Persistence of Perorally Inoculated Entomopoxvirus Spindles in the Midgut of Bombyx Mori Larvae

Peroral inoculation of entomopoxvirus (EV) spindles, microstructures composed of the protein fusolin, enhances the infectivity of some insect viruses by disrupting the physical barrier against microbe infection, the peritrophic matrix, in the insect midgut. Here, we examined the temporal persistence of spindles of Anomala cuprea EV (ACEV) that infect Coleopteran species in Bombyx mori larva midgut because spindle solubility over time in the midgut is closely associated with the degree of the enhancement of microbe infectivity by fusolin. A number of ACEV spindles fed to B. mori larvae were retained in the digestive systems even 20 h after the completion of feeding spindles, and a number of spindles were found to be excreted still almost intact in feces under a light microscope. In an in vitro experiment, most ACEV spindles remained intact in B. mori midgut juice 1 h after the start of incubation and some of spindles appeared even overnight in contrast to Bombyx mori nucleoplyhedrovirus polyhedra, which were immediately dissolved in midgut juice. These results suggest spindles are not generally dissolved readily in the midgut of many insects. The difficulty in solubility of ACEV spindles is considered to be mainly due to that fusolin contains many cysteine residues that form a 3D network of disulfide bonds between fusolin dimers. To use spindles at a low cost as additives in microbial insecticides, increasing the solubility of spindles by protein engineering is important to utilize full spindles inoculated.