Development of qPCR assays for nitrification and denitrification genes in catfish aquaculture ponds

Authors: Older, Caitlin; GRIFFIN, MATT - Mississippi State University; WARE, CYNTHIA - Mississippi State University; Ott, Brian

Submitted to: Microbiology Spectrum
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2025
Publication Date: 5/22/2025
Citation: Older, C.E., Griffin, M.J., Ware, C., Ott, B.D. 2025. Development of qPCR assays for nitrification and denitrification genes in catfish aquaculture ponds. Applied and Environmental Microbiology. e03088-24. https://doi.org/10.1128/spectrum.03088-24.
DOI: https://doi.org/10.1128/spectrum.03088-24

Interpretive Summary: Nitrogenous waste products, primarily composed of fish waste and excess feed, are toxic to fish and thus their removal is a critical process in aquaculture production. In earthen pond production systems, such as those used in the catfish industry, phytoplankton act as the dominant sink for these waste products, however bacteria can also play roles in transforming the initial forms of nitrogenous waste into less toxic forms through processes known as denitrification and nitrification. As the United States catfish industry continues to intensify, the bacterial communities relevant in removal of nitrogenous waste will become more integral to efficient production and necessitate further research. In an effort to better understand the dynamics of these communities, scientists at the USDA ARS Warmwater Aquaculture Research Unit in Stoneville, MS, in collaboration with Mississippi State University researchers, developed quantitative PCR (qPCR) assays targeting denitrification and nitrification pathways. Quantitative PCR assays were successfully developed for four targets: amoA, nxrB, napA, and nirK. Testing of these assays in sediment and water samples collected from ponds with low or high dissolved oxygen, or no fish (control) suggest they may be useful for studies evaluating the effect of pond management practices on nitrifying and denitrifying bacterial populations.

Technical Abstract: Nitrogenous waste products are toxic to fish, and their removal is a critical process in aquaculture production. In earthen pond production systems, such as those used in the catfish industry, phytoplankton act as the dominant sink for ammonia; however, bacteria can also play roles in denitrification and nitrification, particularly when excess ammonia exists. As the US catfish industry continues to intensify, the bacterial communities relevant in the removal of nitrogenous waste will become more integral to efficient production and necessitate further research. Here, quantitative PCR (qPCR) assays targeting four genes covering the denitrification and nitrification pathways present in catfish aquaculture ponds were developed. Twenty-four existing primer sets were used to amplify relevant genes in samples obtained from catfish pond water and sediment and then subjected to high-throughput sequencing to identify the sequence variants present in the environment. Five conventional PCR assays yielded sequencing results conducive to qPCR primer design. Quantitative PCR assays were successfully developed for four targets: amoA, nxrB, napA, and nirK. Application of these assays to sediment and water samples collected from ponds with low or high dissolved oxygen, or no fish (control), demonstrated significant differences in the abundance of amoA, nxrB, and nirK genes. Significantly higher abundances of these genes were found in ponds with low and high dissolved oxygen. In water samples, NO3-N exhibited significant positive correlations with the abundance of three genes (nxrB, napA, and nirK) encoding enzymes that either produce or utilize nitrite.