Endogenous viral elements and PIWI gene evolution in Spodoptera: diversity, conservation, and piRNA production

: Endogenous viral elements (EVEs) are viral sequences that have integrated into host genomes. While these elements can be considered molecular fossils of past infections, in some cases, they are co-opted and contribute to host fitness. For instance, in arthropods, some EVEs derived from non-retro-transcribing RNA viruses (nrEVEs) generate PIWI-interacting RNAs (piRNAs), and in Aedes mosquitoes at least, they act as immune effectors. Here, we investigated the diversity and function of nrEVEs across four Spodoptera species (Lepidoptera: Noctuidae), which are notorious agricultural pests. Comparative genomic analyses revealed 21-27 nrEVEs per genome, with a strong enrichment on the Z chromosome. Most nrEVEs originated from (-)ssRNA viruses, particularly Rhabdoviridae, Orthomyxoviridae, Nyamiviridae, and Phenuiviridae. We identified species-specific integrations as well as a conserved orthologous nrEVE likely dating back ∼15 million years. Small RNA sequencing showed that only a subset of nrEVEs produced piRNAs, primarily in ovarian tissues, with a minority displaying sequence complementarity compatible with antiviral activity. Specifically, in Spodoptera frugiperda, we detected abundant nrEVE-derived piRNAs with high similarity to a currently known circulating virus, the Spodoptera frugiperda rhabdovirus (SfRV). Among them, we observed a piRNA peak with seed complementarity to the SfRV genome that correlated with the absence of infection. In parallel, analysis of PIWI-subfamily Argonaute genes, which codify for the proteins involved in the piRNA biogenesis, revealed lineage-specific expansions and signatures of positive selection in Spodoptera, exhibiting a distinct evolutionary trajectory compared to other lepidopteran families. Our results broaden our understanding of insect-virus coevolution suggesting a dual role for nrEVEs in noctuid moths: while most likely persist as genomic fossils, a subset displays features compatible with potential antiviral response through piRNA production.