Prevalence, replication, and tissue-specific localization of Euschistus heros virus (EhV) across life stages of the Neotropical brown stinkbug Euschistus heros (Hemiptera: Pentatomidae)

The Neotropical brown stink bug, Euschistus heros (Hemiptera: Pentatomidae), is a major agricultural pest in Brazilian soybean crops, exhibiting increasing insecticide resistance in field. This study investigates the dynamics of Euschistus heros virus (EhV), a multispecies iflavirus, in laboratory populations of E. heros, revealing a persistent, systemic virus infection across all life stages. Screening of 99 adults showed 100 % EhV genome prevalence in mated individuals, while virgin males and females had an 80 % infection rate. Viral loads peaked in freshly laid eggs (590,462 ± 27,426 copies/ng RNA), suggesting likely maternal transmission, before declining sharply in nymphs with an 18.08-fold reduction by the fifth instar. Adults exhibited sex- and mating-status-dependent variation, with virgin females harboring the highest copy number (227,380 ± 144,938 copies/ng RNA), which is 2.87-fold higher than mated females. Tissue-specific analysis revealed elevated EhV genome accumulation in legs/wings (221,723 ± 102,120 copies/ng RNA in males) and heads, with significantly lower loads in thoraces and abdomens. Strand-specific RT-qPCR suggested that EhV may exhibit replicative activity across developmental stages, as indicated by approximately balanced levels of genomic and replicative strands. Additionally, transmission electron microscopy of antennal tissues from an infected male individual revealed cytoplasmic icosahedral virions (∼30 nm), providing the first ultrastructural evidence of EhV assembly. These findings suggest that EhV may establish a stable, replicative association with E. heros and point to vertical transmission as a plausible mechanism of persistence. Nonetheless, confirmation of this pattern will require additional analyses with larger sample sizes, field collected insects, and complementary experimental approaches.