Frontiers in Microbiology 01 frontiersin.org
Stably transmitted deﬁned
microbial community in
honeybees preserves Hafnia alvei
inhibition by regulating the
1, Haoyu Lang
1, Wenhao Zhang
2, Yifan Zhai
3,4, Hao Chen
3,4, Yan Liu
3,4 and Hao Zheng
1 College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China,
2 Faculty of Agriculture and Food, Kunming University of Science and Technology, Kunming, China,
3 Shandong Academy of Agricultural Sciences, Institute of Plant Protection, Jinan, China, 4 Key
Laboratory of Natural Enemies Insects, Ministry of Agriculture and Rural Aairs, Jinan, China
The gut microbiota of honeybees is highly diverse at the strain level and
essential to the proper function and development of the host. Interactions
between the host and its gut microbiota, such as speciﬁc microbes regulating
the innate immune system, protect the host against pathogen infections.
However, little is known about the capacity of these strains deposited in
one colony to inhibit pathogens. In this study, we assembled a deﬁned
microbial community based on phylogeny analysis, the ‘Core-20’ community,
consisting of 20 strains isolated from the honeybee intestine. The Core-20
community could trigger the upregulation of immune gene expressions
and reduce Hafnia alvei prevalence, indicating immune priming underlies
the microbial protective eect. Functions related to carbohydrate utilization
and the phosphoenolpyruvate-dependent sugar phosphotransferase system
(PTS systems) are represented in genomic analysis of the deﬁned community,
which might be involved in manipulating immune responses. Additionally,
we found that the deﬁned Core-20 community is able to colonize the
honeybee gut stably through passages. In conclusion, our ﬁndings highlight
that the synthetic gut microbiota could oer protection by regulating the host
immune system, suggesting that the strain collection can yield insights into
host-microbiota interactions and provide solutions to protect honeybees
from pathogen infections.
Apis mellifera, colonization resistance, Hafnia alvei, immune system, gut microbiota
e host intestinal tract is a complex ecosystem oering niches for benecial symbionts
that aid in food digestion and disease resistance (Engel and Moran, 2013b; Pereira and
Berry, 2017). Imbalanced gut microbiota driven by the antibiotic treatment could lead to
metabolism changes, potentially pathogenic bacteria blooming, epithelial barrier
TYPE Original Research
PUBLISHED 01 December 2022
Shenyang Agricultural University, China
Chinese Academy of Sciences (CAS), China
Nanjing Agricultural University,
Ningbo University, China
This article was submitted to
a section of the journal
Frontiers in Microbiology
RECEIVED 19 October 2022
ACCEPTED 14 November 2022
PUBLISHED 01 December 2022
Wang J, Lang H, Zhang W, Zhai Y, Zheng L,
Chen H, Liu Y and Zheng H (2022) Stably
transmitted deﬁned microbial community
in honeybees preserves Hafnia alvei
inhibition by regulating the immune
Front. Microbiol. 13:1074153.
© 2022 Wang, Lang, Zhang, Zhai, Zheng,
Chen, Liu and Zheng. This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that
the original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
Wang et al. 10.3389/fmicb.2022.1074153
Frontiers in Microbiology 02 frontiersin.org
disruption, and increased susceptibility to infections (Bue etal.,
2015; Raymann etal., 2017; Fünaus etal., 2018; Lang et al.,
2022). erefore, the gut microbiota can preclude infections of
enteric pathogens, which is one of the most widespread benets
to its host (Spees etal., 2013; Kim etal., 2017). Considering the
complexity of interactions between the microbiota and the host,
the underlying basis of this protection, or ‘colonization resistance’,
is still insuciently understood.
Honeybees (Apis mellifera) harbor about ve core host-
specic bacterial genera, which probably have co-evolved with
social bees for over 80 million years (Koch etal., 2013; Kwong and
Moran, 2016). ey include Snodgrassella, Gilliamella,
Bidobacterium, Bombilactobacillus Firm-4, and Lactobacillus
Firm-5 (Martinson et al., 2011; Kwong and Moran, 2016).
Additionally, the genus Apilactobacillus, Frischella,
Commensalibacter, Bartonella, and Bombella are less prevalent,
which occupy particular niches and engage in host health
maintenance (Engel etal., 2016; Liu etal., 2022). With relatively
simple gut microbiota, honeybees present opportunities to
investigate gut community dynamics and host–microbe
interaction as an experimental system (Zheng etal., 2018). Recent
research has demonstrated the honeybee gut microbiome
contributes to metabolism, development, and protection against
pathogens (Engel etal., 2016; Raymann and Moran, 2018). Some
species belonging to Bombilactobacillus Firm-4, Lactobacillus
Firm-5, and Bidobacterium can inhibit the growth of other
microorganisms in vitro (Forsgren etal., 2010; Vásquez etal.,
2012; Butler etal., 2013; Killer etal., 2014). Members of bee gut
microbiota, such as Snodgrassella alvi and Gilliamella apis, could
lower gut lumen pH and oxygen levels (Zheng et al., 2017),
compete for nutrients (Martinson etal., 2012; Wu etal., 2021), and
antagonize with type VI secretion system (Steele etal., 2017) to
inhibit pathogen virulence and growth.
e colonization resistance conferred by the gut microbiota
through stimulating the host’s innate immune system was
supported by increasing evidence (Lawley and Walker, 2013). e
innate immune system of honeybees comprises the Toll and Imd
pathways (Lourenço etal., 2013, 2018; Danihlík et al., 2015),
which primarily regulate the production of antimicrobial peptides
(AMPs), such as abaecin, apidaecin, defensin, and hymenoptaecin,
during pathogen infection (Evans etal., 2006; Guo etal., 2021).
