Manipulation of coral photosymbionts for enhancing resilience to environmental change

Ocean warming is occurring at an unprecedented rate. Only a small increase in seawater temperature can disrupt the symbiotic relationship between corals and their photosynthetic algae causing coral bleaching. The bleaching threshold of corals is largely dependent on the microalgae they host. Some studies have indicated the ability for small increases in corals' tolerance to environmental change through shifts in their symbiont communities. However, the increase in frequency of severe bleaching events that have led to worldwide loss of coral cover indicate that this is not enough for coral persistence. This thesis investigates the feasibility and efficacy of manipulating algal symbiont populations associated with corals to enhance their stress tolerance in an era of rapid environmental change. Due to their comparatively short generation time, coral algal symbionts have the potential to evolve more rapidly to environmental changes than their coral host. Chapter two investigates the thermal tolerance of the most common algal symbiont of Great Barrier Reef corals, Cladocopium goreaui, after ~80 asexual generations (2.5 years) of in vitro directed laboratory evolution at an elevated temperature. Using a reciprocal transplant design, I show that the upper temperature tolerance range of the selected C. goreaui increased, evidenced by superior photophysiological performance, growth rates and lower levels of extracellular reactive oxygen species, relative to wild-type cells. In comparison, wild-type C. goreaui cells were unable to photosynthesise or grow at elevated temperature. The enhanced thermal tolerance of the selected C. goreaui in hospite was less apparent. Two of three coral species tested showed positive growth when harbouring the selected C. goreaui at elevated temperature, compared to those that hosted the wild-type cells that had negative growth at elevated temperature. Despite this, recruits of the three coral species bleached regardless of whether they hosted the thermally selected or the wild-type C. goreaui. Important next steps were to decipher the genetic basis underlying enhanced thermal tolerance in the selected algal C. goreaui. Therefore, Chapter three investigates the differences in gene expression between the wild-type and selected cells during the reciprocal transplant experiment. Samples were taken at three time points over 35 days and a de novo transcriptome was assembled. Comparative transcriptomics revealed significant differences in gene expression between the wild-type and selected C. goreaui at elevated temperature. The wild type cells displayed an unstable transcriptomic response of upregulated genes over time, involving large changes in the numbers of genes upregulated and their associated functions. Down-regulated genes, however, were consistently photosynthesis-related, concurrent with their inferior photosynthetic performance at elevated temperature as detailed in chapter 2. The thermally selected C. goreaui shared very few differentially expressed genes with the wild-type cells, having a more stable transcriptomic response to elevated temperature over time. Upregulated genes largely involved those encoding DNA transcription and initiation processes. Although some photosynthetic-related genes were downregulated during one time point, the majority of downregulated genes were involved in the regulation of cell projection organisation. Chapters two and three investigate the most common Great Barrier Reef species of coral photosymbiont, C. goreaui. However, the family that they belong to, the Symbiodiniaceae, is genetically diverse and studies have found wide phenotypic differences between species, with differing thermal tolerances. This led me to testing whether thermal selection experiments could be used successfully across a range of species in the Symbiodiniaceae. Therefore, in chapter four I examine the response of five genetically distinct strains of the Symbiodiniaceae, belonging to four genera, over the course of approximately one year. For three genera I observed a stable adaptive change after only 41-69 asexual generations, where selected cells grew faster and in some cases had higher photosynthetic efficiencies than their wild-type counterparts at elevated temperature. The observed increases in growth rates are comparable with evolutionary experiments in other microalgae, where thermally selected populations have been exposed to elevated temperatures for up to 400 generations. The Symbiodiniaceae are not the only algae to be associated with corals. Apicomplexan-like microalgae were discovered in 2008 and the phylum Chromerida was created. Chromerids have been isolated from corals and contain a functional photosynthetic plastid. Their discovery opens a new avenue of research into the use of alternative/additional photosymbionts of corals. Furthermore, not only do global environmental changes pose a threat to marine organisms but also the simultaneous effects of local stressors such as herbicide additions to coastal systems that often coincide with high summer temperatures. Diuron is one of the most commonly applied herbicides in the catchments of the Great Barrier Reef, acts to inhibit photosynthesis in plants and algae and has been directly linked to coral bleaching. In chapter 5 I test the performance of four chromerid populations as well as C. goreaui in response to elevated temperature, diuron and their combined exposure. Three of the four chromerid strains exhibited high thermal tolerances and two exceptional herbicide tolerances, greater than any observed for photosynthetic microalgae. I subsequently investigate the ability of the chromerids to form a symbiosis with larvae of two common GBR coral species under ambient and stress conditions. Chromerid uptake by coral larvae was low compared to C. goreaui. I did not observe any overall negative or positive larval fitness effects of the infection with chromerid algae vs. C. goreaui. However, the possibility that chromerid algae may have more important roles in later coral life stages or with other species of coral cannot be excluded. The research presented in this thesis is among the first to test the possibility of experimental evolution to enhance the thermal tolerance of coral symbionts belonging to the Symbiodiniaceae, as well as the use of the potentially alternative symbionts, the chromerids. My results show that it is possible to experimentally evolve cultured Symbiodiniaceae strains across multiple species and highlight the genetic and molecular pathways that underpin thermal tolerance in the most common Great Barrier Reef species, C. goreaui. Despite increased thermal tolerance of the thermally-selected Symbiodiniaceae in vitro and the high thermal and diuron tolerance of some chromerid populations, these were unable to significantly enhance the upper thermal limit or diuron tolerance of the coral-algal symbiosis. Therefore, further work into the algal-coral association and bleaching response is required to assess whether algal symbiont manipulation has the potential to be a valuable tool in coral reef conservation and restoration initiatives in a rapidly changing ocean

