The microbial ecology of marine sponge-associated microorganisms

Abstract

Sponges, phylum Porifera, are amongst the most ancient metazoans. As prodigious filter feeders, they influence both benthic and pelagic ecosystems and their association with a diverse range of microorganisms raises many questions in the ecology, evolution and biotechnology realms. Despite this recent attention, much remains unknown about the microbial communities associated within marine sponges. The research described within this thesis utilised DNA (16S rRNA-gene) and/or RNA (16S rRNA) next-generation sequencing technologies to address the specificity, stability and community-level ecology of sponge-microbe associations. Numerous 16S rRNA-based studies have identified microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so-called sponge-specific clusters (SCs) or sponge- and coral-specific clusters (SCCs). With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) available, the aim was to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. These results strongly support the existence of sponge-specific microbes, with the ARB database generated during this work providing a valuable resource for further studies of rare and abundant sponge symbionts. A systematic evaluation of sponge tissue preservation (RNAlater, liquid nitrogen, lyophilized, frozen) and nucleic acid extraction (CTAB-based DNA extraction, RNA isolation via TRIzol and two coextraction methods) protocols, for the recovery of DNA and/or RNA, was assessed using the New Zealand sponge Ancorina alata. Although all protocols resulted in sufficiently high DNA and/or RNA quantity and quality for downstream applications, there were significant differences in yield of nucleic acids extracted. Denaturing gradient gel electrophoresis (DGGE) analysis of community 16S rRNA gene- and 16S rRNA-derived fragments revealed no major changes attributable to either preservation or extraction method. All protocols performed favourably and therefore the choice of protocol can be made based on practical considerations, including ease of use, time availability and cost. Based on these assessments, RNAlater and a Qiagen co-extraction kit were used for all subsequent experiments of this thesis. Understanding variability in microbial communities is a fundamental goal when examining any microbe-host association. RNA (16S rRNA)–based amplicon pyrosequencing was used to investigate the temporal stability of sponge-associated bacterial communities. Temporal variation between a host and its symbiotic community can also be tracked by isotopic composition (δ15N and δ13C values) of the host tissue. Two New Zealand sponge species (A. alata and Tethya stolonifera) were sampled over a two year period. A total of 4468 operational taxonomic units (OTU), at the 97% level, were identified from both sponge species and seawater samples, affiliated with 26 bacterial phyla in total: 24 from seawater, 20 from T. stolonifera and 15 from A. alata samples. Bacterial symbionts of both sponge species were remarkably stable throughout the monitoring period, driven by a small number of OTUs that were persistent and dominated communities over time. Variability of sponge-associated bacterial communities was driven by OTUs that were low in abundance or transient over sampling time points. The similarity of δ15N and variability of δ13C values between sponge species suggested that only some carbon and nitrogen pathways are shared between the two sponge species. Considering the specificity and complexity of sponge microbial symbioses, coupled with the immense volumes of seawater to which sponges are exposed, it was hypothesised that the microbial communities within sponges will be sensitive to environmental perturbations. Using DNA (16SrRNA-gene) and RNA (16S rRNA)–based amplicon pyrosequencing the effects of sub-lethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile were assessed. Sub-lethal thermal stress (up to 31°C) had no effect on the present and/or active portions of the bacterial community, but a shift in the bacterial assemblage was observed in necrotic sponges. More than two-thirds of DNA and RNA sequences could be assigned to SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health at temperature above 31°C suggests that R. odorabile may be highly vulnerable to the effects of global climate change. Based on assessed thermal thresholds, R. odorabile was exposed to ambient (27°C) and sub-lethal (31°C) seawater temperatures, combined with a range of elevated nutrient levels (including 10 μmol/L total nitrogen). The symbiotic microbial community, analyzed by 16S rRNA gene amplicon pyrosequencing, was highly conserved for the duration of the experiment at both phylum and 97% OTU level. Additionally, elevated nutrients and temperatures did not alter the archaeal associations in R. odorabile, with sequencing of 16S rRNA gene libraries revealing similar Thaumarchaeota diversity and DGGE revealing consistent amoA gene patterns, across all experimental treatments. A conserved eukaryote community was also identified across all nutrient and temperature treatments by DGGE. These highly stable microbial associations indicate that R. odorabile symbionts are capable of withstanding short-term exposure to elevated nutrient concentrations and sub-lethal temperatures. This thesis contributes to the field of sponge microbiology by advancing our understanding of the specificity, stability and community level ecology of sponge-microbe associations. The studies described here are among the first to utilise next-generation sequencing technologies to address ecologically significant questions about marine sponges and the microbes which inhabit them.