Identifying Functions of Bacteria Linked to Differential Success in Juvenile Oysters

Proposed Research Project: This project aims to characterize bacterial functions linked to improved oyster performance. We proposed metagenomic sequencing of bacteria from juvenile oysters from a single genetic family line. We deployed these oysters at four sites in the Patuxent and South Rivers from October 2023 to February 2024 and monitored their performance.

Working in collaboration with Dr. Tsvetan Bachvaroff (UMCES-IMET), metagenomes will be characterized for five oysters from each of the four deployment sites and five “baseline” oysters sampled before all deployments for a total of 25 oysters. DNA will be extracted from whole juvenile oysters and checked for quality. We will then enrich for bacterial DNA to minimize the non-target oyster DNA in the sample. Metagenomes will be sequenced using the nanopore GridION (IMET BAS lab) because it provides long reads that improve sequencing quality and depth, overall improving the reliability of bioinformatic analyses for inferring bacterial functions.

Researcher: Dr. Allison Tracy

Expected Outcomes

Best case scenario: The results of this study will identify functions of bacterial communities that are linked to higher performance in valuable juvenile oysters, thus providing novel information on why juvenile oysters have greater success at certain sites. For example, we may find more bacterial genes focused on metabolism in oysters with better growth. We also collected samples to characterize bacterial communities of adult oysters that we deployed at the four sites at the same time, though the adult dataset will not provide functional information. Interpreting both datasets together will maximize the value of both. For example, the functional bacterial information from the juvenile oysters will help us better interpret the performance of the adult oysters and the role of their bacterial associates.

Worst case scenario: If the extracted DNA is not of sufficient quality for GridION sequencing. In that case, we will use the NextSeq platform that has newly arrived at the IMET BAS lab. There may also be challenges with minimizing non-target oyster DNA in the sample, but we have identified techniques from previous studies as a starting point and oyster sequences can be filtered against the existing oyster genome.

Probable Ancillary Benefits

• Improved knowledge of the functions of bacterial symbionts in oysters that informs research, hatchery science, and restoration/ aquaculture applications, for example: hatchery breeding and rearing decisions, deployment practices, and site selection
• Enhanced IMET, Tracy lab, and Bachvaroff lab capacity for cutting-edge GridION sequencing
• New and stronger collaborations between IMET researchers and other UMCES labs
• Professional skills development for a technician working on this for her Master’s thesis (Tracy
  lab) and a PhD student providing additional support (Bachvaroff lab)

Results

We have piloted the methods for two juvenile oysters, each with three technical replicates, to identify the best methods for the full dataset. This has included testing the DNA extraction method and the Short Read Eliminator (SRE) to retain the longest DNA reads we need for Nanopore GridION sequencing. We were successful in extracting long reads of DNA from the tissue. Results of the SRE indicated two samples were ready for library preparation and sequencing on the Nanopore MinION.

The sequencer generated large quantities of DNA reads, but most of those reads were from the oyster host rather than the bacteria. This was a challenge that we expected, and we are now testing three methods to enhance the extraction of bacterial DNA vs. oyster host DNA: (1) swabbing oyster tissues for bacterial associates, (2) using the oyster pallial fluid rather than tissue, and (3) applying saponin to reduce oyster host DNA. Thus far, the saponin digest is most promising given the higher quantity of high-quality DNA. Our next steps are to continue evaluating the saponin treatment and to explore sequencing methods that help us focus on bacterial DNA.