Thermal and Herbicide Tolerances of Chromerid Algae and Their Ability to Form a Symbiosis With Corals

Reef-building corals form an obligate symbiosis with photosynthetic microalgae in the family Symbiodiniaceae that meet most of their energy requirements. This symbiosis is under threat from the unprecedented rate of ocean warming as well as the simultaneous pressure of local stressors such as poor water quality. Only 1°C above mean summer sea surface temperatures (SSTs) on the Great Barrier Reef (GBR) can trigger the loss of Symbiodiniaceae from the host, and very low concentrations of the most common herbicide, diuron, can disrupt the photosynthetic activity of microalgae. In an era of rapid environmental change, investigation into the assisted evolution of the coral holobiont is underway in an effort to enhance the resilience of corals. Apicomplexan-like microalgae were discovered in 2008 and the Phylum Chromerida (chromerids) was created. Chromerids have been isolated from corals and contain a functional photosynthetic plastid. Their discovery therefore opens a new avenue of research into the use of alternative/additional photosymbionts of corals. However, only two studies to-date have investigated the symbiotic nature of Chromera velia with corals and thus little is known about the coral-chromerid relationship. Furthermore, the response of chromerids to environmental stressors has not been examined. Here we tested the performance of four chromerid strains and the common dinoflagellate symbiont Cladocopium goreaui (formerly Symbiodinium goreaui, ITS2 type C1) in response to elevated temperature, diuron and their combined exposure. Three of the four chromerid strains exhibited high thermal tolerances and two strains showed exceptional herbicide tolerances, greater than observed for any photosynthetic microalgae, including C. goreaui. We also investigated the onset of symbiosis between the chromerids and larvae of two common GBR coral species under ambient and stress conditions. Levels of colonization of coral larvae with the chromerid strains were low compared to colonization with C. goreaui. We did not observe any overall negative or positive larval fitness effects of the inoculation with chromerid algae vs. C. goreaui. However, we cannot exclude the possibility that chromerid algae may have more important roles in later coral life stages and recommend this be the focus of future studies

Thermal and Herbicide Tolerances of Chromerid Algae and Their Ability to Form a Symbiosis With Corals

