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

Abstract

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.

Fig 1. Survivorship of honeybees orally exposed to E. faecalis.

(A) Schematic illustration of the experimental design for the treatment of MF honeybees. Survivorship of A. mellifera (B) and A. cerana (C) after oral exposure to E. faecalis was monitored and recorded each day for 12 days. (D) Schematic illustration of the experimental design for the treatment of CV honeybees. 5-day-old CV or MF bees were orally exposed to E. faecalis. Survivorship of A. mellifera (E) and A. cerana (F) were monitored and recorded each day for 7 days. n = 25 for each treatment group with three replicate experiments. *, P<0.05; **, P < 0.01; ***, P < 0.001 (Mantel-Cox test).

Fig 2. Hemolymph infection of E. faecalis leading to the death of honeybees.

Absolute abundance of E. faecalis in the gut (A) and hemolymph (B) of A. mellifera and A. cerana in the MF group after 12 days post oral inoculation with E. faecalis. ***, P < 0.001; ns, not significance (Mann-Whitney u test). (C) Schematic illustration showing the experimental design for hemolymph injection of E. faecalis. Serial dilutions of E. faecalis in PBS were injected into the hemolymph of newly emerged MF honeybees. Survivorship of A. mellifera (D) and A. cerana (E) were monitored and recorded every 2 h for 16 h. n = 12 for each treatment group with three replicate experiments. *, P<0.05; **, P < 0.01; ***, P < 0.001 (Mantel-Cox test).

Fig 3. A. mellifera and A. cerana exhibit differential immune gene expression following E. faecalis inoculation.

(A) The expression of immune regulatory genes in the Imd and Toll pathways did not show obvious differences between A. mellifera and A. cerana. (B) The expression of AMP genes was significantly increased in A. cerana. Gene expression was determined 24 h post infection. *, P < 0.05; **, P < 0.01 (Tukey honest method).

Fig 4. The positive selection of Spz4 gene in A. cerana.

(A) Violin plots showing dN/dS ratios for different categories of immune genes in A. mellifera and A. cerana. Black solid lines show medians of orthologus group values, and white dotted lines show the limits of the upper and lower quartiles. (B) The predicted structural domain of the Spz4 protein in A. mellifera and A. cerana. (C) Maximum likelihood estimations of dN/dS at each site of Spz4, together with estimated profile confidence intervals (if available). The dN/dS = 1 (neutrality) is depicted as a horizontal gray line. Boundaries between partitions (if present) are shown as vertical dashed lines. Predicted regions corresponding to glycosyltransferases are highlighted in red shadow (D). The predicted structure of the Spz4 protein in A. mellifera and A. cerana using the AlphaFold. The positive selection sites on the structure of Spz4 protein in A. cerana are represented in red, ranging from amino acids 188 to 206; the same sites are marked green in A. mellifera. *, P < 0.05 (Wilcoxon rank sum test).

Fig 5. The immune response against E. faecalis in RNAi-treated A. cerana.

(A) Schematic illustration of the experimental design. Knockdown of Spz4 or Spz5 gene expression in A. cerana was achieved by feeding nanoparticle-mediated dsRNA. Bees were then inoculated with E. faecalis. (B)The expression of Spz4 and Spz5 in A. cerana after oral treatment with dsSpz4 or dsSpz5 for two days. The expression level of AMP genes in the gut (C) and the survival rate (D) of A. cerana in the control (MF) and RNAi (MF + dsSpz4 or MF + dsSpz5) groups infected with E. faecalis. **, P < 0.01; ***, P < 0.001 (Tukey honest method). *, P<0.05; ns, not significance (Mantel-Cox test).