Cooperative Fish and Wildlife Research Units Program: West Virginia
Education, Research and Technical Assistance for Managing Our Natural Resources

Smith, C.R., Ottinger, C.A., Walsh, H.L., Mazik, P.M. & Blazer, V.S. 2023. Application of a lipopolysaccharide (LPS)-stimulated mitogenesis assay in Smallmouth Bass (Micropterus dolomieu) to augment wild fish health studies. Fishes, 8(3), 159.


A mitogenesis assay was developed for smallmouth bass (Micropterus dolomieu) which distinguishes between proliferation of immunoglobulin M (IgM+ and IgM-) lymphocytes and utilizes imaging flow cytometry and advanced machine learning for cell cycle analyses. Mitogenesis was detected by 5-ethynyl-2’-deoxyuridine (EdU) incorporation into replicating DNA and lymphocytes were differentiated with an anti-smallmouth bass IgM monoclonal antibody. The assay was field tested in fish collected from three sites, in two river drainages within West Virginia, as part of fish health assessments occurring in spring before spawning and fall during recrudescence. Leukocytes were isolated from anterior kidney tissue and exposed to the mitogens concanavalin A (Con A), phytohemagglutinin (PHA-P) and lipopolysaccharide (LPS) from E.coli O111:B4 for the mitogenesis assay. Mitogenic responses were dependent upon specific mitogen used, collection site, and season in which the fish were collected. To provide a better understanding of factors affecting these responses, tissue parasites and macrophage aggregates were quantified. Wild fish are inherently exposed to stressors, including disease, parasites, and fluctuations in environmental parameters such as temperature, water quality and contaminants. The differences in lymphocyte mitogenesis among smallmouth bass populations observed in this study validate the utility of this improved method for future ecotoxicological studies and fish health assessments. It also emphasizes the need for integrated assessments to determine not only environmental parameters but also infectious agents that may modulate the immune response.