Coral-associated bacterial communities in early coral life stages: transmission mode and scope for manipulation

© 2019 Katarina DamjanovicGlobal impacts of climate change and other anthropogenic disturbances are causing massive declines in coral reef ecosystems. As reef-forming scleractinian corals provide essential resources to a large part of the population, their degradation has severe ecological, social and economic consequences. Efforts are therefore urgently required to limit human impacts (e.g., by drastically reducing emissions of greenhouse gases), and also to assist coral adaptation to climate change. In this context, favourably adjusting coral-associated microbial communities could potentially benefit the host, as microbial symbionts are known to play critical roles in coral health. Probiotics have already proven effective in other organisms such as plants to increase crop yields, and humans to treat various bowel conditions. The successful application of probiotics in corals is contingent on the feasibility to manipulate the coral microbiome. Therefore, a central question of this thesis is whether the coral microbiome can be influenced by targeted bacterial inoculation in the laboratory. Initially, understanding how corals acquire and maintain their bacterial associates will assist in predicting whether probiotics could be taken up and retained across generations. The transmission mode of bacteria in corals with different reproductive strategies therefore constitutes an additional focus of this thesis. I start the thesis by providing information on the ecological importance of coral reefs, the threats that they are currently facing in the context of climate change and approaches that have been proposed to accelerate the adaptation of corals to environmental disturbances (Chapters 1 and 2). Within that scope, I focus on coral-associated microorganisms, highlight their roles for coral health and discuss the potential of microbial biotechnology to mitigate coral reef degradation. Chapter 2 includes experimental data providing proof-of-concept that the bacterial microbiome of juvenile corals can be influenced by exposing coral larvae to the mucus (which is known to contain a diverse microbiome) extracted from adult colonies of different coral species (Chapter 2). In Chapters 3 and 4, I investigate the transmission mode of coral-associated bacteria using 16S rRNA gene metabarcoding and fluorescence in situ hybridisation (FISH) microscopy. No evidence of direct vertical transmission of internalised bacteria was observed in the broadcast spawner Acropora tenuis (Chapter 3). However, metabarcoding shows that the gametes were already associated with diverse bacteria upon release and that early coral life stages successively associated with different bacterial communities, probably acquired from the environment. It is possible that the coral-associated mucus is removed during the FISH fixation procedure and therefore bacteria in the mucus would not be visualised by FISH microscopy. Parental colonies may thus drive the transfer of certain bacteria by releasing them to the water column upon spawning (horizontal transmission) and/or through the mucus layer coating the gametes while these are still inside the coral polyp (vertical transmission). Clear evidence for vertical transmission is present in the brooder, Pocillopora acuta, where newly released larvae contained internalised bacterial aggregates (Chapter 4). DNA metabarcoding provides evidence for vertical transmission as well as horizontal uptake of bacteria in this coral species. In Chapters 5 and 6, I explore the possibility of manipulating coral-associated bacterial communities by exposing coral recruits to fragments of adult corals (Chapter 5), as well as to a cocktail comprising a small number of pure bacterial cultures (Chapter 6). In the former approach, rearing P. acuta coral recruits in the vicinity of adult fragments of P. acuta and Platygyra daedalea under a flow-through system did not result in different bacterial associates developing in juveniles compared to control corals. The temporal changes of bacterial assemblages in early recruits suggest that their microbiome is dynamic, which may facilitate uptake of bacterial strains in an inoculum but also challenge their retention over time. In Chapter 6, I rear A. tenuis and P. daedalea recruits in the same aquaria and repeatedly inoculate them with a bacterial consortium generated in the laboratory. The seven bacterial strains present in the inoculum were enriched in inoculated recruits of the two coral species compared to the no-inoculum controls, and there is a significant effect of the inoculum on the coral-associated bacterial communities. Additionally, some of the structuring in the bacterial microbiomes is explained by host species, highlighting the role of host factors in shaping bacterial community composition. These results support proof-of- concept for the feasibility of coral microbiome manipulation as a first step towards developing probiotics aimed at enhancing coral climate resilience. In Chapter 7, the general discussion, characteristics of microbial inoculation strategies that could be implemented to corals are discussed, as well as their advantages and shortcomings. Emphasis is also drawn to current knowledge gaps and research priorities for the field of microbiome engineering in corals

Intra- and interspecific social challenges modulate the levels of an androgen precursor in a seasonally territorial tropical damselfish.