Reef-building corals form an obligate symbiosis with photosynthetic microalgae in the family Symbiodiniaceae that meet most of their energy requirements. This symbiosis is under threat from the unprecedented rate of ocean warming as well as the simultaneous pressure of local stressors such as poor water quality. Only 1°C above mean summer sea surface temperatures (SSTs) on the Great Barrier Reef (GBR) can trigger the loss of Symbiodiniaceae from the host, and very low concentrations of the most common herbicide, diuron, can disrupt the photosynthetic activity of microalgae. In an era of rapid environmental change, investigation into the assisted evolution of the coral holobiont is underway in an effort to enhance the resilience of corals. Apicomplexan-like microalgae were discovered in 2008 and the Phylum Chromerida (chromerids) was created. Chromerids have been isolated from corals and contain a functional photosynthetic plastid. Their discovery therefore opens a new avenue of research into the use of alternative/additional photosymbionts of corals. However, only two studies to-date have investigated the symbiotic nature of Chromera velia with corals and thus little is known about the coral-chromerid relationship. Furthermore, the response of chromerids to environmental stressors has not been examined. Here we tested the performance of four chromerid strains and the common dinoflagellate symbiont Cladocopium goreaui (formerly Symbiodinium goreaui, ITS2 type C1) in response to elevated temperature, diuron and their combined exposure. Three of the four chromerid strains exhibited high thermal tolerances and two strains showed exceptional herbicide tolerances, greater than observed for any photosynthetic microalgae, including C. goreaui. We also investigated the onset of symbiosis between the chromerids and larvae of two common GBR coral species under ambient and stress conditions. Levels of colonization of coral larvae with the chromerid strains were low compared to colonization with C. goreaui. We did not observe any overall negative or positive larval fitness effects of the inoculation with chromerid algae vs. C. goreaui. However, we cannot exclude the possibility that chromerid algae may have more important roles in later coral life stages and recommend this be the focus of future studies

Thermal and herbicide tolerances of chromerid algae and their ability to form a symbiosis with corals

Reef-building corals form an obligate symbiosis with photosynthetic microalgae in the family Symbiodiniaceae that meet most of their energy requirements. This symbiosis is under threat from the unprecedented rate of ocean warming as well as the simultaneous pressure of local stressors such as poor water quality. Only 1°C above mean summer sea surface temperatures (SSTs) on the Great Barrier Reef (GBR) can trigger the loss of Symbiodiniaceae from the host, and very low concentrations of the most common herbicide, diuron, can disrupt the photosynthetic activity of microalgae. In an era of rapid environmental change, investigation into the assisted evolution of the coral holobiont is underway in an effort to enhance the resilience of corals. Apicomplexan-like microalgae were discovered in 2008 and the Phylum Chromerida (chromerids) was created. Chromerids have been isolated from corals and contain a functional photosynthetic plastid. Their discovery therefore opens a new avenue of research into the use of alternative/additional photosymbionts of corals. However, only two studies to-date have investigated the symbiotic nature of Chromera velia with corals and thus little is known about the coral-chromerid relationship. Furthermore, the response of chromerids to environmental stressors has not been examined. Here we tested the performance of four chromerid strains and the common dinofiagellate symbiont Cladocopium goreaui (formerly Symbiodinium goreaui, ITS2 type C1) in response to elevated temperature, diuron and their combined exposure. Three of the four chromerid strains exhibited high thermal tolerances and two strains showed exceptional herbicide tolerances, greater than observed for any photosynthetic microalgae, including C. goreaui. We also investigated the onset of symbiosis between the chromerids and larvae of two common GBR coral species under ambient and stress conditions. Levels of colonization of coral larvae with the chromerid strains were low compared to colonization with C. goreaui. We did not observe any overall negative or positive larval fitness effects of the inoculation with chromerid algae vs. C. goreaui. However, we cannot exclude the possibility that chromerid algae may have more important roles in later coral life stages and recommend this be the focus of future studies

Can trans-generational experiments be used to enhance species resilience to ocean warming and acidification?

