Transmisssion Dynamics of Enterococcus spp. Throughout the Heliconius erato phyllis (Lepidoptera; Nymphalidae) Life Cycle

Enterococcus spp. are members of the gut microbiota of Heliconius butterflies, although their transmission remains poorly characterised. We used pulsed-field gel electrophoresis (PFGE) to compare Enterococcus strains from developmental stages of Heliconius erato phyllis and larval diet leaves, assessing their presence across the life cycle. Two female butterflies were collected, and 432 enterococci were isolated from eggs, larval instars, pupae, adults and larval diet leaves. Species were identified by MALDI-TOF MS, and antimicrobial susceptibility profiles were determined. Enterococcus faecalis and Enterococcus casseliflavus predominated across the butterfly life cycle and larval diet leaves, with isolates showing diverse resistance profiles. To minimise redundancy, isolates from the same sample, belonging to the same species and showing identical antimicrobial resistance profiles were considered closely related; therefore, 52 of 432 enterococci (24 E. casseliflavus and 28 E. faecalis) were selected for genetic characterisation. PFGE revealed genetic relatedness between leaf- and larva-derived strains, consistent with diet-mediated horizontal transmission. In addition, related profiles between isolates from developmental stages may suggest persistence of enterococci across the life cycle. These findings demonstrate the prevalence of E. faecalis and E. casseliflavus throughout the life cycle of H. erato phyllis and suggest that diet-associated horizontal transmission may contribute to their maintenance.