Differential nitric oxide synthesis and host apoptotic events correlate with bleaching susceptibility in reef corals

Coral bleaching poses a threat to coral reefs worldwide. As a consequence of the temperature-induced breakdown in coral–dinoflagellate symbiosis, bleaching can have extensive effects on reef communities. However, our understanding of bleaching at a cellular level is limited, and this is particularly true regarding differential susceptibility among coral species. Recent work suggests that bleaching may represent a host innate immune-like response to symbiont dysfunction that involves synthesis of the signalling compound nitric oxide (NO) and the induction of host apoptotic-like cell death. In this study, we examined the activity of apoptosis-regulating enzymes alongside oxidised NO accumulation (a proxy for NO synthesis) in the reef corals Acropora millepora, Montipora digitata, and Pocillopora damicornis during experimental thermal stress. P. damicornis was the most sensitive species, suffering mortality (tissue sloughing) after 5 days at 33 °C but non-lethal bleaching after 9 days at 31.5 °C. A. millepora bleached at 33 °C but remained structurally intact, while M. digitata showed little evidence of bleaching. P. damicornis and A. millepora both exhibited evidence of temperature-induced NO synthesis and, after 5 days of heating, levels of oxidised NO in both species were fivefold higher than in controls maintained at 28.5 °C. These responses preceded bleaching by a number of days and may have occurred before symbiont dysfunction (measured as chlorophyll a degradation and oxidised NO accumulation). In A. millepora, apparent NO synthesis correlated with the induction of host apoptotic-like pathways, while in P. damicornis, the upregulation of apoptotic pathways occurred later. No evidence of elevated NO production or apoptosis was observed in M. digitata at 33 °C and baseline activity of apoptosis-regulating enzymes was negligible in this species. These findings provide important physiological data in the context of the responses of corals to global change and suggest that early events in the host may be important in the collapse of the coral–dinoflagellate symbiosis.

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

The functioning of coral reef systems, as biodiversity hotspots, is largely dependent on the symbiotic association between dinoflagellate symbionts (Symbiodinium spp.) and scleractinian coral hosts. The breakdown of this symbiosis (coral bleaching), as a result of global warming and other stressors, therefore has profound implications for the tropical marine environment. Corals associate with a variety of Symbiodinium genotypes, and it is this mosaic nature that contributes to the variable stress thresholds of corals. Research over the past 25 years has established that the generation and scavenging of reactive oxygen species (ROS) in both partners, under light and thermal stress, is a fundamental element of the bleaching response. However, while the existence of more thermally susceptible and tolerant symbiont types has been recognized, the differences in the antioxidant systems that may accompany these properties have received less attention. The purpose of this thesis was to explore the role of the antioxidant network in explaining the different thermal susceptibilities of various symbiont types and how the activity of key antioxidants in both partners under thermal stress relates to bleaching patterns in different corals. Thus, the specific objectives were to: (1) assess the antioxidant network response in different Symbiodinium types; (2) investigate the activity and structural diversity of key enzymatic antioxidants in different Symbiodinium types; (3) examine the regulation of these antioxidants at the transcriptomic and proteomic levels; and (4) contrast the symbiont’s and host’s antioxidant responses under bleaching conditions. Symbiodinium types in culture were found to differ significantly with regards to the concentration and activity of specific antioxidants, exhibiting magnitude scale differences in some of them. However, the response of the main removal pathway, involving superoxide dismutase (SOD) and ascorbate peroxidase (APX), under lethal thermal stress was fairly similar. Instead, the typespecific differences were found to lie in more downstream systems, and particularly in those associated with the maintenance of the glutathione redox state. A declining glutathione redox state was the common feature of the three thermally susceptible Symbiodinium types: B1, C1, and E. Indeed, in comparison to the most sensitive type (B1), the tolerant type F1 exhibited stronger antioxidant up-regulation and the successful preservation of the highly reduced glutathione pool. Comparing antioxidant gene orthologues from members of different Symbiodinium clades (A-E) revealed a higher degree of sequence variation at the amino acid level for peroxidases, which reflected the genetic radiation of the genus. In contrast, primary defences in the form of SOD isoforms were highly conserved. Sequence variations between Symbiodinium types involved residues that constitute binding sites of substrates and co-factors, and therefore likely affect the catalytic properties of these enzymes. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between transcriptomic expression and proteomic activity due to high variability in expression between replicates, and little response in their enzymatic activity over three days. In contrast to previous findings, up-regulation of antioxidant defences was not evident in Symbiodinium cells inside the host (i.e. in hospite). In fact, oxidative stress in the thermally sensitive corals Acropora millepora and Pocillopora damicornis was only apparent from increased host catalase activity, which interestingly preceded photosynthetic dysfunction of their symbionts. Baseline antioxidant activities of thermally tolerant and susceptible host species showed no differences, though the scavenging activities of the hosts were considerably higher than those of the symbionts. Baseline activities for the symbionts were different, however, with Symbiodinium C15 from the thermally tolerant coral Montipora digitata exhibiting the lowest activities for SOD and catalase peroxidase. This thesis provides significant findings with respect to the variability in antioxidant activity, structure, and network response in different Symbiodinium types in culture, and how these relate to thermal tolerance. What effect these differences have on the response in the intact symbiosis remains unclear, however, as the findings contradict the classic bleaching model of photoinhibition and symbiont-derived ROS. I argue, using previously published data, that heating rates might profoundly affect the way we perceive the antioxidant response of both partners to thermal stress, and that host antioxidant defences might not be as easily overwhelmed by symbiont ROS as suggested previously. This thesis reports important findings on the antioxidant system in different Symbiodinium types, but also raises new questions about the antioxidant response of the intact coral.

Victoria University of Wellington2014

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 world's 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. © 2011 Pernice et al.

Public Library of Science2011

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

Public Library of Science2011

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

Thomas Krüger

Victoria University of Wellington2014