Studies on different vertebrate groups have provided evidence that androgen levels in males increase after competitive social interactions during the breeding season, as postulated by the Challenge Hypothesis. However, social modulation of androgen levels may vary with latitude and may differ between species holding seasonal versus year-round territories. Here, we tested the Challenge Hypothesis on a seasonal tropical damselfish, Abudefduf sexfasciatus, where males temporarily defend territory and eggs against both intra- and interspecific individuals. Carrying out simulated territorial intrusions (STIs) in the laboratory, we document for the first time a consistent increase in the plasma level of the androgen precursor 11-ketoandrostenedione (11KA) in fish confronted to either intra- or interspecific challenges. Collecting samples in the field also revealed higher 11KA levels in fish facing frequent territorial interactions than in non-territorial individuals. Levels of 11-ketotestosterone (11KT) were high in territorial males in the field, but were not incremented after simulated territorial intrusions in the laboratory. Plasma levels of cortisol and testosterone were not affected by challenges but were different in wild and captive specimens. Although the endocrine responses to STIs did not differ between intra- and interspecific challenges, agonistic displays expressed by resident fish were more intense towards intraspecific intruders. Taken together, our study emphasizes the need to incorporate androgen precursor concentrations to advance our understanding on the physiology of territorial interactions

No scope for social modulation of steroid levels in a year-round territorial damselfish.

Both latitude and mating system have been proposed to shape relationships between steroid hormone levels and social behavior. Recently it has been postulated that species with long lasting non-seasonal territorial behavior have low androgen responsiveness. Tropical damselfishes are an ideal family to test this proposition because they show a large variety in mating systems. Here we contribute to the comparative dataset by measuring the response in steroid levels after social modulation in the banded sergeant, Abudefduf septemfasciatus, a species with non-seasonal territoriality. In highly territorial and brooding males, we found low androgen and cortisol levels that did not increase after experimental intraspecific simulated territorial intrusions (STI tests). No relationship was found between the variation in steroid hormone levels and territorial responses to naturally occurring territorial intrusions. Although steroid levels were low, male A. septemfasciatus were highly territorial both to STI challenges and to fishes that passed the territory. They often chased intruders for several meters away from the territory. This indicates that during nest defence in a non-seasonal territorial damselfish species, territorial behaviors are shown independent of variation in androgen and cortisol levels

Growth of osteosarcoma cells in a three-dimensional bone-like matrix alters their susceptibility to adeno-associated virus

Osteosarcoma cells U2OS are partially susceptible to adeno-associated virus (AAV)-2 infection, allowing efficient synthesis of Rep proteins and, in a low percentage of cells, capsid production. It is not clear if this partial susceptibility to infection is due to the bone-cell-like nature of these cells or is a result of their transformed properties. Here, we grew osteosarcoma cells in a biomimetic three-dimensional bone-like matrix composed of calcium phosphate and chitosan, and tested whether this would increase or reduce their permissiveness to virus. The osteosarcoma cells grew in the matrix and began to express the alkaline phosphatase bone cell differentiation marker. This was accompanied by a block to their infection by AAV, as indicated by Rep and capsid production. Infection of cells growing in three-dimensional tissue-like matrices could be, in a wider context, a practical way to mimic in vivo conditions

Experimental inoculation of coral recruits with marine bacteria indicates scope for microbiome manipulation in Acropora tenuis and Platygyra daedalea

Coral-associated microorganisms are essential for maintaining the health of the coral holobiont by participating in nutrient cycling and protecting the coral host from pathogens. Under stressful conditions, disruption of the coral prokaryotic microbiome is linked to increased susceptibility to diseases and mortality. Inoculation of corals with beneficial microbes could confer enhanced stress tolerance to the host and may be a powerful tool to help corals thrive under challenging environmental conditions. Here, we explored the feasibility of coral early life stage microbiome manipulation by repeatedly inoculating coral recruits with a bacterial cocktail generated in the laboratory. Co-culturing the two species Acropora tenuis and Platygyra daedalea allowed us to simultaneously investigate the effect of host factors on the coral microbiome. Inoculation cocktails were regularly prepared from freshly grown pure bacterial cultures, which were hence assumed viable, and characterized via the optical density measurement of each individual strain put in suspension. Coral early recruits were inoculated seven times over 3 weeks and sampled once 36 h following the last inoculation event. At this time point, the cumulative inoculations with the bacterial cocktails had a strong effect on the bacterial community composition in recruits of both coral species. While the location of bacterial cells within the coral hosts was not assessed, metabarcoding using the 16S rRNA gene revealed that two and six of the seven bacterial strains administered through the cocktails were significantly enriched in inoculated recruits of A. tenuis and P. daedalea, respectively, compared to control recruits. Despite being reared in the same environment, A. tenuis and P. daedalea established significantly different bacterial communities, both in terms of taxonomic composition and diversity measurements. These findings indicate that coral host factors as well as the environmental bacterial pool play a role in shaping coral-associated bacterial community composition. Host factors may include microbe transmission mode (horizontal versus maternal) and host specificity. While the long-term stability of taxa included in the bacterial inocula as members of the host-associated microbiome remains to be evaluated, our results provide support for the feasibility of coral microbiome manipulation, at least in a laboratory setting

