While thermal priming and the relative role of epigenetic modifications have been widely studied in terrestrial plants, their roles remain unexplored in seagrasses so far. Here, we experimentally compared the ability of two different functional types of seagrass species, dominant in the Southern hemisphere, climax species Posidonia australis and pioneer species Zostera muelleri, to acquire thermal-stress memory to better survive successive stressful thermal events. To this end, a two-heatwave experimental design was conducted in a mesocosm setup. Findings across levels of biological organization including the molecular (gene expression), physiological (photosynthetic performances and pigments content) and organismal (growth) levels provided the first evidence of thermal priming in seagrasses. Non-preheated plants suffered a significant reduction in photosynthetic capacity, leaf growth and chlorophyll a content, while preheated plants were able to cope better with the recurrent stressful event. Gene expression results demonstrated significant regulation of methylation-related genes in response to thermal stress, suggesting that epigenetic modifications could play a central role in seagrass thermal stress memory. In addition, we revealed some interspecific differences in thermal responses between the two different functional types of seagrass species. These results provide the first insights into thermal priming and relative epigenetic modifications in seagrasses paving the way for more comprehensive forecasting and management of thermal stress in these marine foundation species in an era of rapid environmental change.

Symbiosis between reef-building corals and unicellular algae (Symbiodiniaceae) fuels the growth and productivity of corals reefs. Capacity for Symbiodiniaceae to fix inorganic carbon (Ci) and translocate carbon compounds to the host is central to coral health, but how these processes change for corals thriving in environmental extremes remains largely unresolved. We investigate how a model coral -Pocillopora acuta -persists from a reef habitat into an adjacent extreme mangrove lagoon on the Great Barrier Reef. We combine respirometry and photophysiology measurements, Symbiodiniaceae genotyping, and 13 C labelling to compare P. acuta metabolic performance across habitats, in relation to the Ci uptake and 27 translocation capacity by symbionts' autotrophy. We show that differences in P. acuta 28 metabolic strategies across habitats align with a shift in dominant host-associated 29 Symbiodiniaceae taxon, from Cladocopium in the reef to Durusdinium in the mangroves. This 30 shift corresponded with a change in 'photosynthetic strategy', with P. acuta in the mangroves 31 utilising absorbed light for photochemistry over non-photochemical quenching. Mangrove 32 corals translocated similar proportions of carbon compared to the reefs, despite a lower Ci uptake. These trends indicate that coral survival in mangrove environments occurs through sustained translocation rate of organic compounds from coral symbionts to host. Introduction 36The ecological success of reef-building corals resides on their ability to establish and 37 maintain metabolic exchanges through an effective symbiotic association with dinoflagellates 38 from the family Symbiodiniaceae. Symbiodiniaceae fuel their hosts with organic carbon by fixing inorganic carbon (Ci) through photosynthesis (Davy et al. 2012). While Ci uptake rates by the algal symbionts have rarely been measured, they appear strongly regulated by environmental factors, such as availability of CO2 (pCO2) (Suggett et al. 2012b; Brading et al. 42 2013) and temperature (Oakley et al. 2014). Recent work on cultured Symbiodiniaceae 43 revealed that different environmental optima primarily drive variation in Ci uptake rates (Ros 44 et al. 2020). Within reef systems where Symbiodiniaceae are hosted within cnidarian tissues, symbiont cells are typically carbon-limited (Smith and Muscatine 1999; Doherty 2009; Towanda and Thuesen 2012); as such, cnidarians can exhibit a stimulated carbon metabolism under naturally higher pCO2 (more acidic) environments (Suggett et al. 2012b). The efficiency 48 of Symbiodiniaceae carbon metabolism across environments thus appears an important trait in 49 supporting their host's survival, and a means to cope with stressful conditions. 50 51 Associations between the cnidarian host and specific genera, species or strains of 52 Symbiodiniaceae profoundly influence the stress resilience of their coral host (Berkelmans and 53