When honeybees were colonized with conventional gut
microbiota or mono-colonized with strains from S. alvi, the
immune system of honeybees was stimulated to inhibit potential
pathogens such as Serratia marcescens (Horak et al., 2020).
However, substantial strain-level diversity was found within the
bee gut microbiota, where individual strains harbor unique genes
and distinct functional capabilities (Ellegaard etal., 2019; Brochet
et al., 2021; Lang et al., 2022). In addition to understanding
individual strains involved in interactions determining
colonization resistance, how bacterial combinations by multiple
strains from dierent species control colonization resistance still
need to beinvestigated.
Hafnia alvei, a specic pathogen in bees, could cause
septicemia with a mortality rate of over 90% by injection and
inammation of the intestinal tract by oral (Møller, 1954; Erban
etal., 2017; Grabowski and Klein, 2017). Leveraging previous
work, Lactobacillus apis W8171 could inhibit H. alvei infection
and prevent severe mucosal architecture damage in the honeybee
rectum (Lang et al., 2022). In this study, we established a
consortium based on phylogeny analysis, the ‘Core-20’
community, consisting of 20 strains isolated from the honeybee
intestine that provide colonization resistance against H. alvei.
Interestingly, the higher complex and biodiversity community
displays advantages in promoting the expression of regulators and
AMPs of the immune system. e comparative genomic analysis
revealed that the phosphoenolpyruvate-dependent sugar
phosphotransferase system (PTS system) could potentially
be involved in manipulating immune responses. In addition,
wetransmitted the Core-20 community for four passages and
found that the Core-20 could colonize steadily. us, the Core-20
community serves as a stable and functional microbiota that can
beused for detailed investigation of host-microbe and microbe-
microbe interactions in honeybees.
Materials and methods
Characterization of stains in the Core-20
community designed by the phylogeny
of honeybee gut microbiota
To establish dened minimal microbiota that recapitulates
healthy honeybee gut microbiota stably and functionally, the
integral intestine homogenization of conventional honeybee was
cultured on a rich, non-selective culture medium. About 110
strains were mono-cloned and identied by whole genome
sequencing (WGS), representing conventional bacterial strains.
e quality-controlled reads were assembled with the
SOAPdenovo2 genome assembler. e completeness and
contamination of genomes were assessed by CheckM (>96%
completeness, <0.6% contamination). Phylogenetic analysis by
WGS shows that strains assorted into dierent clusters according
to gANI identities referred to as species-level (Su etal., 2021; Wu
etal., 2021). Six strains representing the six most prevalent and
abundant genera of honeybee gut microbiota are selected for a
bacterial consortium named “Core-6,” and 20 strains at the
species-level form “Core-20” bacterial community (Figure1).
Within the Proteobacteria phylum, four members of the
Core-20 community were assigned to the genus Gilliamella, one
strain to Snodgrassella, and three strains to Bartonella. Two abundant
species clusters in the Firmicutes phylum are Bombilactobacillus
Firm-4 and Lactobacillus Firm-5, including two strains and four
strains, respectively. Additionally, Apilactobacillus kunkeei, which
proved its ability to protect honeybees from pathogens, was added
as an essential functional part (Daisley etal., 2020a,b).
Wang et al. 10.3389/fmicb.2022.1074153
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Composition of honeybee gut microbiota and strains of the Core-20 and Core-6 community. (A) Maximum-likelihood tree inferred by GTDB-tk
based on the amino acid sequences of bacterial marker genes. (B) Detailed information on strain classiﬁcation and grouping. The Core-6
community consists of six strains representing the six most prevalent and abundant genera of honeybee gut microbiota, and the Core-20 is
composed of 20 strains at the species level. Rounds mark the strains of the Core-20, triangles mark the members of the Core-6 and stars mark
strains used in the mono-colonization experiments. Color bars indicate the classiﬁcation of honeybee gut microbiota.