The contribution of microbial biotechnology to mitigating coral reef degradation

The decline of coral reefs due to anthropogenic disturbances is having devastating impacts on biodiversity and ecosystem services. Here we highlight the potential and challenges of microbial manipulation strategies to enhance coral tolerance to stress and contribute to coral reef restoration and protection

EGFR mutation testing from pleural effusions of non-small cell lung cancer patients at the institute for oncology and radiology of Serbia

Background: The use tumor-derived cell-free DNA extracted from body fluids is being evaluated for genetic testing in lung cancer. The aim of this study was to explore the feasibility and utility of implementation of EGFR molecular testing from pleural effusions in non-small cell lung cancer in the clinical diagnostics workflow. Patients and methods: This study included patients diagnosed with primary lung adenocarcinoma in the period July 2016 to June 2023. EGFR mutation testing was performed by qPCR (Cobas®) and dPCR. Testing was performed from 211 plasma samples when tissue was unavailable at diagnosis, and from 301 plasma samples and 18 pleural effusions at progression on first/second generation of EGFR TKIs. Descriptive methods of statistical analysis were used to summarize the sample data. Fisher's exact test, McNemar's test, Cohen's kappa tests were used for statistical analyses. Two-sided p-values <0.05 were considered statistically significant. Results: A significantly higher detection rate of the T790M mutation in pleural effusion was obtained compared to blood (50% and 20%, p=0.047). When comparing the detection success rate of the resistant T790M mutation in pleural effusion and blood, a statistically significant difference was obtained in favor of pleural effusion (50% vs. 21.87%, p=0.01). Conclusions: Superior performance of pleural effusions compared to blood plasma was shown both in the analysis of success rate and in the detection of the resistant T790M mutation, at progression on EGFR TKIs. Pleural effusion should be considered in this setting whenever available, especially in countries with limited health resources

High germline mutation rates, but not extreme population outbreaks, influence genetic diversity in a keystone coral predator.

Lewontin's paradox, the observation that levels of genetic diversity (π) do not scale linearly with census population size (Nc) variation, is an evolutionary conundrum. The most extreme mismatches between π and Nc are found for highly abundant marine invertebrates. Yet, the influences of new mutations on π relative to extrinsic processes such as Nc fluctuations are unknown. Here, we provide the first germline mutation rate (μ) estimate for a marine invertebrate in corallivorous crown-of-thorns sea stars (Acanthaster cf. solaris). We use high-coverage whole-genome sequencing of 14 parent-offspring trios alongside empirical estimates of Nc in Australia's Great Barrier Reef to jointly examine the determinants of π in populations undergoing extreme Nc fluctuations. The A. cf. solaris mean μ was 9.13 x 10-09 mutations per-site per-generation (95% CI: 6.51 x 10-09 to 1.18 x 10-08), exceeding estimates for other invertebrates and showing greater concordance with vertebrate mutation rates. Lower-than-expected Ne (~70,000-180,000) and low Ne/Nc values (0.0047-0.048) indicated weak influences of population outbreaks on long-term π. Our findings are consistent with elevated μ evolving in response to reduced Ne and generation time length, with important implications for explaining high mutational loads and the determinants of genetic diversity in marine invertebrate taxa

Conceptual diagram illustrating the relationships between evolutionary parameters underlying genetic diversity.

Under mutation-drift equilibrium, pairwise genetic diversity (π) reflects the balance between new mutations (μ) and the loss of variation via genetic drift, reflected by effective population size (Ne). In natural populations, π does not increase linearly with Ne and Ne is often smaller than census population size (Nc). μ is unknown for marine invertebrate taxa, thus the contributions of new mutations to π remain unknown. The drift-barrier hypothesis, a leading explanation for μ variation, proposes that selection against high μ is less efficient in small Ne species. This leads to an inverse relationship between μ and Ne (μ ~1/2Ne for diploid organisms) and the evolution of high μ in small Ne populations. Key evolutionary and ecological processes or traits affecting the magnitude of each parameter are shown. Several evolutionary processes act in combination to reduce Ne and thus, constrain π, decoupling it from Nc. This decoupling leads to a disparity between the range of π and Nc variance observed across taxa, known as Lewontin’s paradox. π can be measured in natural populations using DNA sequencing to calculate pairwise differences between sampled individuals and Nc can be approximated most accurately from direct observations of organisms from field surveys. μ and Ne are inferred parameters from polymorphism data.</p