Seagrasses are a diverse group of angiosperms that evolved to live in shallow coastal waters, an environment regularly subjected to changes in oxygen, carbon dioxide and irradiance. Zostera muelleri is the dominant species in south-eastern Australia, and is critical for healthy coastal ecosystems. Despite its ecological importance, little is known about the pathways of carbon fixation in Z. muelleri and their regulation in response to environmental changes. In this study, the response of Z. muelleri exposed to control and very low oxygen conditions was investigated by using (i) oxygen microsensors combined with a custom-made flow chamber to measure changes in photosynthesis and respiration, and (ii) reverse transcription quantitative real-time PCR to measure changes in expression levels of key genes involved in C metabolism. We found that very low levels of oxygen (i) altered the photophysiology of Z. muelleri, a characteristic of C mechanism of carbon assimilation, and (ii) decreased the expression levels of phosphoenolpyruvate carboxylase and carbonic anhydrase. These molecular-physiological results suggest that regulation of the photophysiology of Z. muelleri might involve a close integration between the C and C, or other CO concentrating mechanisms metabolic pathways. Overall, this study highlights that the photophysiological response of Z. muelleri to changing oxygen in water is capable of rapid acclimation and the dynamic modulation of pathways should be considered when assessing seagrass primary production.

Predation by heterotrophic protists drives the emergence of adaptive traits in bacteria, and often these traits lead to altered interactions with hosts and persistence in the environment. Here we studied adaptation of the cholera pathogen, Vibrio cholerae during long-term co-incubation with the protist host, Acanthamoeba castellanii. We determined phenotypic and genotypic changes associated with long-term intra-amoebal host adaptation and how this impacts pathogen survival and fitness. We showed that adaptation to the amoeba host leads to temporal changes in multiple phenotypic traits in V. cholerae that facilitate increased survival and competitive fitness in amoeba. Genome sequencing and mutational analysis revealed that these altered lifestyles were linked to non-synonymous mutations in conserved regions of the flagellar transcriptional regulator, flrA. Additionally, the mutations resulted in enhanced colonisation in zebrafish, establishing a link between adaptation of V. cholerae to amoeba predation and enhanced environmental persistence. Our results show that pressure imposed by amoeba on V. cholerae selects for flrA mutations that serves as a key driver for adaptation. Importantly, this study provides evidence that adaptive traits that evolve in pathogens in response to environmental predatory pressure impact the colonisation of eukaryotic organisms by these pathogens.

The availability of the first complete genome sequence of the marine flowering plant Zostera marina (commonly known as seagrass) in early 2016, is expected to significantly raise the impact of seagrass proteomics. Seagrasses are marine ecosystem engineers that are currently declining worldwide at an alarming rate due to both natural and anthropogenic disturbances. Seagrasses (especially species of the genus Zostera) are compromised for proteomic studies primarily due to the lack of efficient protein extraction methods because of their recalcitrant cell wall which is rich in complex polysaccharides and a high abundance of secondary metabolites in their cells. In the present study, three protein extraction methods that are commonly used in plant proteomics i.e., phenol (P); trichloroacetic acid/acetone/SDS/phenol (TASP); and borax/polyvinyl-polypyrrolidone/phenol (BPP) extraction, were evaluated quantitatively and qualitatively based on two dimensional isoelectric focusing (2D-IEF) maps and LC-MS/MS analysis using the two most abundant Australian seagrass species, namely Zostera muelleri and Posidonia australis. All three tested methods produced high quality protein extracts with excellent 2D-IEF maps in P. australis. However, the BPP method produces better results in Z. muelleri compared to TASP and P. Therefore, we further modified the BPP method (M-BPP) by homogenizing the tissue in a modified protein extraction buffer containing both ionic and non-ionic detergents (0.5% SDS; 1.5% Triton X-100), 2% PVPP and protease inhibitors. Further, the extracted proteins were solubilized in 0.5% of zwitterionic detergent (C7BzO) instead of 4% CHAPS. This slight modification to the BPP method resulted in a higher protein yield, and good quality 2-DE maps with a higher number of protein spots in both the tested seagrasses. Further, the M-BPP method was successfully utilized in western-blot analysis of phosphoenolpyruvate carboxylase (PEPC—a key enzyme for carbon metabolism). This optimized protein extraction method will be a significant stride toward seagrass proteome mining and identifying the protein biomarkers to stress response of seagrasses under the scenario of global climate change and anthropogenic perturbations.