Honeybee-Gilliamella synergy in carbohydrate metabolism enhances host thermogenesis in cold acclimation

Abstract

How gut symbionts contribute to host adaptation remains largely elusive. Studying co-diversified honeybees and gut bacteria across climates, we found cold-adapted species (Apis mellifera, A. cerana) exhibit enhanced genomic capacity for glucose, pyruvate, lipid and glucuronate production versus tropical species. Metagenomics revealed Gilliamella as the most enriched gut bacterium in cold-adapted bees. Germ-free honeybees inoculated with the Gilliamella from A. cerana showed increased activity, body temperature and fat storage upon cold exposure. Saccharide metabolomics demonstrated higher hindgut glucose levels in Gilliamella-colonized A. mellifera versus germ-free bees, and in A. cerana versus three sympatric tropical species. Although Gilliamella can hydrolyze β-glucan into glucose, cultural experiments suggest it preferentially degrades glucuronate to pyruvate. In turn, monocolonized bees upregulated hindgut glucose/pyruvate utilization while increasing glucuronate provision, suggesting nutritional complementarity. Gilliamella's transporter genes predominantly target ascorbate (a glucuronate derivative), which is elevated in inoculated hindguts. Accordingly, Gilliamella converts ascorbate to D-xylulose-5P (promoting lipogenesis), while showing reduced growth on glucuronate/ascorbate versus glucose, potentially minimizing glucose competition with hosts. We revealed a highly coordinated host-symbiont metabolic synergy enhancing host energy acquisition for cold adaptation.

Fig. 1. Phylogeny and distribution of Apis honeybees.

A Schematic diagram of the honeybee phylogeny. The six species studied in the present work are shown with photos. Four of them have been examined previously, characterized by varied capacities in heat production (numbers represent heat production per mass, W/kg). B Native distributions of honeybee species. The same honeybee species is marked in Panels (A, B) using the same color code. YN Yunnan Province, China; JL Jilin Province, China.

Fig. 2. Gilliamella is most significantly enriched in cold-adapted honeybee hindguts, promoting host thermogenesis.

A Principal component analysis of gut bacteriome showing bacteria most strongly separating different bees. B Significantly enriched gut bacteria identified in the three honeybee subgenera. LDA: linear discriminant analysis. C Apis cerana workers with (Gc) or without (GF) Gilliamella colonization were subject to thermo imaging after cold exposure (10 °C) for 30 min. AV, average temperature for the framed area; HS and CS denote the highest and lowest temperatures for the entire area, respectively. D The same sets of bees were measured for abdominal temperature after 4 h cold exposure, using a thermocouple (the needle-like instrument touching the bee’s abdomen, as shown in the right photograph of the middle insert). Acer, A. cerana; Amel, A. mellifera; Ador, A. dorsata; Alab, A. laboriosa; Aand, A. andreniformis; Aflo, A. florea.

Fig. 3. Characteristic carbohydrate utilization in Gilliamella.

A Maximum-likelihood phylogeny of Gilliamella strains, annotated with the number of genes involved in carbohydrate metabolism: enzymes involved in degradation modules of glucuronate (M00061), galacturonate (M00631), and ascorbate (M00550); proteins transporting only glucose, glucose and maltose, or multiple sugars including glucose; primary hydrolases targeting polysaccharides. Circle and triangle sizes indicate gene copy number. B Schematic metabolic pathways inferred from Gilliamella and honeybee genomes. The Gilliamella-generated substrates can be potentially utilized by the host in lipogenesis. G3P glyceraldehyde 3-phosphate, LCFAs long-chain fatty acids, ELOVL6 elongation of very long chain fatty acids protein 6, ChREBP carbohydrate-responsive element binding protein, PRPP 5-phosphoribosyl diphosphate. C Gene clusters involved in polysaccharide deconstruction and further utilization generating pyruvate in the strain Gilliamella B3835. D Differential growth of Gilliamella on varied carbon sources demonstrates its preference for glucuronate, resulting in lower cell density. The Gilliamella B3835 strain was cultured in Brain Heart Infusion (BHI) broth (minus glucose) supplemented with 1, 10 or 20 mM of glucose, glucuronate, or ascorbate, respectively. Data are presented as means ± SD.

Fig. 4. Gut microbiome comparison in carbohydrate metabolism across honeybees.

A The gene abundance of glucose transporters in gut metagenomes contributed by varied bacteria. The y-axis is the gene abundance of transporters capable of glucose uptake. Pearson correlation coefficient (R) and P value (p) are annotated. Gilliamella rarely encodes glucose transporters, especially glucose-specific transporters (glucU and ptsG), across all metagenome samples (upper panel). And its abundance is negatively associated with the abundance of all glucose transporters in metagenomes (lower panel). The abundance comparison of genes encoded by three gut bacteria in the (B) degradation module of glucuronate, galacturonate and ascorbate, and (C) PRPP production from D-xylulose-5P shows Gilliamella’s superior degradation capacity in alternative carbon sources whereas lower consumption of D-xylulose-5P. ns: no significant difference, **P value < 0.01, ****P value < 0.0001 by Mann–Whitney U test (B–D). Each dot denotes one gut metagenome sample. D Glucose proportions across gut tissues in four sympatric honeybees, A. cerana (Acer), A. dorsata (Ador), A. andreniformis (Aand) and A. florea (Aflo), examined by saccharide metabolomics show significantly more glucose in Acer rectums. Two-way ANOVA and Tukey test were used for the comparison. Data are presented as means ± SD.

Fig. 5. Honeybee-Gilliamella coadaptation to coldness via enhanced lipid synthesis.

A Glucose contents in A. mellifera hindguts colonized by Gilliamella (Gc) versus germ-free ones (GF), showing significant glucose increase following bacterial inoculation. B The glycogen synthesized in gut cells was stained purple using Periodic-Acid Schiff (PAS) staining, suggesting that the hindgut can absorb glucose like the midgut. C The up-regulated genes in Gc versus GF indicate potential cross-feeding between the host and Gilliamella. D Enzymes involved in glucose, pyruvate, lipid, and glucuronate productions contain more gene copies in cold-adapted honeybees. E Maximum-likelihood tree based on the ELOVL6 gene sequences with motifs annotated, suggesting historical gene duplication in cold-adapted honeybees and bumblebees. F Gilliamella inoculation resulted in significantly elevated abdominal triacylglycerol (TAG) on day 10 post-inoculation. G Histological comparison shows Gilliamella colonization affects host lipid storage. HE (hematoxylin and eosin), PAS, and ORO (Oil Red O) were used for cell, glycogen, and lipid staining, respectively. Black arrows indicate multilocular lipid droplets in Gc bees; red arrows indicate unilocular droplets in GF bees. H Hindgut ascorbate is significantly increased in A. mellifera and A. cerana 48 h after Gilliamella inoculation.

Fig. 6. Schematic summary of the honeybee-Gilliamella crosstalk in carbohydrate metabolism, which collectively improves host cold adaptation.

Codes 1–10 denote enzymes shown in Fig. 5D. Dashed lines present putative metabolic flows. Vc ascorbate, Xu5P D-xylulose-5P.