Wang et al. 10.3389/fmicb.2022.1074153
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Bacterial strains were isolated from the guts of A. mellifera and
stored at –80°C with 25% (v/v) glycerol PBS solution. e glycerol
stocks were plated on heart infusion agar supplemented with 5%
(vol/vol) debrinated sheep’s blood (Solarbio, Beijing, China),
MRS agar (Solarbio, Beijing, China) or TPY agar (Solarbio,
Beijing, China) incubated at 35°C in 5% CO2 for 2–3 days. e
culture conditions of strains used in this study were described by
Wu et al. (2021). Conrmed by PCR with universal bacterial
primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R
(5′-TACGACTTAACCCCAATCGC-3′), individual strains were
mixed with 25% glycerol PBS solution. e dened bacterial
communities were generated by mixing equal volumes of bacterial
suspensions with adjusted OD600 = 1.
Honeybee collection, containment, and
Microbiota-free (MF) bees were obtained as described by
Zheng et al. (Zheng etal., 2018). All bees were kept in an
incubator (35°C, RH 50%). For the H. alvei challenging
experiment, newly emerged MF bees (Day 1) were divided into
several groups, with 25 MF bees in one cup cage. For each
colonization group, bees lived on the 1 ml bacterial suspensions
mixed with 1 ml sucrose solution (50%, w/v) and 0.5 g sterilized
pollen for 24 h. For the MF group, 1 ml of 1 × PBS was mixed with
1 ml of sucrose solution (50%, w/v) and 0.5 g sterilized pollen.
Aer 24 h inoculation, all groups were fed regular diets, sucrose
solution (50%, w/v), and sterilized pollen. To precisely control the
infection amount of H. alvei cells, bees from the colonization and
MF groups were all orally inoculated with H. alvei SMH01
individually on Day 7 (Lang etal., 2022). Aer ve-day regular
diets, the load of H. alvei was determined by qPCR.
For inoculation and sampling in passaging line, newly
emerged MF bees (Day 1) were randomly assigned to three cups
and living on the 1 ml the Core-20 bacterial suspension mixed
with 1 ml sucrose solution (50%, w/v) and 0.5 g sterilized pollen
for 24 h, with 25 MF bees in one cup cage. Five days aer the nal
oral inoculation, the whole gut of each bee was sampled,
immediately placed into a sterile 1.5 ml tube individually, and
ground with sterile 25% glycerol PBS solution. ree guts from
each cup were pulled together to prepare inoculation for the
following passage, and the other guts were stored at −80°C for
DNA extraction and sequencing.
16S rRNA gene amplicon sequencing and
DNA was extracted from gut homogenates using the CTAB
method (Powell et al., 2014; Zheng et al., 2018). Targeted
amplicons of the V3-4 region of the 16S rRNA gene were
generated with primers 341F and 806R (Caporaso etal., 2011).
Sequencing libraries were generated with NEBNext Ultra II DNA
Library Prep Kit for Illumina (New England Biolabs, Ipswich,
UnitedStates). ey were sequenced at Novogene Bioinformatics
Technology Co. Ltd., Beijing, China, on the Illumina NovaSeq6000
platform (2 × 250 bp). Bioinformatic analysis was implemented
using Mothur (version 1.40.5; Schloss etal., 2009; Kozich etal.,
2013; Schloss, 2020). Aer primer trimming and quality control,
sequences were split into groups corresponding to their taxonomy
at the level of species and then assigned to operational taxonomic
units (OTUs) at a 1% dissimilarity level based on the reference
database consisting of aligned 16S rRNA sequences of our 20
strains (Supplementary Figure S1; Xue et al., 2019). Relative
abundances were then calculated based on the read numbers.
Principal coordinates analysis (PCoA) and alpha diversity indices
were visualized in R (version 3.6.1). Raw sequence reads have been
deposited at the NCBI SRA database under the BioProject
accession number PRJNA891025.
Quantitative PCR of bacterial 16S rRNA
genes and immune-related genes
DNA was extracted from gut homogenates using the CTAB
method (Powell etal., 2014; Zheng etal., 2018). DNA concentration
was determined with the Qubit 4 Fluorometer (ermo Fischer
Scientic; Waltham, MA, UnitedStates). H. alvei loads and immune-
related gene expressions were determined by qPCR using the
ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech,
Nanjing, China). Primer sets specic to H. alvei and immune-related
genes are listed in Supplementary Table S1 (Horak etal., 2020; Lang
etal., 2022). e primers of spaetzle 4 (Spz4; XM_028668966.1)
were designed based on the nucleotide sequence available in
GenBank: forward 5′-CAACGAATTCAGGGACGAGG-3′, reverse
5′-AGTAGTGCCGGGGAAATTCA-3′. All qPCRs were performed
in 96-well microplates on a QuantStudio 1 real-time PCR system
(ermo Fischer Scientic). Melting curves were generated aer
each run (95°C for 15 s, 60°C for 20 s, and increments of 0.3°C until
reaching 95°C for 15 s). Each reaction was performed in triplicates
on the same plate. e data was analyzed using the QuantStudio
Design and Analysis Soware. Aer calculating gene copies,
normalization was performed to reduce the eect of gut size
variation and extraction eciency using the host’s actin gene
(Kešnerová etal., 2020).
