Early Queen Development in Honey Bees: Social Context and Queen Breeder Source Affect Gut Microbiota and Associated Metabolism

Abstract

The highly social honey bee has dense populations but a significantly reduced repertoire of immune genes relative to solitary species, suggesting a greater reliance on social immunity. Here we investigate immune gene expression and gut microbial succession in queens during colony introduction. Recently mated queens were placed into an active colony or a storage hive for multiple queens: a queen-bank. Feeding intensity, social context, and metabolic demand differ greatly between the two environments. After 3 weeks, we examined gene expression associated with oxidative stress and immunity and performed high-throughput sequencing of the queen gut microbiome across four alimentary tract niches. Microbiota and gene expression in the queen hindgut differed by time, queen breeder source, and metabolic environment. In the ileum, upregulation of most immune and oxidative stress genes occurred regardless of treatment conditions, suggesting postmating effects on gut gene expression. Counterintuitively, queens exposed to the more social colony environment contained significantly less bacterial diversity indicative of social immune factors shaping the queens microbiome. Queen bank queens resembled much older queens with decreased Alpha 2.1, greater abundance of Lactobacillus firm5 and Bifidobacterium in the hindgut, and significantly larger ileum microbiotas, dominated by blooms of Snodgrassella alvi. Combined with earlier findings, we conclude that the queen gut microbiota experiences an extended period of microbial succession associated with queen breeder source, postmating development, and colony assimilation. IMPORTANCE In modern agriculture, honey bee queen failure is repeatedly cited as one of the major reasons for yearly colony loss. Here we discovered that the honey bee queen gut microbiota alters according to early social environment and is strongly tied to the identity of the queen breeder. Like human examples, this early life variation appears to set the trajectory for ecological succession associated with social assimilation and queen productivity. The high metabolic demand of natural colony assimilation is associated with less bacterial diversity, a smaller hindgut microbiome, and a downregulation of genes that control pathogens and oxidative stress. Queens placed in less social environments with low metabolic demand (queen banks) developed a gut microbiota that resembled much older queens that produce fewer eggs. The queens key reproductive role in the colony may rely in part on a gut microbiome shaped by social immunity and the early queen rearing environment.

Keywords: Apis mellifera; Bombella apis; honey bee; immune training; metabolism; microbiota; oxidative stress; queen breeder; vitellogenin.

FIG 1

Relative abundance of early queen microbiota by gut tissue and treatment. Color-coded bars represent relative abundance corrected by species-specific 16S rRNA gene copy number. The panel displays the 10 OTUs with greatest relative abundance by gut niche (x axis) and sampling environment (y axis). “Time zero” represents queens sampled upon receipt from the queen breeder prior to treatment conditions (n = 30), “colony” represents queens exposed to the colony environment for 3 weeks (n = 20) with high metabolic demand (HMD), and “queen bank” represents queens placed in queen banks for 3 weeks (n = 20) with low metabolic demand (LMD).

FIG 2

Normalized microbiota abundance of the honey bee queen hindgut. Figure represents relative abundance normalized by BactQuant results and species-specific 16S rRNA gene copy number. The key lists the seven most abundant OTU’s presented by hindgut niche and sampling environment. Queen breeder is shown vertically in the center* of the figure as black or red. Note scale differences by hindgut tissue on the x axis. For comparison, the size of mouthpart and midgut microbiotas (not shown) averaged 1.8 × 10⁵ and 5.0 × 10⁵ 16S rRNA gene copies, respectively. Other details as in Fig. 1.

FIG 3

Principal-component analysis by niche based on relative abundance of the top 11 OTUs. Data were transformed to log-ratio abundance among all OTUs using a centered log-ratio transformation prior to cluster analysis. The colored symbols in the top right box represent the sampling environment. Taxa in order of absolute abundance: Alpha 2.1 (A2.1); an unnamed Acetobacteraceae related to Commensilibacter, Lactobacillus firm5 (L.F5); a large and diverse phylotype composed of many species, Bombella apis (Bo. apis); a fungal inhibiting Acetobacteraceae that dominates social environments including the queen mouth and midgut, Snodgrassella alvi (S. alvi); a species intimately tied to worker ileum function, Lactobacillus kunkeei (L. kunk); queen- and worker- associated bacteria that populates social environments, Bifidobacterium (Bifido) and Lactobacillus firm4 (L. F4); both core rectum bacteria of workers, Gilliamella apicola (G. api); another highly diverse species group associated with worker ileum function; and finally, Delftia (D), Rhizobiales (R), and Caulobacter (C), three OTUs unknown in honeybees that require methodological validation. Percent variation explained by principal components (first/second) shown in upper left of each panel.

FIG 4

Queen fat body gene expression analyzed with ANOVA and corrected with a Tukey’s test for multiple comparisons. Colors defined in the key represent significant expression differences by time (time zero versus time one) and treatment (time zero versus colony or queen bank).

FIG 5

Principal-component analysis of fat body gene expression. Sixty-three percent of the variation was explained by the first two principle components. Time one saw a significant increase in vitellogenin and insulin-like peptide-1 and a concurrent decrease of Toll and antimicrobial peptide expression independent of treatment. While colony queens had decreased catalase expression, queen bank queens increased expression of both SOD genes and GST-1 indicating increased oxidative stress.