Human-assisted, trans-generational exposure to ocean warming and acidification has been proposed as a conservation and/or restoration tool to produce resilient offspring. To improve our understanding of the need for and the efficacy of this approach, we characterized life-history and physiological responses in offspring of the marine polychaete Ophryotrocha labronica exposed to predicted ocean warming (OW: + 3 degrees C), ocean acidification (OA: pH -0.5) and their combination (OWA: + 3 degrees C, pH -0.5), following the exposure of their parents to either control conditions (within-generational exposure) or the same conditions (trans-generational exposure). Trans-generational exposure to OW fully alleviated the negative effects of within-generational exposure to OW on fecundity and egg volume and was accompanied by increased metabolic activity. While within-generational exposure to OA reduced juvenile growth rates and egg volume, trans-generational exposure alleviated the former but could not restore the latter. Surprisingly, exposure to OWA had no negative impacts within-or trans-generationally. Our results highlight the potential for trans-generational laboratory experiments in producing offspring that are resilient to OW and OA. However, trans-generational exposure does not always appear to improve traits and therefore may not be a universally useful tool for all species in the face of global change

Microbial contributions to the persistence of coral reefs

On contemplating the adaptive capacity of reef organisms to a rapidly changing environment, the microbiome offers significant and greatly unrecognised potential. Microbial symbionts contribute to the physiology, development, immunity and behaviour of their hosts, and can respond very rapidly to changing environmental conditions, providing a powerful mechanism for acclimatisation and also possibly rapid evolution of coral reef holobionts. Environmentally acquired fluctuations in the microbiome can have significant functional consequences for the holobiont phenotype upon which selection can act. Environmentally induced changes in microbial abundance may be analogous to host gene duplication, symbiont switching / shuffling as a result of environmental change can either remove or introduce raw genetic material into the holobiont; and horizontal gene transfer can facilitate rapid evolution within microbial strains. Vertical transmission of symbionts is a key feature of many reef holobionts and this would enable environmentally acquired microbial traits to be faithfully passed to future generations, ultimately facilitating microbiome-mediated transgenerational acclimatisation (MMTA) and potentially even adaptation of reef species in a rapidly changing climate. In this commentary, we highlight the capacity and mechanisms for MMTA in reef species, propose a modified Price equation as a framework for assessing MMTA and recommend future areas of research to better understand how microorganisms contribute to the transgenerational acclimatisation of reef organisms, which is essential if we are to reliably predict the consequences of global change for reef ecosystems

Identification Of A Molecular Basis For The Juvenile Sleep State

Sleep is a highly conserved behavior critical to nervous system function. Across species, sleep amounts are highest in young animals and taper with maturity. These ontogenetic changes to sleep have long been thought to facilitate brain maturation. Indeed, developmental sleep abnormalities are highly prevalent across neuropsychiatric disorders. However, knowledge of the genetic and molecular factors controlling early life sleep is lacking. Here, we use Drosophila to investigate the juvenile sleep state. Detailed behavioral characterization supports the idea that juvenile sleep represents a distinct behavioral state, uniquely evolved for the needs of a developing nervous system. Consistent with this, we find that sleep ontogenetic change is preserved in all studied short and long sleeping Drosophila mutants, suggesting that the molecular determinants of adult sleep duration do not regulate developmental sleep transitions. Through an RNAi-based screen, we identified the transcription factor pdm3 as the first known genetic regulator of sleep ontogeny. Increased sleep in young flies is normally driven by elevated activity of the sleep-promoting dorsal fan shaped body (dFSB). Loss of PDM3 leads to a premature increase in inhibitory wake-promoting dopaminergic (DA) synapses in the dFSB, reducing dFSB activity and preventing young flies from achieving the high sleep amounts normally observed. Pdm3 acts during the mid-pupal developmental stage to control DA projection ingrowth to this sleep region. Transcriptional analysis indicated that pdm3-mediated repression of the synaptogenesis gene Msp300 controls sleep ontogeny. Overall, this work demonstrates that juvenile sleep is a unique behavior subject to distinct genetic regulation, and provides the first insight into molecular cues governing sleep ontogeny. Further, our findings support the existence of a new class of developmentally-expressed sleep genes that orchestrate sleep circuit formation, raising the possibility that some primary sleep disorders are of neurodevelopmental origin