Functional genomics analysis
Input les were assembled and annotated genomes of the
Core-20 (Su etal., 2021; Wu etal., 2021). H. alvei reference protein
sequence (GCF_011617105.1) was downloaded from NCBI and
annotated by KAAS1 (Moriya etal., 2007). Articial metagenomes
were created by merging the contigs of each genome into a
Wang et al. 10.3389/fmicb.2022.1074153
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multi-fasta le (Brugiroux et al., 2016). KEGG mapping was
performed using the online version
(Kanehisa etal., 2022). e
comparison and analysis of orthologous clusters among genomes
were performed at3 (Xu etal., 2019).
Statistical analysis was performed using one-way ANOVA
(ANalysis Of VAriance) with post-hoc Tukey HSD (honestly
signicant dierence) using package “multcomp” in R (version
3.6.1). p-value of less than 0.05 (two-tailed) was considered
statistically signicant (*p < 0.05, **p < 0.01, ***p < 0.001).
Resistance of the Core-20 community
against honeybee opportunistic
pathogen Hafnia alvei
To evaluate the potential of the dened communities to
protect against H. alvei infection, we rst colonized MF
honeybees with the Core-20, the Core-6, and strains from the
genus Snodgrassella, Bartonella, Bombilactobacillus Firm-4,
Apilactobacillus and Bidobacterium (Figure1). At Day 7, all
honeybee were orally infected with H. alvei individually
(106 CFU per bee; Figure 2A). Successful microbiota
colonization was conrmed by 16S rRNA gene V3-V4 amplicon
sequencing at Day 12. Compositional analysis showed that
observed species in the Core-20, compared to the Core-6, were
increased, and the relative abundance of each taxonomy group
diers (Figure2B). Strains W8131, B14384H2 and W8123 from
the genus Gilliamella and strains W8093, W8171, and W8173
from the genus Lactobacillus Firm-5, which are specic to the
Core-20, exhibit substantial improvement in species
abundances, showing their tness in honeybee gut environment
and ability to coexist with the complex bacterial community.
Aer 5 days of infection, H. alvei loads were measured by
qPCR. Among mono-colonized bees, only B. choladocola
B10834H15 and B. choladohabitans W8113 signicantly inhibited
the growth of H. alvei invivo compared with the MF group at Day
12 (Figure2C). According to our previous research, H. alvei loads
in the bees with L. apis W8172, and Gilliamella apicola W8136
(the same species as G. apicola B14384G12) were also signicantly
lower than MF bees (Lang etal., 2022). Interestingly, bees treated
with the Core-6, including all these strains demonstrating the
ability to inhibit pathogens, did not show a signicant reduction
of H. alvei, while the Core-20 reduced the H. alvei loads by 78
times. Taken together, the presence of particular species did
inhibit H. alvei. Still, this microbiota-induced prevention of
pathogen infection possibly changes with the gut microbiota
composition, suggesting a complex dynamic balance between
microbe-host and microbe-microbe interaction.
Immune expression response induced by
the deﬁned community
Intestinal homeostasis maintenance depends on dynamic
interactions between gut bacteria and the host’s innate immune
systems (Yoo et al., 2020). Commensal gut microbiota could
prevent pathogen colonization and infection by enhancing the
mucosal barrier and promoting innate immune responses. e gut
microbial symbionts of the honeybee can induce antimicrobial
immune responses in the host, like AMPs (Kwong etal., 2017).
We assessed the relative expression of genes from Toll and Imd
pathways by qPCR 24 h following inoculation with the Core-20 and
Core-6. e Toll and Imd pathways include the receptors (spz4,
toll; pgrp-lc), the regulators (cactus; dredd), and the transcription
factors (dorsal; relish), respectively. On Day 2, bees colonized with
the Core-20 signicantly upregulated pgrp-lc, dredd, and relish
from the Imd pathway as well as toll and cactus-2 from the Toll
pathway relative to MF bees (Figure3A). Furthermore, wefocused
on the expression of genes encoding AMPs, and remarkably,
we discovered that bees with the Core-6 exhibited a notable
reduction of AMPs abaecin, apidaecin, hymenoptaecin and
defensin-1 (Figure3B), which might indicate an immunosuppressive
ability of the Core-6. To nd out whether the Core-20 could
consistently upregulate host-producing AMPs in response to
H. alvei infection, the expression of AMPs genes was measured
again on Day 7, right before H. alvei inoculation. Interestingly, bees
with the Core-20 showed a signicant increase of AMPs abaecin,
apidaecin, hymenoptaecin, and defensin-1 (Figure3C), indicating
abilities of the Core-20 to stimulate innate immune response
preventing the colonization by pathogenic H. alvei.
Our ndings showed the Core-6 community exhibited
diminished production of antimicrobial peptides, while the
Core-20 community upregulated the host immune system,
including regulators in innate immune pathways and AMPs
expression. Apidaecin, the most susceptible AMP against H. alvei
invitro (Lang etal., 2022), expressed much higher in the Core-20
condition. Overall, our ndings suggested that a primary
mechanism by which Core-20 provides colonization resistance is
that it can trigger host immune responses.
