Symbionts as major modulators of insect health: lactic acid bacteria and honeybees

Abstract

Lactic acid bacteria (LAB) are well recognized beneficial host-associated members of the microbiota of humans and animals. Yet LAB-associations of invertebrates have been poorly characterized and their functions remain obscure. Here we show that honeybees possess an abundant, diverse and ancient LAB microbiota in their honey crop with beneficial effects for bee health, defending them against microbial threats. Our studies of LAB in all extant honeybee species plus related apid bees reveal one of the largest collections of novel species from the genera Lactobacillus and Bifidobacterium ever discovered within a single insect and suggest a long (>80 mya) history of association. Bee associated microbiotas highlight Lactobacillus kunkeei as the dominant LAB member. Those showing potent antimicrobial properties are acquired by callow honey bee workers from nestmates and maintained within the crop in biofilms, though beekeeping management practices can negatively impact this microbiota. Prophylactic practices that enhance LAB, or supplementary feeding of LAB, may serve in integrated approaches to sustainable pollinator service provision. We anticipate this microbiota will become central to studies on honeybee health, including colony collapse disorder, and act as an exemplar case of insect-microbe symbiosis.

Figure 1. Phylogenetic tree of LAB in bee crops.

Phylogenetic tree based on a distance matrix of positions 56–1470 (Escherichia coli numbering) in the 16S rRNA gene of Lactobacillus and Bifidobacterium spp. bacteria from Apis species (red) and the stingless bees Melipona beechii (turquoise), Meliponula bocandeii (green) and Trigona sp (blue). Previously characterized bacterial phylotypes from Apis mellifera are in bold print (red). Bacterial type strains are in bold characters (black). Phylotypes in the framework (right) represent different clusters belonging to a characterised or possibly novel species. Bifidobacterium group is the out-group. Bar: 5 base pair changes. 16S rRNA gene sequences deposited in GenBank HM534742–HM534842 (in parenthesis).

Figure 2. In vitro visualization of lactobacilli.

In vitro visualization of lactobacilli attached to the wall of a honeybee crop using SEM. A, B and C show different areas of a honey crop at similar magnification with visible attachment structures resembling extracellular polymeric substances (EPS). D shows a larger part of the crop with attached bacteria. Photographer Lennart Nilsson.

Figure 3. In vivo visualization of LAB biofilm.

The red fluorescence shows live-stained bacteria in a LAB biofilm attached to a honey crop. The green fluorescence shows the nuclei of the honeybee crop cells. The visualized tissue shows a projection of 112 confocal z-sections (through a 37.6 µm z-depth, covering a xy-area of 246×246 µm).

Figure 4. LAB rescue of honeybee larvae from European Foulbrood.

Proportion dead larvae in both replicates (total number dead after 21 days). Data presented as a mean for the three groups fed M. plutonius (10⁷, 10⁶ and10⁵ bacteria ml⁻¹), and the 3 groups fed M. plutonius and LAB. Irrespective of infectious dose, the overall effect from the LAB supplement was a significantly reduced mortality in the treated groups. Mortality in the uninfected control groups was <7%.