Lema, Anaïs Kimberley (2014) Diversity, stability, and uptake of diazotrophic bacterial communities associated with corals of the Great Barrier Reef. PhD thesis, James Cook University.

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Abstract

Bacteria play crucial roles in biogeochemical cycles and are common symbiotic partners in many living organisms. Although bacteria associated with corals are known to be abundant, diverse and specific, their ecological and functional roles within the coral holobiont are still poorly understood. Diazotrophic prokaryotes, the only organisms capable of fixing gaseous nitrogen (N₂) and converting it to biological usable forms like ammonium (NH₄), are the link between the largest reservoir of nitrogen in the atmosphere and downstream nitrogen pathways. Nitrogen fixation is particularly important in nitrogen-limited ecosystems like coral reefs, and therefore represents a potentially important, but virtually unexplored, functional role for coralassociated bacteria. Consequently, my overall aim in this thesis is to enhance current understanding of the diversity, abundance, stability and initiation of coral-diazotroph relationships using advanced molecular and imaging techniques.

Through profiling of the conserved subunit of the gene encoding for the dinitrogenase iron protein, nifH, I explored the association of coral-bacteria that are capable of carrying out nitrogen fixation (i.e diazotrophs) : 1) within three coral species, Acropora millepora, Pocillopora damicormis and Acropora muricata, at different locations on the Great Barrier Reef and among different micro-niches (mucus and tissue) (chapter 2); 2) in Acropora millepora throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef (Chapter 3); and 3) throughout early life history stages of A. millepora (Chapter 4). Initially, nifH clone libraries were used to explore diazotroph diversity (Chapter 2), followed by application of amplicon pyrosequencing of the nifH gene, including development of computational techniques for analysis of such datasets (Chapters 3 and 4).

Comparisons of diazotrophic assemblages (clone libraries) revealed that corals harbour diverse nifH phylotypes that differ between tissue and mucus microhabitats (Chapter 2). Coral mucus nifH libraries displayed high heterogeneity, many of which overlapped with bacterial groups found in seawater. In contrast, the dominant diazotrophic assemblages associated with tissues within each coral species were the same across all reefs studied, indicating that coral-diazotrophic associations are species-specific. Notably, dominant diazotrophic assemblages within tissues of all three coral species, and across A. millepora from different reefs, seasons, and early life stages, were all dominated with sequences closely related to Rhizobiales, which represented over 50% of the total sequences retrieved across most samples (Chapters 2, 3, and 4). In addition, Bradyrhizobia spp.-affiliated sequences (>50% of rhizobia sequences), dominated diazotrophic assemblages of both early life stages and adults of A. millepora suggesting that they are a continuous component of the coral microbiome (Chapters 3 and 4).

To evaluate the relative abundance of rhizobia in the coral holobiont, overall bacterial communities were analysed using amplicon sequencing of the general bacterial 16S rRNA gene (Chapters 3 and 4). Rhizobia phylotypes represented a variable and generally small fraction of overall bacterial communities in adult colonies of A. millepora from inshore and midshelf reefs throughout all seasons (Chapter 3). However, the consistent presence of rhizobia throughout all early life stages of A. millepora, representing 2 to 12% of 16S rRNA gene sequences across all samples from aposymbiotic (i. e. Symbiodinium-free) larvae to 12 month-old symbiotic juveniles, demonstrate that these functionally important bacteria are present and remain stable throughout the coral's early life stages (Chapter 4).

Phylogenetic analyses of general bacterial communities (16S rRNA genes) associated with A. millepora revealed that inshore corals are dominated by bacteria from the Oceanospirillales family, particularly Endozoicomonas spp., whereas offshore corals have more diverse communities (Chapter 3). Roseobacter-affiliated sequences dominate the early development stages of lab-reared A. millepora larvae and 1 week-old juveniles; whereas bacterial communities of older juveniles are diverse and significantly shift in the field (Chapter 4). Together, these two chapters indicate that the environment also influences coral-associated bacterial communities.

Finally, diazotrophic bacteria were co-localised using FISH (Fluorescent In Situ Hybridization) and confocal microscopy, and translocation of fixed nitrogen was observed within Acropora millepora larvae using NanoSIMS (nano scale secondary ion mass spectrometry) (Chapter 5). Coral larvae were experimentally incubated with a diazotroph derived from A. millepora juveniles and most similar to Vibrio diazotrophicus, and grown with ¹⁵N gas as sole source of nitrogen. After a 4 hour incubation, ¹⁵N enriched Vibrio sp.( Acc. Num.: KF691569) cells (average isotopic enrichment of ~1.4 ¹⁵N atom %; +/- SE 0.08) were observed in the aboral ectodermal layer of coral larvae, through FISH and NanoSIMS analyses. Other bacterial members were also observed in ectodermal layers using FISH, although not solely at the aboral end.

In summary, this thesis significantly advances knowledge about diazotrophic assemblages in corals, particularly Acropora millepora, on the Great Barrier Reef. The presence of consistent and dominant populations of rhizobia across all nifH datasets suggests that, as in terrestrial plants, rhizobia could have developed a mutualistic relationship with corals, and may be essential in nitrogen cycling within the coral holobiont. The development of methods for the observation of coral-bacteria nutritional interactions through advanced imaging techniques (FISH and NanoSIMS) will highly contribute to future studies exploring nutritional pathways in coral symbiotic relationships.

Item ID:	39865
Item Type:	Thesis (PhD)
Keywords:	bacterial cultures; coral reef; corals; diazotrophic; ecology; genetics; Great Barrier Reef; larvae; marine bacteria; microbial; microorganisms; nitrogen-fixing; rhizobia; Queensland; reef
Related URLs:	http://researchonline.jcu.edu.au/22180/ http://researchonline.jcu.edu.au/36705/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 2: Lema, Kimberley A., Willis, Bette L., and Bourne, David G. (2012) Corals form characteristic associations with symbiotic nitrogen-fixing bacteria. Applied and Environmental Microbiology, 78 (9). pp. 3136-3144. Chapter 3: Lema, Kimberley A., Willis, Bette L., and Bourne, David G. (2014) Amplicon pyrosequencing reveals spatial and temporal consistency in diazotroph assemblages of the Acropora millepora microbiome. Environmental Microbiology, 16 (10). pp. 3345-3359.
Date Deposited:	12 Aug 2015 04:19
FoR Codes:	06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060503 Microbial Genetics @ 34% 05 ENVIRONMENTAL SCIENCES > 0501 Ecological Applications > 050102 Ecosystem Function @ 33% 06 BIOLOGICAL SCIENCES > 0603 Evolutionary Biology > 060309 Phylogeny and Comparative Analysis @ 33%
SEO Codes:	96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960808 Marine Flora, Fauna and Biodiversity @ 50% 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50%
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