Role of ergosterol biosynthesis in growth, drug sensitivity and host colonization of honey bee trypanosomatid parasite, Lotmaria passim

Abstract Lotmaria passim is a widespread trypanosomatid parasite of the honey bee gut, yet the metabolic pathways underpinning its growth and infection remain poorly defined. Like other kinetoplastids, L. passim synthesizes ergosterol as its principal membrane sterol, but the functional significance of this pathway in an insect-associated lineage has not been experimentally assessed. We investigated ergosterol biosynthesis in L. passim through phylogenetic analyses, targeted gene disruption, sterol profiling, and honey bee infection assays. Early sterol biosynthetic enzymes displayed non-canonical evolutionary affiliations, whereas late stage enzymes localized to the endoplasmic reticulum. Genetic analyses showed that sterol C-8 isomerase appears to be essential for parasite viability, while sterol C-5 desaturase (SC5D) is dispensable in vitro. SC5D-null mutants (LpΔSC5D) accumulated ergosta-7,22-dienol, confirming its role in the final step of ergosterol synthesis. Although LpΔSC5D grew normally at optimal temperature, they exhibited temperature-sensitive growth defects, abnormal morphology, amphotericin B resistance, and a marked reduction in honey bee gut colonization. These findings demonstrate that while L. passim can tolerate altered sterol composition in culture, intact ergosterol biosynthesis is critical for environmental resilience and successful host infection. Sterol metabolism thus emerges as a key determinant of fitness and infection in this insect‑associated trypanosomatid.