Evaluating nanopore sequencing for microbial community characterization in catfish pond water

Authors: Older, Caitlin; Richardson, Brad; Wood, Monica; Waldbieser, Geoffrey - Geoff; WARE, CYNDI - Mississippi State University; GRIFFIN, MATT - Mississippi State University; Ott, Brian

Submitted to: Journal of the World Aquaculture Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/12/2023
Publication Date: 7/3/2023
Citation: Older, C.E., Richardson, B.M., Wood, M.L., Waldbieser, G.C., Ware, C., Griffin, M.J., Ott, B.D. 2023. Evaluating nanopore sequencing for microbial community characterization in catfish pond water. Journal of the World Aquaculture Society. 55:289-301. https://doi.org/10.1111/jwas.13002.
DOI: https://doi.org/10.1111/jwas.13002

Interpretive Summary: In the United States, catfish are primarily farmed in earthen ponds, resulting in an aquatic environment influenced both by management practices and natural ecological processes. Issues related to pond ecology, such as high concentrations of ammonia or off-flavor compounds, have direct impacts on fish health and production efficiency. Bacteria are important components of pond ecology and therefore, identifying which bacteria are present in ponds can be useful for understanding what conditions may lead to bacterial communities associated with disease or off-flavor and could inform management practices. Scientists at the USDA, ARS, Warmwater Aquaculture Research Unit in Stoneville, MS, along with scientists at Mississippi State University identified appropriate methods for bacterial community profiling of catfish pond water. Optimal methods for long-read DNA sequencing technology, known as nanopore sequencing, were identified by using a mixture of DNA from multiple bacteria of relevance to aquaculture. Appropriate methods were then used on samples collected monthly from three experimental catfish ponds throughout a single growing season (May through September 2020). These results showed that each pond has a unique bacterial community, even when the ponds are in close proximity and under the same management. However, the time of sample collection had more influence on what bacteria were present in the pond water. Now that appropriate methods have been identified, this technique can be used for further work on bacteria-associated issues in catfish aquaculture ponds to improve catfish production.

Technical Abstract: In the United States, catfish are primarily farmed in earthen ponds, resulting in an aquatic environment influenced both by management practices and natural ecological processes. Issues related to pond ecology, such as high concentrations of ammonia or off-flavor compounds, have direct impacts on fish health and production efficiency. Bacteria are important components of pond ecology and play integral roles in nutrient cycling, disease, and the dissolved oxygen budget of commercial catfish ponds. Profiling pond water microbiota can be useful for understanding what conditions may lead to microbial communities associated with disease or off-flavor and could inform management practices. The aim of this study was to identify appropriate methods for bacterial community profiling of catfish pond water with nanopore sequencing of the 16S rRNA gene. To this end, two forward primers (27F, 27F_mod), two reverse primers (1492R, 1525R), and three different polymerase chain reaction (PCR) cycle numbers (10, 15 and 20 cycles) were tested on a mock community consisting of aquaculture-relevant bacteria. Results indicated a combination of forward primer 27F_mod and reverse primer 1492R, and minimizing the number of PCR cycles, yielded results most consistent with the theoretical composition of the mock. The optimized protocol was then applied to water samples obtained from three experimental catfish ponds sampled in May, June, August, and September 2020. Applying these methods to pond samples demonstrated individual ponds represent distinct microcosms composed of unique bacterial communities, although this pond effect was secondary to the influence of sampling month on pond community dynamics.