Quantitative PCR assays to measure the HPI axis neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) in channel catfish (Ictalurus punctatus)

Authors: Ott, Brian; Torrans, Eugene; GRIFFIN, MATT - Mississippi State University; ALLEN, PETER - Mississippi State University

Submitted to: Aquaculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2022
Publication Date: 4/21/2022
Citation: Ott, B.D., Torrans, E.L., Griffin, M.J., Allen, P.J. 2022. Quantitative PCR assays to measure the HPI axis neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) in channel catfish (Ictalurus punctatus). Aquaculture. 555:738253. https://doi.org/10.1016/j.aquaculture.2022.738253.
DOI: https://doi.org/10.1016/j.aquaculture.2022.738253

Interpretive Summary: Catfish and other animals initiate the stress response through a series of events that occur in the brain that can be measured using molecular biology techniques. Scientists at the USDA-ARS Warmwater Aquaculture Research Unit collaborated with a scientist from Mississippi State University to create a quantitative polymerase chain reaction (qPCR) assay to measure the concentration of mRNA from genes associated with the stress response in the channel catfish hypothalamus. The performance of the assay and detailed procedural instructions are provided in this manuscript. This qPCR assay was used in numerous USDA-ARS WARU experiments and will provide a useful tool for assessing the stress response of catfish for aquaculture and fish biology scientists.

Technical Abstract: Central to the stress response of fishes is the upregulation of the hypothalamus-pituitary-interrenal (HPI) axis. This pathway is initiated by an increase of corticotropin-releasing factor (CRF) and urotensin I (UI) transcripts which translate into hormones stimulating the production of cortisol in the anterior kidney. Quantifying changes in CRF and UI transcription may provide molecular-level evidence of the initiation of the stress response. To determine changes in CRF and UI transcription in the channel catfish (Ictalurus punctatus) hypothalamus, two duplex quantitative PCR (qPCR) assays were developed, optimized, and tested for both CRF and UI as targets. Performance of each respective qPCR was validated as a singleplex reaction and a duplex using a-tubulin as a reference. There were no significant declines in qPCR efficiency observed in the duplex format for either assay. These duplex qPCR assays are advantageous over single-gene reactions by reducing the number of wells used on the PCR plate, reducing reagent consumption, and minimizing sources of experimental error. Further, these assays have potential to serve as valuable tools evaluating the stress response of channel catfish at the molecular level.