Supplementary tables for Honey Bee symbiont buffers larvae against nutritional stress and supplements lysine

Honey bees have suffered dramatic losses in recent years, largely due to multiple stressors underpinned by poor nutrition. Nutritional stress especially harms larvae, who mature into workers unable to meet the needs of their colony. In this study, we characterize the metabolic capabilities of a honey bee larvae-associated bacterium, Bombella apis (formerly Parasaccharibacter apium), and its effects on the nutritional resilience of larvae. We found that B. apis is the only bacterium associated with larvae that can withstand the antimicrobial larval diet. Further, we found that B. apis can synthesize all essential amino acids and significantly alters the amino acid content of synthetic larval diet, largely by supplying the essential amino acid lysine. Analyses of gene gain/loss across the phylogeny suggest that four amino acid transporters were gained in recent B. apis ancestors. In addition, the transporter LysE is conserved across all sequenced strains of B. apis. Finally, we tested the impact of B. apis on developing honey bee larvae subjected to nutritional stress and found that larvae supplemented with B. apis are bolstered against mass reduction despite limited nutrition. Together, these data suggest a novel role of B. apis as a nutritional mutualist of honey bee larvae.

Methods
To define orthologs, protein sequences were extracted from NCBI annotated sequence files for the listed accesions (Supplementary Table 3). Reciprocal best BLAST hits were calculated, and genes clustered into ortholog groups using complete linkage. Conserved core orthologs were used to generate the species tree for these genera and this was used, in conjunction with GLOOME to infer branch-specific gene gain/loss events (Supplementary Table 2). To define presence/absence of amino acid biosynthesis genes (Supplementary Table 1), ortholog representatives were run against GapMind to find amino acid biosynthetic genes in the proteomes. Additionally, annotation based on NCBI’s PGAP was used, in conjunction with DOE’s IMG/M, to confirm the putative function of orthologous groups of genes.