Pathway independence: Bacteria-derived algicide IRI-160AA functions independently of ammonium toxicity in Karenia species.

The brevetoxin-producing microalga Karenia brevis blooms nearly annually along the US Gulf Coast, severely impacting human and ecosystem health. The marine bacterium Shewanella sp. IRI-160 produces an algicidal exudate, IRI-160AA, that has demonstrated control properties against harmful dinoflagellates including Karenia spp. The algicide contains ammonium (NH₄⁺) and several polyamines, which were previously shown to act synergistically to kill dinoflagellates. This algicidal activity, however, does not fully explain the observed effects of the algicide. The study presented here employed a pathway discrimination analysis to further explore relationships between the direct growth-inhibitory effects of NH4+ and the algicidal activity of IRI-160AA. Two strains of K. brevis and two strains of K. mikimotoi were conditioned to varying NH₄⁺ concentrations (50-150 µM) before applying identical acute stressor treatments consisting of NH₄⁺ alone (60 µM) or 2.5% IRI-160AA (∼60 µM NH₄⁺ equivalent). Response ratios quantified whether NH₄⁺ conditioning and acute exposure to either NH₄⁺ or IRI-160AA produced independent or interactive effects. Pathway discrimination analysis showed threshold-dependent growth inhibitory effects of acute NH₄⁺ exposure with conditioning, where antagonistic effects were observed at moderate and high levels of conditioning (response ratios 0.68-0.88 and 0.57-0.71, respectively). IRI-160AA exposure provides evidence consistent with pathway independence across all conditioning levels for most species Karenia strains tested (response ratios 0.95-1.15), suggesting that NH₄⁺ and other algicidal components act through separate cellular pathways. These findings support that IRI-160AA contains multiple bioactive compounds that operate through distinct, non-interacting mechanisms and indicates that the algicide has potential for consistent performance across different nutrient conditions in coastal waters.