The gypsy moth, Lymantria dispar, is a polyphagous forest pest worldwide. The baculovirus, Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) is a natural pathogen of L. dispar. The Toll-like receptors (TLR) pathway plays a crucial role in both innate and adaptive immunity in animals. However, The TLR pathway and its underlying immune mechanism against baculovirus in L. dispar have not been explored. In this study, eleven TLRs and five downstream TLR pathway components were identified and characterized from L. dispar. Structural analysis indicated that intracellular Toll/interleukin-1 receptor (TIR) domains of LdTLRs and LdMyD88 contained three conserved motifs, and the 3D structures of TIR domains of LdTLRs possessed similar patterns in components arrangement and spatial conformation. The TLR proteins of L. dispar were placed into five monophyletic groups based on the phylogenetic analysis. LdTLR1, 2, 5, 6, 7, 8 and all identified downstream TLR pathway factors were highly induced upon LdMNPV infection, indicating that the TLR pathway of L. dispar was activated and might play a role in the immune response to LdMNPV infection. Collectively, these results help elucidate the crucial role of the TLR pathway in the immune response of L. dispar against LdMNPV, and offer a foundation for further understanding of innate immunity of the pest.

Certain parasites manipulate host behavior following infection to enhance their own dispersal and transmission. Lepidopteran larvae infected with baculoviruses exhibit increased locomotion, ascending to the apex of their host plant where they ultimately die in a characteristic inverted, liquefied posture suspended by their prolegs—a phenomenon termed “tree-top disease”. Although numerous studies have investigated the underlying causes of this behavior, the precise mechanism governing tree-top disease formation remains unresolved. In this study, Lymantria dispar larvae were infected with Metarhizium anisopliae and Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). We compared symptom profiles across infection modes and assessed virulence, demonstrating that M. anisopliae infection alters the hyperactive state induced by LdMNPV in larvae exhibiting tree-top disease. Specifically, M. anisopliae promoted tree-top disease behavior during early infection stages but suppressed it during later stages. Furthermore, the symptomatology of larvae co-infected with both pathogens differed significantly from that observed in larvae infected with either M. anisopliae or LdMNPV alone. Co-infected larvae also exhibited accelerated mortality compared to those infected with a single pathogen. The above findings indicate that L. dispar larvae, when co-infected with LdMNPV and M. anisopliae may change behavioral responses that could further modulate the pathogenesis of LdMNPV-induced tree-top disease. Furthermore, a synergistic interaction between M. anisopliae and LdMNPV was observed in the biocontrol of L. dispar.