Healthy Marine Ecosystems Rely on Their Tiniest Inhabitants

The health of ocean habitats relies on more than the activities of our finned friends. Just as human health is proving to be linked to the microbial communities in our guts, marine health is influenced by the bacteria in its ecosystems.

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Coral reefs are particularly important ecosystems, harbouring approximately 30% of the known aquatic species and supporting the productivity of ~25% of marine fisheries. Their collection of microorganisms — including the dinoflagellate alga Symbiodinium, fungi, viruses and bacteria — -are even considered to be a distinct ecological unit called a “holobiont.”

It is believed that the large, diverse populations of bacteria harbored within corals have co-evolved with their hosts, and are are likely to serve beneficial roles in provisioning and cycling of carbon, nitrogen and sulfur in coral reefs. They may also be involved in protecting their hosts against pathogenic microbes by preventing their colonisation through physical occupation of otherwise available niches or through the production of antibacterial compounds.

Despite the importance of bacteria to coral hosts, there have been very few studies that focused on the identification and characterisation of species-specific association between corals and bacteria.

A group of Thai scientists from the National Center for Genetic Engineering and Biotechnology (BIOTEC) and the Phuket Marine Biological Center are taking deep dives into the marine microbiology of coral reefs in the Gulf of Thailand and the Andaman Sea to glean the role bacteria might play in the health of the habitat and its responses to environmental stressors, such as elevated seawater temperature.

In one study, published in January in the open access journal Microbiology, the team, led by lead author Sithichoke Tangphatsornruang and first author Wirulda Pootakham, investigated the dynamics of coral-associated microbiomes during a recent thermal bleaching event in the Andaman Sea.

Previous studies had suggested that an intact bacterial consortium can provide coral holobionts with resilience against thermal stress, and experiments using antibiotics to artificially manipulate the resident bacterial community have demonstrated that undisturbed bacterial consortium ameliorated the thermal stress response and promoted the recovery of their hosts from bleaching events.

Examining the shifts in both bacterial and algal populations associated with Porites lutea corals during a natural bleaching event in 2016, the Thai team observed dramatic shifts in structure and diversity of associated bacterial communities when corals were exposed to elevated seawater temperature.

Contrary to what was expected, the composition and diversity of Symbiodinium communities remained unaltered throughout the bleaching event. Rather, the balance of the symbiosis shifted from a predominant association between corals and Gammaproteobacteria to a predominance of Alphaproteobacteria, and to a lesser extent Betaproteobacteria, following the bleaching event.

“It appears that the switching and/or shuffling of Symbiodinium types may not be the primary mechanism used by P. lutea to cope with increasing seawater temperature,” the authors wrote. “The shifts in the structure and diversity of associated bacterial communities may contribute more to the survival of the coral holobiont under heat stress.”

The New Gold Standard

Even though the contribution of coral-associated bacteria to the overall fitness and long-term survival of coral hosts have been recognized, very little is known about the composition and structure of bacterial communities across P. lutea reefs, and the Thai study was the first to examine shifts in bacterial and algal communities associated with P. lutea during a thermal stress.

One of the reasons why: The high cost and low-throughput nature of traditional environmental 16S rRNA gene profiling limited the amount of sequences done per sample and was insufficient to capture the complete diversity of the population.

The 16S rRNA gene is a highly conserved component of the transcriptional machinery of all DNA-based life forms and thus is highly suited as a target gene for sequencing DNA in samples containing up to thousands of different species. It consists of both conserved and variable regions, and sequencing the variable regions allows discrimination between different microorganisms such as bacteria, archaea and microbial eukarya.

Originally performed using clone-based Sanger sequencing, the traditional 16S method has gradually been replaced by various short-read next generation sequencing platforms due to their economy of scale and the orders of magnitude higher sequencing throughput.

The Thai team opted for a different approach: PacBio Single Molecule, Real-Time (SMRT) Sequencing. They detailed the approach and its results in another study, published in Nature Scientific Reports in June.

“A resurgence of full-length 16S rRNA sequences used as gold standards, through the adoption of the PacBio circular consensus sequencing (CCS) technology, has the potential to transform microbial community profiling studies by increasing the accuracy of taxonomic assignments for both known and novel species,” the authors wrote.

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The full-length sequence data obtained via SMRT Sequencing was especially useful in mitigating issues relating to classification discrepancies/misclassification. The benefits in throughput achieved via other sequencing methods have come at the cost of read length, the authors argue.

“This tradeoff has inevitably resulted in less accurate classification of partial 16S sequences, especially at the genus or species level,” Pootakham et al wrote. “Previous studies have shown that taxonomic assignment and phylogenetic placement were highly sensitive to the region of the 16S rRNA gene sequenced as well as the length of the region sequenced.”

Full-length 16S sequences had the advantage of covering all hypervariable regions of the 16S rRNA genes, and could provide higher taxonomic resolution compared to partial 16S sequences, the authors said.

“In addition to providing correct taxonomic assignment, full-length 16S rRNA gene sequences yielded more accurate estimates of species richness compared to short hypervariable fragments obtained from next generation sequencing,” they wrote.

With tens of thousands of full-length 16S reads obtained from each sample, the team was able to thoroughly examine the core microbiome harboured by P. lutea and compared the diversity and composition of bacterial communities associated with corals from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, they identified a conserved community of bacteria that were present consistently across diverse reef habitats.

The team declared the PacBio full-length 16S rRNA sequences as “superior” in resolving taxonomic uncertainty of coral associates at the species level. They also found that using barcodes enabled multiplexing of different samples into a single SMRTbell library, reducing the overall sequencing cost.

“Recent advances in high-throughput PacBio sequencing and its ability to generate CCS reads with higher than 99% accuracy have provided an economical approach to investigate the diversity and structure of microbial communities in environmental samples using full-length 16S rRNA sequences,” the authors concluded.

The generation of full-length 16S rRNA sequences will help the research community expand the microbial 16S rRNA gene catalogue as new full-length sequences from underrepresented or previously undiscovered taxa can be deposited into the reference databases, the authors stated.

Together with the findings of the bleaching study, the data are important in furthering our understanding of coral habitat resilience in times of increased climate change, they added.

We’re also looking forward to what Rutgers researcher Alexander Shumaker will find when he investigates the microbiome of samples from a reef in the Hawaiian Archipelago during a natural temperature-induced coral bleaching event as part of his winning 2017 Microbial SMRT Grant proposal. Using shotgun metagenome sequencing, he hopes to characterize the symbiotic microbial community of M. capitata and understand whether differences in the coral microbiome may be a factor in the recovery of colonies that survive.

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