Potential to regulate host immune
Protection against H. alvei by the Core-20 community supports
immune regulation as a factor in pathogen defense. To gain insights
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into the potential functional capabilities of the Core-20 to activate
immunologic responses, wesequenced and annotated the individual
genomes of the 20 strains and mapped the predicted protein
sequences against the KEGG database. Articial metagenomes of the
Core-6 and Core-20 were generated by merging the contigs of
individual strains. e presence and completeness of KEGG modules
were determined for individual genomes of 20 strains, the Core-6
and Core-20 (Figure4). Aer hierarchical clustering, weobserved
dierent functional groups depending on their phylogenetic
relatedness incidentally. e majority of strains share highly
conserved modules, including phosphate acetyltransferase-acetate
kinase pathway (M00579), PRPP biosynthesis (M00005), F-type
ATPase (M00157), various carbohydrate metabolism pathways and
multiple amino-acid and nucleoside biosynthesis pathways.
Additionally, modules more prominent in Gilliamella strains
comprised pyridoxal-p biosynthesis (M00916) and carbohydrate
degradation modules, such as ascorbate, D-glucuronate, and
D-galacturonate (M00550, M00061, M00631). We also found
Core-20 leads to protection against oral H. alvei infection. (A) Experimental design for honeybees colonized with speciﬁc microbes challenging
with H. alvei. (B) Relative abundance of the Core-6 and Core-20 community on Day 12. 16S rRNA V3-V4 amplicons were sequenced and
analyzed, showing successful microbiota colonization and composition dierences between the Core-6 and the Core-20. (C) Absolute
abundance of H. alvei in dierent treatment groups 5 days post-infection. Single strains, such as B. choladocola B10834H15 and B.
choladohabitans W8113, signiﬁcantly inhibited the growth of H. alvei. The Core-20 community, which is much more complex than the Core-6,
signiﬁcantly reduced the H. alvei loads.
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nucleotide sugar biosynthesis (M00554), galactose degradation
(M00632), and beta-oxidation (M00086) modules enriched in
Bidobacterium strains. In total, the comparison of the Core-6 and
Core-20 shows functional similarity. However, there were still several
modules enriched in the Core-20, including cobalamin anaerobic
biosynthesis (M00924), beta-oxidation (M00087), propanoyl-CoA
metabolism (M00741), d-galactonate degradation(M00552), pectin
degradation (M00081), and hydroxyproline degradation (M00948).
Additionally, wealso estimated the complement of KEGG modules
for the genome of H. alvei, and we found highly overlapping
functions with the Core-20 community, indicating its tness and
potential virulence. At the same time, several pathways were found
enriched in H. alvei, such as glycogen biosynthesis (M00854),
undecaprenylphosphate alpha−L − Ara4N biosynthesis (M00761),
fumarate reductase (M00150), cysteine biosynthesis (M00338),
menaquinone biosynthesis (M00116), ubiquinone biosynthesis
(M00117), and multiple pathways of biotin biosynthesis. Overall,
wespeculated that receptors or products from carbohydrate, fatty
acid, and amino acid metabolism could probably display a key role
in regulating the immune system.
Next, weinvestigated the genes specic to the Core-20 but not
present in the Core-6, potentially associated with the capacity to
trigger the immune system. e comparative analysis found that
3,206 genes unique to the Core-20 were enriched in 1,231 Gene
Ontology clusters (Figure5A). Notably, the enriched GO among
all identied clusters was the phosphoenolpyruvate-dependent
sugar phosphotransferase system (PTS, GO:0009401), a complex
enzyme system functioning in the detection, transport, and
phosphorylation of various sugar substrates (Kotrba etal., 2001;
Gabor et al., 2011). e PTS is comprised of two general
cytoplasmic components, enzyme I(EI) and histidine phosphoryl
carrier protein (HPr), and membrane-bound sugar-specic
multidomain enzymes II (EII). Each EII complex consists of one
or two hydrophobic integral membrane domains (domains C and
D) and two hydrophilic domains (domains A and B; Figure5B).
Mannose/fructose/sorbose family PTS system was observed, and
four genes, including EIIAB, EIIB, EIIC, and EIID, were shared in
four strains from the genus Lactobacillus Firm-5 (Figure 5C).
Interestingly, W8173, W8093, and W8171, three stains specic to
the Core-20, harbored their unique clusters of EIIA, EIIB, EIIC,
and EIID (Figure5D). Taken into account that both IIC and IID
components of the mannose phosphotransferase system are
involved in recognition of antimicrobial peptides (Kjos etal., 2010;
Zhou etal., 2016), our results indicated that membrane-bound EII
of phosphotransferase system could probably trigger an immune
response, causing protection in the Core-20 bees.
