of the cell, providing energy in the form of ATP, and controlling cellular pathway activation and ROS generation. The overall morphology of host mitochondria was compared to that of associated symbionts under an experimental thermal stress using confocal and electron microscopy. The results demonstrate that hyperthermic stress induces the degradation of cnidarian host mitochondria that is independent of symbiont cellular deterioration. The potential sites of host mitochondrial disruption were also assessed by measuring changes in the expression of genes associated with electron transport and ATP synthesis using quantitative RT-PCR. The primary site of degradation appeared to be downstream of complex III of the electron transport chain with a significant reduction in host cytochrome c and ATP synthase expression. The consequences of reduced expression could limit the capacity of the host to mitigate ROS generation and maintain both organelle integrity and cellular energy supplies. The disruption of host mitochondria, cellular homeostasis, and subsequent cell death irrespective of symbiont integrity highlights the importance of the host response to thermal stress and in symbiosis dysfunction that has substantial implications for understanding how coral reefs will survive in the face of climate change.

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.

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.

A lipidomic approach to understanding free fatty acid lipogenesis derived from dissolved inorganic carbon within cnidarian-dinoflagellate symbiosis.

The cnidarian-dinoflagellate symbiosis is arguably one of the most important within the marine environment in that it is integral to the formation of coral reefs. However, the regulatory processes that perpetuate this symbiosis remain unresolved. It is essential to understand these processes, if we are to elucidate the mechanisms that support growth and resource accumulation by coral host, and conversely, recently observed reduction and/or mortality of corals in response to rapid environmental change. This study specifically focused on one area of metabolic activity within the symbiosis, that of free fatty acid synthesis within both the dinoflagellate symbionts and cnidarian host. The main model system used was Aiptasia pulchella and Symbiodinium sp. in combination with aposymbiotic A. pulchella, the symbiotic coral Acropora millepora system and dinoflagellate culture. Fatty acids (FAs) were selected because of their multiple essential roles inclusive of energy storage (resource accumulation), membrane structure fluidity and cell signaling. The study addressed free FA lipogenesis by using a new method of enriched stable isotopic (13C) incorporation from dissolved inorganic carbon (DI13C) combined with HPLC-MS. FAs derived from DI13C aligned with a mixture of known lipogenesis pathways with the addition of some unusual FAs. After 120 hr, 13C-enriched FA synthesis rates were attributed to only a complex integration of both n–3 and n–6 lipogenesis pathways within the dinoflagellate symbionts. Furthermore, there was no detectible evidence of symbiont derived enriched isotope fatty acids, catabolized 13C derivatives or DI13C being directly utilized, in host late n–6 pathway long-chain FA lipogenesis. These findings do not align with a popular mutualistic translocation model with respect to the use of translocated symbiont photoassimilates in host long-chain FA lipogenesis, which has important connotations for linking nutrient sources with metabolite production and the dynamic regulation of this symbiosis.

Differential Regulation by Heat Stress of Novel Cytochrome P450 Genes from the Dinoflagellate Symbionts of Reef-Building Corals

ABSTRACT Exposure to heat stress has been recognized as one of the major factors leading to the breakdown of the coral-alga symbiosis and coral bleaching. Here, we describe the presence of three new cytochrome P450 (CYP) genes from the reef-building coral endosymbiont Symbiodinium (type C3) and changes in their expression during exposure to severe and moderate heat stress conditions. Sequence analysis of the CYP C-terminal region and two conserved domains, the “PERF” and “heme-binding” domains, confirmed the separate identities of the CYP genes analyzed. In order to explore the effects of different heat stress scenarios, samples of the scleractinian coral Acropora millepora were exposed to elevated temperatures incrementally over an 18-h period (rapid thermal stress) and over a 120-h period (gradual thermal stress). After 18 h of gradual heating and incubation at 26°C, the Symbiodinium CYP mRNA pool was approximately 30% larger, while a further 6°C increase to a temperature above the average sea temperature (29°C after 72 h) resulted in a 2- to 4-fold increase in CYP expression. Both rapid heat stress and gradual heat stress at 32°C resulted in 50% to 90% decreases in CYP gene transcript abundance. Consequently, the initial upregulation of expression of CYP genes at moderately elevated temperatures (26°C and 29°C) was followed by a decrease in expression under the greater thermal stress conditions at 32°C. These findings indicate that in the coral-alga symbiosis under heat stress conditions there is production of chemical stressors and/or transcriptional factors that regulate the expression of genes, such as the genes encoding cytochrome P450 monooxygenases, that are involved in the first line of an organisms chemical defense.

A nanoscale secondary ion mass spectrometry study of dinoflagellate functional diversity in reef-building corals

Nutritional interactions between corals and symbiotic dinoflagellate algae lie at the heart of the structural foundation of coral reefs. Whilst the genetic diversity of Symbiodinium has attracted particular interest because of its contribution to the sensitivity of corals to environmental changes and bleaching (i.e. disruption of coral-dinoflagellate symbiosis), very little is known about the in hospite metabolic capabilities of different Symbiodinium types. Using a combination of stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), we investigated the ability of the intact symbiosis between the reef-building coral Isopora palifera, and Symbiodinium C or D types, to assimilate dissolved inorganic carbon (via photosynthesis) and nitrogen (as ammonium). Our results indicate that Symbiodinium types from two clades naturally associated with I. palifera possess different metabolic capabilities. The Symbiodinium C type fixed and passed significantly more carbon and nitrogen to its coral host than the D type. This study provides further insights into the metabolic plasticity among different Symbiodinium types in hospite and strengthens the evidence that the more temperature-tolerant Symbiodinium D type may be less metabolically beneficial for its coral host under non-stressful conditions.

Photoreactivation is the main repair pathway for UV-induced DNA damage in coral planulae

SUMMARY The larvae of most coral species spend some time in the plankton, floating just below the surface and hence exposed to high levels of ultraviolet radiation (UVR). The high levels of UVR are potentially stressful and damaging to DNA and other cellular components, such as proteins, reducing survivorship. Consequently, mechanisms to either shade (prevent) or repair damage potentially play an important role. In this study, the role of photoreactivation in the survival of coral planulae was examined. Photoreactivation is a light-stimulated response to UV-damaged DNA in which photolyase proteins repair damaged DNA. Photoreactivation rates, as well as the localization of photolyase, were explored in planulae under conditions where photoreactivation was or was not inhibited. The results indicate that photoreactivation is the main DNA repair pathway in coral planulae, repairing UV-induced DNA damage swiftly (K=1.75 h–1 and a half-life of repair of 23 min), with no evidence of any light-independent DNA repair mechanisms, such as nucleotide excision repair (NER), at work. Photolyase mRNA was localized to both the ectoderm and endoderm of the larvae. The amount of cell death in the coral planulae increased significantly when photoreactivation was inhibited, by blocking photoreactivating light. We found that photoreactivation, along with additional UV shielding in the form of five mycosporine-like amino acids, are sufficient for survival in surface tropical waters and that planulae do not accumulate DNA damage despite being exposed to high UVR.