Discussion

Combining FISH and colonization experiments, we revealed the colonization specificity of Apibacter and its distribution in the bee gut. Comparative genomic analyses of 30 genomes from theFlavobacteriaceae family, including 14 newly sequencedApibacter genomes from this study and publicly available genomes for the outgroups, we characterized gene signatures underlying lifestyle transition and adaptation to bee gut symbionts.
FISH visualization indicates that Apibacter coinhabit withSnodgrassella and colonize the epithelium of the bee gut. As core members of gut bacteria in A. cerana , both Apibacter andSnodgrasella are microaerophilic, sharing nutritional sources (Zheng, Powell, Steele, Dietrich, & Moran, 2017). We showed thatApibacter isolated from A. cerana were able to colonizeA. mellifera , although at a significantly lower colonization rate. These results suggest that host incompatibility is probably not the constraining factor responsible for the rarity of Apibacterin A. mellifera . However, it is not yet possible to examine inter-host competition between Apibacter isolated from different honeybee species, because isolates from A. mellifera were not available to us.
Comparative genomic analysis revealed key gene functions potentially associated with the adaptation to bee gut niche. In a typical symbiotic system, benefits provision was considered crucial for the establishment of a mutualistic relationship (EWALD, 1987; Sachs et al., 2013). Our findings reveal that Apibacter are indeed providing beneficial traits to the host. For example, genes involved in amino acid biosynthesis are preserved in Apibacter spp., at a background of overall genome reduction, which echoes those previously reported in other bee gut symbionts (Kwong et al., 2014). Furthermore, theApibacter group retained the mannose catabolic gene, which was responsible for monosaccharide detoxification in the honeybee therefore broadening food choice for the host (Zheng et al., 2016).
Polysaccharides utilization is a prominent property carried by bee gut symbionts including Gilliamella , Bifidobacterium andLactobacillus (Bonilla-Rosso & Engel, 2018; Engel, Martinson, & Moran, 2012; Kešnerová et al., 2017). However, relevant genes are substantially lost in the Apibacter group. Interestingly, the core bacterial species Snodgrasella that coinhabit withApibacter at A. cerana gut epithelium also lack the capacity to utilize polysaccharides (Kwong et al., 2014). We speculate that polysaccharides might be limited in the niche that they share.
The gut lumen is mainly anaerobic, where the dominant symbiotic anaerobes inhabit. However, oxygen can diffuse from the intestinal epithelium cells and create a microaerobic environment for facultative anaerobes (He et al., 1999; Zheng, Powell, et al., 2017). A previous study found both cytochrome bd and cbb3 in the Apibacter genome, which were presumably involved in microaerobic respiration (Kwong et al., 2018). In the present work, we identified additional anaerobic respiration NAR operon that was conserved within the Apibacter group and in the coinhabitingSnodgrassella , but absent from the other four core bee gut bacteria species. These observations suggest that the NAR pathway might be important for the microbiome to colonize intestinal epithelium. Such respiratory flexibility might enable Apibacter to survive altered oxygen tensions. This finding is congruent with the observation in mouseE. coli , where they require both microaerobic and anaerobic respirations for successful colonization (Jones et al., 2007). A further study proved that the NAR pathway played a key role in E. colicolonization of the mouse gut, because the NarG mutant showed colonization deficiency for both commensal bacteria and pathogenicE. coli (Jones et al., 2011). These results are in line with the observation that nitrate reduction could facilitate the growth of gut microaerobic bacteria at low oxygen conditions (Tiso & Schechter, 2015). Therefore, we conclude that the NAR operon is an important genetic signature for Apibacter adaptation to the bee gut.
Genes that are shared between the LCAs of Clade C and Clade E but absent from the Apibacter group, contain functions either deleterious to the mutualistic relationship with the host, or redundant for the symbiotic lifestyle. Histidine biosynthesis is one of the most energy consuming processes for bacteria, such that the degradation of histidine as carbon and nitrogen sources is strictly regulated (Bender, 2012). The histidine catabolism is limited in bee gut environments, as oxygen is required for the activation of the Hut operon (Goldberg & Hanau, 1980). Considering that the bee gut is mostly anoxic, the Hut pathway is highly likely to be malfunctioning inApibacter and is susceptive to be lost. In addition, histidine degradation is important for pathogens to recognize eukaryotic hosts and to activate virulence factors (Zhang et al., 2014).
In conclusion, combining molecular and colonization experiments, for the first time, we visualized and quantified the distribution ofApibacter spp. inside the bee gut, and proved thatApibacter isolates of A. cerana could survive in A. mellifera . Genomic comparisons with relatives living on other lifestyles revealed that host beneficial traits and respiration nitrate reduction (NAR pathway) were key functions for adaptation to the bee gut environment.