Honeybee gut microbiota plays an important role in host physiology and metabolism. Recent studies have shown that the influence of the resident microorganisms in the regulation of honeybee immune system is profound, which protects against the pathogen Serratia marcescens. However, only few of the core gut members in the regulation of immune functions have been studied. Here, we explored how different bee gut bacterial species aided in the clearance of the pathogenic Hafnia alvei, which causes bee septicemia with a high mortality rate. We found that both Gilliamella apicola W8136 and Lactobacillus apis W8172 protect honeybees from the opportunistic pathogen, while two other strains from Gilliamella and Lactobacillus did not affect the invasion of H. alvei. Transcriptomic analysis revealed that gut species induced different expression profiles in the gut. Specifically, two regulator genes from the Toll pathway, PGRP-S3 recognizing Gram-positive and Spätzle that bind to the Toll protein for the downstream signal transduction, were elevated by L. apis. Correspondingly, multiple genes encoding antibacterial proteins were also stimulated by L. apis. Interestingly, we found an increased expression of apidaecin, which also exhibited a high in vitro inhibitory effect on H. alvei. To elucidate the difference of strains in the host’s immune regulation, comparative genomic analyses indicate that the S-layer proteins unique to L. apis are potentially involved in honeybee Toll signaling and the activation of antibacterial protein production.

IMPORTANCE Honeybees are essential pollinators supporting global agricultural economies and food supplies. Recent honeybee decline has been linked to several factors, while pathogen infection is considered one of the most significant contributing factors. Although a limited number of bacterial pathogens have been identified, Hafnia alvei is one of the pathogens causing septicemia in adult bees. In this study, we showed that two bee gut members, Gilliamella and Lactobacillus, can clear H. alvei from invasion. Mono-colonization of specific strains can stimulate the host Toll signaling pathway and the downstream expression of AMPs. Specifically, apidaecin upregulated by the gut symbionts is more effective against the pathogen. Moreover, our genomic analysis suggests that the surface-layer proteins specific to Lactobacillus strains are an important driver of Toll signaling, highlighting the variation of bee gut strains in regulating the host immune system.

The honey bee, Apis mellifera, is the most critical managed agricultural pollinator and its recent colony losses is of global concern. Bacterial pathogens are one of the causes attributed to the decline of the bee population. Serratia marcescens is an opportunistic pathogen with a broad host range, capable of affecting various plants and animals. S. marcescens can invade various tissues of the honey bee, including the gut and hemolymph, leading to severe infections and compromising bee health. S. marcescens is often associated with secondary infections, and can rapidly multiply forming biofilms, making it particularly challenging to control and eliminate. Understanding the interactions between S. marcescens and the honey bee, as well as exploring potential solutions such as probiotics, is crucial for bee health improvement. In this study, we assessed the effect of Lactobacillus kunkeei, a bee gut associated bacterium on honey bee survival, protection against the opportunistic pathogen S. marcescens, and activation of bee immune responses. Newly emerged worker bees were divided into groups: MD- no treatment, GH-inoculated with hive bees’ gut homogenate and Lk- inoculated with L. kunkeei. After five days of inoculation, bees in each respective group were exposed to the opportunistic pathogen S. marcescens. Our results show that L. kunkeei increased bee survival after S. marcescens infection, inhibited its proliferation in the gut, and reduced antimicrobial peptide genes expression in the gut epithelium compared to microbe-deprived bees in a laboratory setting. Our findings highlight the potential use of the probiotic to enhance bee health.

FUNDING This research was funded by the Newton-Utafiti grant from the British Council (grant 275898413), the Swiss National Science Foundation Postdoc Mobility (grants P300PA_174329 and P2ELP3_165168) and icipe core funding provided by UK Aid from the UK Government, Swedish International Development Cooperation Agency (Sida), Swiss Agency for Development and Cooperation (SDC), Federal Democratic Republic of Ethiopia, and the Government of Kenya. The views expressed herein do not necessarily reflect the official opinion of the donors.