Identification of a Molecular Basis for the Juvenile Sleep State

Sleep is a highly conserved behavior critical to nervous system function. Across species, sleep amounts are highest in young animals and taper with maturity. These ontogenetic changes to sleep have long been thought to facilitate brain maturation. Indeed, developmental sleep abnormalities are highly prevalent across neuropsychiatric disorders. However, knowledge of the genetic and molecular factors controlling early life sleep is lacking. Here, we use Drosophila to investigate the juvenile sleep state. Detailed behavioral characterization supports the idea that juvenile sleep represents a distinct behavioral state, uniquely evolved for the needs of a developing nervous system. Consistent with this, we find that sleep ontogenetic change is preserved in all studied short and long sleeping Drosophila mutants, suggesting that the molecular determinants of adult sleep duration do not regulate developmental sleep transitions. Through an RNAi-based screen, we identified the transcription factor pdm3 as the first known genetic regulator of sleep ontogeny. Increased sleep in young flies is normally driven by elevated activity of the sleep-promoting dorsal fan shaped body (dFSB). Loss of PDM3 leads to a premature increase in inhibitory wake-promoting dopaminergic (DA) synapses in the dFSB, reducing dFSB activity and preventing young flies from achieving the high sleep amounts normally observed. Pdm3 acts during the mid-pupal developmental stage to control DA projection ingrowth to this sleep region. Transcriptional analysis indicated that pdm3-mediated repression of the synaptogenesis gene Msp300 controls sleep ontogeny. Overall, this work demonstrates that juvenile sleep is a unique behavior subject to distinct genetic regulation, and provides the first insight into molecular cues governing sleep ontogeny. Further, our findings support the existence of a new class of developmentally-expressed sleep genes that orchestrate sleep circuit formation, raising the possibility that some primary sleep disorders are of neurodevelopmental origin

Identification of a Molecular Basis for the Juvenile Sleep State

Sleep is a highly conserved behavior critical to nervous system function. Across species, sleep amounts are highest in young animals and taper with maturity. These ontogenetic changes to sleep have long been thought to facilitate brain maturation. Indeed, developmental sleep abnormalities are highly prevalent across neuropsychiatric disorders. However, knowledge of the genetic and molecular factors controlling early life sleep is lacking. Here, we use Drosophila to investigate the juvenile sleep state. Detailed behavioral characterization supports the idea that juvenile sleep represents a distinct behavioral state, uniquely evolved for the needs of a developing nervous system. Consistent with this, we find that sleep ontogenetic change is preserved in all studied short and long sleeping Drosophila mutants, suggesting that the molecular determinants of adult sleep duration do not regulate developmental sleep transitions. Through an RNAi-based screen, we identified the transcription factor pdm3 as the first known genetic regulator of sleep ontogeny. Increased sleep in young flies is normally driven by elevated activity of the sleep-promoting dorsal fan shaped body (dFSB). Loss of PDM3 leads to a premature increase in inhibitory wake-promoting dopaminergic (DA) synapses in the dFSB, reducing dFSB activity and preventing young flies from achieving the high sleep amounts normally observed. Pdm3 acts during the mid-pupal developmental stage to control DA projection ingrowth to this sleep region. Transcriptional analysis indicated that pdm3-mediated repression of the synaptogenesis gene Msp300 controls sleep ontogeny. Overall, this work demonstrates that juvenile sleep is a unique behavior subject to distinct genetic regulation, and provides the first insight into molecular cues governing sleep ontogeny. Further, our findings support the existence of a new class of developmentally-expressed sleep genes that orchestrate sleep circuit formation, raising the possibility that some primary sleep disorders are of neurodevelopmental origin