Gut microbiome drives individual memory variation in bumblebees

The potential of the gut microbiome as a driver of individual cognitive differences in natural populations of animals remains unexplored. Here, using metagenomic sequencing of individual bumblebee hindguts, we find a positive correlation between the abundance of Lactobacillus Firm-5 cluster and memory retention on a visual discrimination task. Supplementation with the Firm-5 species Lactobacillus apis, but not other non-Firm-5 bacterial species, enhances bees’ memory. Untargeted metabolomics after L. apis supplementation show increased LPA (14:0) glycerophospholipid in the haemolymph. Oral administration of the LPA increases long-term memory significantly. Based on our findings and metagenomic/metabolomic analyses, we propose a molecular pathway for this gut-brain interaction. Our results provide insights into proximate and ultimate causes of cognitive differences in natural bumblebee populations.


Supplementary Methods
Our gene mapping method could not with certainty identify individual bacterial species because not all the 31 housekeeping genes were detected and with > 95% sequence identity (Methods, Supplementary Data 10). Therefore, three other approaches (mOTU, MetaPhlAn3 and Kraken2) were performed.
For mOTU, the high-quality sequencing reads obtained from the gene-mapping approach were aligned to the mOTU database with default parameters by the mOTU pipeline (Version 3.0.1) 1 , identifying species (mOTUs) and their relative abundance. The mOTU database contains phylogenetic marker gene sequences extracted from reference genomes, metagenomic samples and metagenome assembled genomes. Like the gene-mapping method, mOTU also identified the four dominant species known to exist in bee guts (Supplementary Data 11 and Supplementary Fig. 9). They were S. alvi, G. apicola, B. bohemicum and Lactobacillus Firm-5 species (L. apis identified by the gene-mapping method and L. bombicola identified by mOTU). In our study, Lactobacillus strains were isolated from the hindguts of bumblebees (Bombus terrestris) using MRS agar. Four of the 20 isolates were L. apis strains, 12 of them were unclassified Lactobacillus, and none of them were L.
bombicola. This indicates that L. apis may be the most abundant classified Lactobacillus species in our bumblebees. Further, for this reason, we cannot be confident that the mOTU method identified individual bacterial species accurately.
The high-quality sequencing reads were also used for species-level taxonomic annotation with another two established tools, MetaPhlAn3 2 and Kraken2 3 . MetaPhlAn3 (Version 3.0.2) was performed with default parameters, which is based on unique clade-specific marker genes. Kraken2 (Version 2.1.1-beta) was conducted for taxonomic annotation with default parameters, which is based on exact k-mer assignments. The Minikraken database (MiniKraken2_v2_8GB) was downloaded (ftp://ftp.ccb.jhu.edu/pub/data/kraken2_dbs/old/minikraken2_v2_8GB_201904.tgz) and used.
The relative abundances of taxa were subsequently calculated by Bracken (Version 3.29) 4 .
Our quantitative real-time PCR results detected the known dominant bacterial species in bumblebee guts, including S. alvi, G. apicola and B. bohemicum, which were shown to be highly abundant ( Supplementary Fig. 10). However, MetaPhlAn3 did not identify S. alvi and G. apicola, and Kraken2 did not identify B. bohemicum (Supplementary Data 11 and Supplementary Fig. 9). Therefore, we again cannot be confident that these two approaches accurately identified bacterial species.
Phylogenetic analyses of Ellegaard et al. 6 indicate that Firm-5 strains isolated from honeybees and bumblebees belong to separate sublineages of the Firm-5 phylotype. Their results suggest that L. apis is specific to honeybees and L. bombicola is specific to bumblebees. However, we isolated 4 L. apis strains (no L. bombicola strains) out of 20 isolates from bumblebee guts, demonstrating the existence of L. apis in bumblebee guts and indicating its possible high abundance among the classified Lactobacillus. The differences found between their study and ours may be because the bumblebees used in each study were different species/subspecies from different regions and environment (wild bumblebees in western Switzerland versus indoor-kept commercial bumblebees from Koppert, China). Different environments and diets will lead to distinct gut microbiota composition, including different bacterial species and their abundance.