Stability transmission of Core-20
community during successive passaging
Due to the potential of the Core-20 to inhibit pathogens and
shape the host immune system, wewonder whether the Core-20
Core-20 triggers host immune gene expression in Imd and Toll pathways (A,B) at 24 h post-colonization and (C) 7 days post-colonization. The
Core-20 displayed a signiﬁcant promotion in regulators of the Toll and Imd pathways on Day 2 and potential ongoing upregulation in AMPs
expression on Day 7. Besides, the Core-6 signiﬁcantly reduced the expression of AMPs on Day 2. All results indicated that the gut microbiota could
stimulate the host’s innate immune system. Gene expression was normalized relative to the housekeeping gene actin. *p< 0.05; **p< 0.01 (Tukey
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community can stably colonize the honeybee gut over several
passages. Microbiota-free bees were inoculated with the frozen
mixtures of the Core-20 colony and sampled the whole gut on
Day 7. e gut microbiota was mixed and passed on throughout
four passages: passage 1 (P1), P2, P3, and P4. At the end of each
passage, bacterial communities were sequenced by amplicon
The presence and completeness of KEGG modules analysis of individual strains, the Core-6, and the Core-20 community. A hierarchical clustering
heat map of KEGG module distribution in the draft genomes and artiﬁcial metagenomes. The comparison of the Core-6 and Core-20 shows
functional similarity. Wealso found highly overlapping functions between H. alvei and the Core-20 community, indicating its ﬁtness and potential
virulence. The color code indicates the presence and completeness of each KEGG module, expressed as a value between module complete (dark
blue) and module absent (white).
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sequencing of the variable regions 3 and 4 of the 16S rRNA gene
(Figures6A,B). All strains except G. sp. W8131 were detected
individually in bee gut samples among passages, indicating W8131
either is below the detection limit or does not colonize. e relative
abundance of Bidobacterium, Snodgrassella, and Apilactobacillus
increased during passaging. Notably, G. apicola W14384G12 and
L. melliventris W8171 were dominant within their genus,
respectively. e relative abundance of Bartonella was maintained
at a relatively stable level during transmission, suggesting the
restriction and regulation of honeybee hosts to gut microbiota.
We also found an overall decrease in alpha diversity over time
across the four passages (Figure6C, Tukey honest method, p < 0.05
for P1-P4, P2-P4) and a signicant dierence between P1 and the
other passages in beta-diversity measured by Bray–Curtis
dissimilarity (Figure 6D, PERMANOVA, p = 0.013 for P1-P2,
p = 0.004 for P1-P3, p = 0.001 for P1-P4). Our ndings indicated
that strains of the Core-20 community display stable coexistence
aer slight uctuations in species abundances and biomass during
P1. In summary, these data suggest that the Core-20 community
maintains stability despite uctuations over the course of passage.
While early culture-based studies demonstrated that honeybee
gut symbionts could becultured in vitro, induce host immune
response, and confer protection against pathogens aer inoculation,
little is known about the capacity of these isolates deposited in one
colony. In this study, weassembled a dened microbial consortium
of honeybees (the Core-20 community) based on the phylogeny
analysis, which strongly inhibits H. alvei. Following exposure,
H. alvei can grow to high loads (109 CFU per gut), produce
inammatory reactions, and potentially result in host mortality.
We focused on the expansion of H. alvei infection, which is
primarily inuenced by the gut microbiota, and carried out
comprehensive investigations on the mechanism of colonization
resistance by the gut microbiota. e Core-20 community could
trigger upregulation of AMPs and precise H. alvei prevalence,
indicating immune priming underlies part of the dened
community protective eect. Functions related to carbohydrate
utilization and the PTS system were represented in genomic analysis
of the Core-20 community, which might play a role in immune
The PTS system enriched in the Core-20 community might trigger the immune response oering protection. (A) The Venn diagram was generated
using OrthoVenn2. Results showed the number of shared orthologous clusters of protein-coding genes between artiﬁcial metagenomes.
(B) Diagrammatic representation of the bacterial phosphotransferase signal transduction pathway (Mannose/fructose/sorbose family PTS system
as an example). General phosphoryl and sugar transport reaction catalyzed by the PTS. Sugars are transported and concomitantly phosphorylated
by the PTS. (C,D) In the genus Lactobacillus Firm-5, gene loci of mannose/fructose/sorbose family PTS system (C), shared in 4 strains or (D),
unique to strains in the Core-20. Homologous genes are connected by gray bars. Wefound that W8173, W8093, and W8171, three stains speciﬁc
to the Core-20, harbored their unique clusters of EIIA, EIIB, EIIC, and EIID, indicating that membrane-bound EII of phosphotransferase system
could probably trigger an immune response.
Wang et al. 10.3389/fmicb.2022.1074153
Frontiers in Microbiology 10 frontiersin.org
system stimulation. Additionally, we found that the Core-20
community is able to colonize the honeybee gut over four passages
stably. Our ndings highlight a dened microbial community could
oer protection via host–microbe interaction (for example,
regulating the host immune system), suggesting that the Core-20
community could beused for gut microbiota research in honeybees.
