Genetic diversity and host-symbiont cophylogenetic analysis of Neorickettsia endobacteria of Fasciola hepatica.

Neorickettsia are obligate intracellular bacteria that persist as endosymbionts of digenean trematodes and include several vertebrate pathogens. However, many digenean-associated lineages remain poorly characterized, including Neorickettsia associated with the liver fluke Fasciola hepatica (nFh). Here, we present a large-scale population genomic analysis of nFh to assess its prevalence, genetic diversity, evolutionary history, and transmission dynamics. We screened whole-genome sequencing data from 502 F. hepatica samples collected across Peru, the UK, Uruguay, and the USA, detecting Neorickettsia infection in all regions and an overall prevalence of approximately 17% in Peru. From these data, we generated 77 reference-guided nFh consensus genomes. nFh exhibited low nucleotide diversity (π = 9.1 × 10-5), consistent with an obligate intracellular lifestyle. Phylogenetic analyses revealed a clear divergence between UK and American nFh lineages and two closely related haplotype clusters within South America. Cophylogenetic analyses showed significant congruence between nFh and host nuclear phylogenies, but not mitochondrial phylogenies, supporting long-term host association with mixed transmission modes beyond strict transovarial inheritance. Analysis of host kinship and endosymbiont genotype sharing further supported both vertical inheritance and horizontal transmission within vertebrate hosts. nFh infection was significantly associated with triclabendazole resistance in F. hepatica from Cusco, Peru, but not from Cajamarca, indicating region-specific associations between endosymbiont infection and drug resistance. Together, these findings establish nFh as a widely distributed endosymbiont of F. hepatica with low genetic diversity, geographic population structure, and mixed transmission modes, with potential implications for the epidemiology of fascioliasis and anthelmintic resistance.