Toll receptor ligand Spätzle 4 responses to the highly pathogenic Enterococcus faecalis from Varroa mites in honeybees

Honeybees play a major role in crop pollination, which supports the agricultural economy and international food supply. The colony health of honeybees is threatened by the parasitic mite Varroa destructor , which inflicts physical injury on the hosts and serves as the vector for variable viruses. Recently, it shows that V . destructor may also transmit bacteria through the feeding wound, yet it remains unclear whether the invading bacteria can exhibit pathogenicity to the honeybees. Here, we incidentally isolate Enterococcus faecalis , one of the most abundant bacteria in Varroa mites, from dead bees during our routine generation of microbiota-free bees in the lab. In vivo tests show that E . faecalis is only pathogenic in Apis mellifera but not in Apis cerana . The expression of antimicrobial peptide genes is elevated following infection in A . cerana . The gene-based molecular evolution analysis identifies positive selection of genes encoding Späetzle 4 (Spz4) in A . cerana , a signaling protein in the Toll pathway. The amino acid sites under positive selection are related to structural changes in Spz4 protein, suggesting improvement of immunity in A . cerana . The knock-down of Spz4 in A . cerana significantly reduces the survival rates under E . faecalis challenge and the expression of antimicrobial peptide genes. Our results indicate that bacteria associated with Varroa mites are pathogenic to adult bees, and the positively selected gene Spz4 in A . cerana is crucial in response to this mite-related pathogen.