A major function of the stable gut microbiota is to provide
colonization resistance, preventing pathogens from colonizing and
causing long-term infection and even mortality. Ghimire et al.
identied Clostridioides dicile-inhibiting strains through single
strain versus pathogen coculture assays in vitro. However, when they
came to investigate how changes in the combinatorial assembly of
bacteria might aect the inhibition capacity, their results
demonstrated that new phenotypes masking the individual strain
phenotype could emerge depending on the composition of the mix.
For instance, bacterial consortia, where all the strains individually
showed inhibition, display the enhancement of C. dicile growth
(Ghimire etal., 2020). Moreover, germ-free mice colonized with
The Core-20 community stably colonized honeybees for four passages. (A) Experimental design for passaging transmission. (B) Relative
abundance of strains in the Core-20 during four passages. All strains except G. sp. W8131 were detected individually in bee gut samples among
passages. (C) Box plots of Shannon’s alpha diversity index at each passage. Results showed a slight decrease in alpha diversity. (D) Principal
coordinates analysis (PCoA) plot of Bray–Curtis dissimilarity among samples. Wefound that the last three passages showed community similarity
except for P1.
Wang et al. 10.3389/fmicb.2022.1074153
Frontiers in Microbiology 11 frontiersin.org
members of the altered Schaedler ora (ASF), a bacterial consortium
consisting of eight mouse-derived strains, provided insucient
colonization resistance to Salmonella enterica serovar
Typhimuriumthe. However, enforced with three facultative
anaerobes in Oligo-MM12 mice prevent infection completely
(Brugiroux etal., 2016). Here, B. choladocola B10834H15 from
Bartonella and B. choladohabitans W8113 from Bidobacterium
signicantly inhibited the growth of H. alvei. In previous studies,
Bidobacterium of honeybees could produce antimicrobial
substances in vitro to inhibit other microorganisms, contributing to
the resistance of pathogenic bacteria for the host (Forsgren etal.,
2010; Vásquez etal., 2012; Butler etal., 2013). In addition, Bombella
apis has been evidenced to benet the larval development of honey
bees and protect larvae against fungal pathogens (Liu etal., 2022).
Notably, the Core-6 community could increase the growth of
H. alvei. In contrast, single strains and the Core-20 eectively
inhibited H. alvei (Figure2C), demonstrating that a dened bacterial
community could oer the inhibition capacity as individual strains.
e microbe-microbe interaction needs to be concerned with
designing dened pathogen-inhibiting bacterial mixtures in vivo.
e mechanisms that regulate the ability of the microbiota to
restrain pathogen growth are complex, including induction of host
immune responses, localization to intestinal niches, and
competitive metabolic interactions (Kamada etal., 2013). AMPs
can maintain gut microbiota homeostasis by selectively inhibiting
foreign bacteria and keeping native symbionts from over-
proliferating (Kwong etal., 2017). e synthesis and secretion of
AMPs is a highly regulated process, mainly controlled by the Toll
and Imd pathways (Lourenço etal., 2013, 2018; Danihlík etal.,
2015). Specic gut symbionts, such as S. alvi, A. kunkeei, Frischella
perrara, and L. apis, have been conrmed to induce honeybee
innate immune response. ey upregulate the Toll and Imd
pathway, leading to AMPs expression (Emery etal., 2017; Daisley
et al., 2020b; Lang etal., 2022). Considering that the Core-6
consisted of microbes that were able to induce the immune
response, the whole gut microbiota balance composition could
bemore important for regulating the immune system. e Core-20,
a high-species-diversity colony, had more signicant upregulation
of the immune regulatory genes and AMPs genes encoding
abaecin, apidaecin, hymenoptaecin, and defensin-1 (Figure 3),
suggesting the ability of the Core-20 community in stimulating
host innate immune system through their regulators and eectors.
Biolm and the outer membrane protein, such as the S-layer
protein unique to L. apis W8172, could bepotential drivers of the
host immune response. Weused KEGG modules to character gene
sets linked to specic metabolic capacities and OrthoVeen2 to
compare and annotate orthologous gene clusters among multiple
genomes (Figures4, 5). Results showed that the PTS system was
signicantly enriched in the Core-20 community. e PTS system
is a highly conserved phosphotransfer cascade whose components
modulate many cellular functions in response to carbohydrate
availability (Houot etal., 2010). Previous studies have elucidated the
importance of bacterial PTS system for honeybees, including
detoxifying specic nectar components (Engel and Moran, 2013a),
nutrient metabolic transformations (Lee etal., 2015), and adaptation
to the diet and gut environment of the honeybee. PTS system of
Enterococcus faecalis could increase proinammatory cytokine
secretion by colon tissue and macrophages to enhance colonization
in mice (Fan etal., 2019). Besides, the PTS system of Vibrio cholerae
display control of carbohydrate transport and activation of biolm
formation on abiotic surfaces (Houot etal., 2010). Additionally, EIIC
and EIID from the mannose/fructose/sorbose family PTS system,
the membrane-banding proteins, is responsible for specic targeting
by antimicrobial peptides, indicating their potential to regulate the
immune system (Diep etal., 2007; Kjos etal., 2010; Zhou etal., 2016).
