Title

Microbial diversity and function, and an exploration of bleaching in the Australian kelp Ecklonia Radiata

Author Identifiers

Charlie Melissa Phelps

http://orcid.org/0000-0003-4375-4550

Date of Award

2022

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Science

First Advisor

Kathryn McMahon

Second Advisor

Megan Huggett

Third Advisor

Peter Steinberg

Fourth Advisor

Thomas Wernberg

Abstract

The kelp, Ecklonia radiata is the dominant, temperate macroalgae in the Great Southern Reef (GSR) of Australia. As a foundation species, the kelp provides a habitat for many marine organisms in the GSR benthic reef ecosystems. Macroalgae such as kelps host a dense biofilm, consisting of a compositionally and functionally diverse consortium of microbiota. The macroalgal host and its associated microbiome constitute a ‘holobiont’, where interactions can contribute towards the success and survival of both the microbial community and host. Despite the importance of E. radiata, and the tightknit relationship with its associated microbial community, describing and interpreting diversity and functionality of the microbial community is in its infancy. There is a need to further understand the dynamics of the microbial community composition and gene function, and how these vary over space and time. An understanding of natural variation is of critical importance when attempting to interpret the impact of environmental perturbations (for example, heat waves and eutrophication), which can cause shifts in microbial community composition in holobionts, and may result in dysbiosis, the proliferation of opportunistic pathogens, and eventually disease. Ecklonia radiata is documented to experience bleaching disease across its habitat range, however there are no comprehensive investigations as to the possible pathogens that are associated with E. radiata bleaching disease. Thus, the overarching aim of this thesis is to investigate the microbial diversity and function associated with E. radiata and the relationship with tissue bleaching on the kelp.

Using 16S rRNA gene amplicon sequencing, Chapter 2 aimed to (1) examine the temporal and spatial variations in the bacterial community of bacterial microbiota on the kelp E. radiata, and (2) investigate the relationship between environmental drivers and the bacterial community on kelp. These analyses revealed that over large geographical distances (∼3,300 kms), kelp microbiomes differed significantly, however within regions the community structure was relatively stable. The environmental factors that were measured somewhat influenced the community structure. A persistent core microbial community was identified with the same members dominating across regions, potentially suggesting these individuals play essential roles in maintaining host health. As such, it is imperative to define the key functional roles in the E. radiata microbiome and establishing key processes that maintain kelp holobiont health.

The E. radiata microbial gene function profile was then investigated through metagenome shotgun sequencing (Chapter 3). Here, I aimed to examine the functional repertoire of the bacterial community of the kelp and to identify what relationship, if any, there is between spatial (four sites in the NSW region) and temporal (seven bi-monthly time points, over a 17-month period) patterns in microbial function and associated environmental conditions. I identified several key functional pathways in the E. radiata microbiome, including pathways associated with defence response mechanisms, biogeochemical cycling and nutrient metabolism. Additionally, spatiotemporal differences in gene function composition were noted, and environmental drivers, in particular temperature, were found to influence the E. radiata microbial gene functional profile. These novel findings are fundamental in identifying the E. radiata holobiont functional processes and are critical in the understanding of the role microbes play in maintaining kelp health and the subsequent reef ecosystem.

The baseline microbial community composition and functioning can be altered, this generally occurs with extreme weather conditions and or anthropological disturbances such as eutrophication. When this occurs, host associated microbial communities can shift to a depreciated state, impacting the host, potentially exacerbating opportunistic pathogens, and facilitating disease. For E. radiata some taxa have been identified to be abundant on bleached kelp compared to healthy, but their role as pathogens has not been investigated. Koch’s postulates are a series of tests that aim to establish a causative relationship between disease and pathogens and are performed through a series of inoculation studies. In marine systems, a similar series of tests have been done in a range of studies to understand the role of individual strains of bacteria in diseases of corals, sponges and macroalgae, however, the methods among studies vary slightly in the way that the inoculation treatments are performed and controlled. In Chapter 4 I aimed to explore 1) the extent of confounding effects from bacterial growth media in inoculation studies of diseases of marine benthic organisms through a literature review; 2) the effect of bacterial growth media and its components on bleaching disease. The review identified confounding factors in previous inoculation studies, based on whether the putative pathogen cells were pre-treated prior to inoculation e.g., centrifuged washed and/or filtered and if a sterile media control was incorporated in the experimental design. I found that nutrient growth media used in inoculation studies contributes to bleaching disease in E. radiata and caused a shift in the overall microbial community composition. Furthermore, the microbial community composition was substantially altered when kelps were held in aquaria, in comparison to undisturbed kelps collected from the reef. These results were summarised into a framework recommending approaches for interpretating previous inoculation studies and for future inoculation research. The recommendations for future inoculation studies include having pre-treatment methods and controls to reduce nutrient addition. It is also recommended that the microbial community composition is assessed via a method such as 16S rRNA amplicon sequencing, to identify any shifts in the microbiome when moved from the reef into the aquaria. The range of knowledge obtained from this research has widened the understanding of kelp holobiont interactions and provided invaluable data and a tool that can advise and inform researchers and managers on changes in kelp health dynamics in projected climate change conditions in the future. Hence, the key findings identified in this thesis are an important contribution for kelp conservation and identify possible concepts for restoration efforts through understanding of the E. radiata holobiont.

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