Spatial distribution of Apibacter in the gut of A. cerana
We characterized the colonization of Apibacter in the gut ofA. cerana using fluorescence in situ hybridization (FISH) and qPCR. Honeybee symbionts Snodgrassella and Gilliamella are dominant in the ileum, with Snodgrassella colonizing the inner wall of ileum (Martinson et al., 2012). These two bacteria were used as reference coordinates to infer the location of Apibacter using species-specific probes (Fig. 1A-D). The spectral images showed thatApibacter co-resided with Snodgrassella in both ileum and midgut (Fig. 1A, C). The signals were stronger at the inner walls, indicating that Apibacter colonized the gut intima as didSnodgrassella . Gilliamella covered on the top ofApibacter and extended into the gut lumen (Fig. 1B, D).Apibacter could not be visualized clearly in the rectum, as the rectum was filled with pollen grains with auto-fluorescent under the excitation wavelength. We also quantified the absolute abundances ofApibacter in different gut compartments (midgut, ileum, rectum) using qPCR. It showed that the absolute abundances of Apibacterincreased from midgut to rectum, with cell numbers ranged from 9.43×105 to 1.74×107 (Fig. 1E).
Host specialization is a common feature for many host-microbiome associations (Oh et al., 2010), which has been demonstrated by the honeybee symbiotic bacteria Snodgrassella (Kwong et al., 2014) and Lactobacillus (Ellegaard et al., 2019). To test if it is also the case for Apibacter , we performed the colonization specificity test. Apibacter sp. strain B3706 isolated from A. ceranawas inoculated to the microbiota-free A. cerana and A. mellifera (see Methods). Six days after inoculation, the cell numbers in the gut were more than 107, which were much higher than in the inoculum (< 106), indicating that strain B3706 was able to colonize the guts of both A. ceranaand A. mellifera . However, the colonization efficiency inA. cerana was significantly higher than that in A. mellifera (Fig. 1F), suggesting that strain B3706 was less adapted to the gut of A. mellifera .