According to Rolf Freter’s nutrient niche theory, a pathogen
can only invade if it is able to use a specic limiting nutrient
more eciently than the rest of the community, which means
colonization resistance against pathogens is aected by ecient
restriction of all available nutrient niches by a complex
microbial community (Freter etal., 1983). Invasion theory
Figures out that biotic selection could be the critical
determinant (Dillon etal., 2005; van Elsas etal., 2012; Mallon
etal., 2015; Ketola etal., 2017). Higher diversity communities
can competitively exclude an invader by reducing the
availability of ecological niches and eciently utilizing
resources (Hromada etal., 2021). us, the protective eect is
probably provided through antagonism between microbes
(Chiu etal., 2017; Ubeda etal., 2017). In the case of an animal
pathogen, three facultative anaerobes potentially prevent
infection in Oligo-MM
mice by lling up the niche space that
is preferred by S. Tm (Brugiroux etal., 2016). Previous studies
showed that H. alvei reduced nitrates and fermented
l-arabinose, glycerol, maltose, d-mannitol, d-mannose,
l-rhamnose, trehalose, and d-xylose (Møller, 1954; Janda etal.,
2005; Tian and Moran, 2016; Erban etal., 2017). Genomic
analysis reveals that H. alvei harbors various carbohydrate
degradation modules and has similar functions as the Core-20
(Figure4), suggesting its ability to grow in the honeybee gut
and compete for multiple carbohydrates. Gilliamella, a primary
polysaccharide degrader in the honeybee gut, utilizes mannose,
arabinose, xylose, or rhamnose (monosaccharides that can
cause toxicity in bees; Zheng et al., 2016, 2017, 2019).
Functions for carbohydrate use and PTS systems are
represented in genomic analysis of the Core-20 community,
which may also promote colonization resistance by competition
for limited nutrients that H. alvei presumably depends on. Our
ndings implied that protection by the Gilliamella and the
Core-20 bees occurs via occupation of niche space (for
example, consumption of carbohydrates) that can no longer
be exploited by H. alvei. Loss of microbial diversity might
create ecological niches that pathogens can use, underlying
why bees colonized with low-complexity gut microbiota, such
as the Core-6, are more susceptible to H. alvei infection.
Whereas, because the Core-20 had 14 strains more than the
Core-6, it is conceivable that the Core-20 community could
Wang et al. 10.3389/fmicb.2022.1074153
Frontiers in Microbiology 12 frontiersin.org
actually ll up the niche space that is preferred by H. alvei and
thereby prevent infection.
e honey bee gut microbiota is dominated by limited
numbers of bacterial phylotypes, commonly with species from
the Gilliamella, Snodgrassella, Lactobacillus Firm-5,
Bombilactobacillus Firm-4, Bidobacterium, and Bartonella
genera. Gut microbial communities inuence host health in
many ways, including food digestion, defense against
pathogens, and modulation of behavior, development, and
immunity (Engel and Moran, 2013a,b). erefore, dysbiosis
(microbial imbalance) may impact honeybee health and
susceptibility to disease. Honeybees treated with tetracycline
severely altered both the size and composition of the gut
microbiome, decreasing the survival rate of bees and increasing
susceptibility to opportunistic pathogens (Raymann et al.,
2017; Lang etal., 2022). Here, the Core-20 consisted of typical
isolates representing species in honeybee gut microbiota,
which demonstrated transmission stability and functional
redundancy during passages. Potentially, consequences of
dysbiosis, such as nutritional impacts or heightened
susceptibility to toxins, could be reduced through the
development of alternative treatment methods, for example,
adding the Core-20 to the bee hive.
In conclusion, wehave assembled a minimal community of
20 bacterial strains that provided colonization resistance against
H. alvei, elucidating the underlying molecular and functional
mechanisms. e native gut symbionts are essential in the
resistance to pathogen invasion. Such strain collections can
yield insights into host-microbiota interactions, hoping to oer
solutions to protect honeybees from pathogen infection.
Data availability statement
e datasets presented in this study can befound in online
repositories. e names of the repository/repositories and
accession number(s) can be found in the article/
JW and HZ designed the research. JW, HL, and WZ
collected the samples. JW, HL, and WZ performed the
experiments and analyzed the data with contributions from
YZ, LZ, HL, and YL. JW and HZ wrote the manuscript. All
authors contributed to the article and approved the
is work was supported by the National Key R&D Program
of China (grant no. 2019YFA0906500).
We thank Jun Guo, Zijing Zhang, and Xiaohuan Mu for their
assistance in sample collection.
Conﬂict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their aliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
e Supplementary material for this article can befound online
SUPPLEMENTARY TABLE S1
qPCR primer sequences for gene expression and bacterial load.
SUPPLEMENTARY FIGURE S1
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