No longer uncertain: the validation of tenebrionid insects as hosts of Blattambidensovirus incertum1 isolates by phylogeny and infection studies

The mealworm ( Tenebrio molitor ) is one of the most commonly mass-reared insects for food and feed. Monitoring the health status of commercially reared mealworm populations is of great importance for the early detection of entomopathogens and for preventing pathogen outbreaks. Metagenomic screening is a suitable and commonly used method for detecting entomopathogens. The approach used here previously enabled the discovery of the Tenebrio molitor densovirus (TmDV) (family Parvoviridae , subfamily Densovirinae ) in symptomatic larvae. In the present study, the search for TmDV was extended to larvae, pupae and adults of T. molitor, including 19 symptomatic and asymptomatic samples obtained from a commercial mealworm mass-rearing facility. The presence of TmDV in all life stages of T. molitor was demonstrated, and its relative abundance was quantified using Nanopore sequencing. The infectivity of TmDV to T. molitor was demonstrated by isolating viral particles from sample LD2 and feeding them to mealworms. The experiment confirmed T. molitor as a susceptible host but showed a rather asymptomatic course of the infection with little effect on larval growth during 56 days of observation. It is hypothesized that this largely covert infection may explain the lack of reports of TmDV in mealworms or other insects, despite its detection in metagenomics surveillance studies of various insectivorous vertebrates. The complete genomes of 15 different TmDV genotypes present in various ratios in the different life stages of T. molitor could be reconstructed. Including these genotype sequences in phylogenetic analyses allowed us to re-evaluate the relationship and diversity of previously reported TmDV and related isolates, all belonging to the species Blattambidensovirus incertum1 . Our findings suggest that T. molitor and possibly other insects are susceptible hosts of viruses of Blattambidensovirus incertum1, while its occasional detection in metagenomic datasets of insectivorous vertebrates may not represent true densovirus host associations.