Antonie van Leeuwenhoek (2021) 114:1293–1305

https://doi.org/10.1007/s10482-021-01602-x (0123456789().,-volV)
( 01234567
89().,-volV)

ORIGINAL PAPER

The composition of bacteria in gut and beebread of stingless

bees (Apidae: Meliponini) from tropics Yunnan, China
Qi-He Tang . Chun-Hui Miao . Yi-Fei Chen . Zhi-Xiang Dong .
Zhe Cao . Shi-Qun Liao . Jia-Xuan Wang . Zheng-Wei Wang .
Jun Guo

Received: 5 January 2021 / Accepted: 1 June 2021 / Published online: 10 June 2021
Ó The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021

Abstract Stingless bees are the main pollinators in
tropical and subtropical regions. However, there are
only a few studies on the structure and composition of
bacteria in the gut and beebread of stingless bees,
especially in China. To address this shortage of
information, we characterized the microbiota of three
common species of stingless bees (Lepidotrigona
terminata, Lepidotrigona ventralis and Tetragonula
pagdeni) and beebread samples of T. pagdeni. The
results showed that the gut of stingless bees contained
Qi-He Tang, Chun-Hui Miao and Yi-Fei Chen have
contributed equally.
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10482-021-01602-x.
Q.-H. Tang
Y.-F. Chen
Z.-X. Dong

Z. Cao
S.-Q. Liao
J.-X. Wang
J. Guo (&)
Faculty of Life Science and Technology, Kunming
University of Science and Technology, Kunming 650500,
China
e-mail: guojun0591@126.com
C.-H. Miao
Sericulture and Apiculture Reserach Institute, Yunnan
Academy of Agriculutral Sciences, Mengzi, China
Z.-W. Wang (&)
CAS Key Laboratory of Tropical Forest Ecology,
Xishuangbanna Tropical Botanical Garden, Chinese
Academy of Sciences, Jinghong 650000, China
e-mail: wangzhengwei@xtbg.ac.cn
a set of dominant bacteria, including Acetobacter-like,
Snodgrassella, Lactobacillus, Psychrobacter, Pseu-
domonas, Bifidobacterium and other species. The gut
microbiota structures of the three stingless bees were
different, and the abundances of bacterial species in
the gut varied between communities of the same bee
species. The reasons for this are manifold and may
include food preference, age and genetic differences.
In addition, the abundances of Lactobacillus, Carni-
monas, Escherichia-Shigella, Acinetobacter and other
species were high in the beebread of stingless bees. In
conclusion, our findings reveal the bacteria composi-
tion and structure of the gut and beebread of stingless
bees in China and deepen our understanding of the
dominant bacteria of the gut and beebread of stingless
bees.
Keywords 16S rRNA sequencing
Beebread
bacteria
Gut bacteria
Microbiome analysis

Stingless bees
Introduction
Bees play a key role in maintaining biodiversity and
generating economic value. They pollinate 73% of
crops and some wild plants (Guimares-Cestaro et al.
2020) and can provide profits of approximately 1.6
billion to 5.7 billion dollars a year (Dong et al. 2020).
However, the health and population of honeybees are

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in great danger, and the diversity and abundance of
wild pollinators have also declined in many agricul-
tural landscapes (Garibaldi et al. 2013; Potts et al.
2010). Global warming (climate change), habitat loss
and fragmentation, pathogen infection, parasite infes-
tation, exposure to pesticides or agrochemicals, and
the interactions between these factors are the main
causes for this decline (Guimares-Cestaro et al. 2020;
Lima et al. 2016; Potts et al. 2010). The gut microbes
of animals can benefit their hosts by helping to digest
food, improve host malnutrition, and protect against
invading parasites, pathogens and diseases (Engel and
Moran 2013; Ribiere et al. 2019). Therefore, honey-
bees have already become model organisms for gut
microbiota research because the bacteria are critical to
the health and wellness of bee hosts (Zheng et al.
2018).
The gut of social bees are inhabited by highly
conserved core bacteria, including Snodgrassela alvi,
Gilliamella apicola, Bifidobacterium aeroides, Lacto-
bacillus Firm-4, Lactobacillus Firm-5, Frischella
perrara, Bartonella apis, Apibacter adventoris,
Parasaccharibacter appium and Alpha2.1 (Bonilla-
Rosso and Engel 2018; Moran et al. 2012; Powell et al.
2014; Romero et al. 2019; Zheng et al. 2018). Because
social bees exchange bacteria through social beha-
viours (such as oral trophallaxis or faecal-oral trans-
mission), almost all social bees have the first five
bacteria listed above in their gut, also known as their
core bacteria (Kwong and Moran 2016). However, on
a long evolutionary time scale, the gut microbiota of
honeybees has been not only conserved but also
diversified. That is, different host bee species carry
different bacterial strains, and specific hosts often
carry related strains. For example, Bombiscardovia
coagulans is mainly found in Bombus spp. (Killer et al.
2010), and Bartonella apis is mainly found in Apis
species but not in Bombus or Meliponini species
(Kwong and Moran 2016). In addition, different
Bifidobacteria were found in honeybees and bumble-
bees (Praet et al. 2015).
Stingless bees (Apidae: Meliponini) are the largest
group of eusocial bees and are found mainly in tropical
and subtropical regions worldwide (Paludo et al.
2019), with 50 genera and 505 named species (Makkar
et al. 2018). Similar to honeybees, stingless bees have
three castes (queen, worker and drone) in the colony,
their labour division is similar to that of the honeybee
colony (Bassindale and Matthews 1995; Ngalimat
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Antonie van Leeuwenhoek (2021) 114:1293–1305
et al. 2020; Wille 1983), and their colonies subsist on
pollen and nectar (Wiebke et al. 2019). To store
pollen, they deposit the collected pollen inside wax
comb cells, and then workers add more secretions
(containing beneficial microorganisms, enzymes, and
honey) to ferment the pollen to form beebread (de
Paula et al. 2021; Detry et al. 2020). Interestingly,
Vollet-Neto et al. (2017) found that 2-day-old sting-
less bees (Scaptotrigona aff. depilis) preferred to
consume fermented beebread over fresh pollen. How-
ever, the bacterial composition of beebread remains to
be revealed in stingless bees.
Throughout the tropics, many stingless bee species
have been cultivated by farmers for their products
(honey, pollen and propolis) and for providing polli-
nation services for fruits and crops. These stingless
bees are important for the pollination of many
flowering plants in tropical ecosystems (Slaa et al.
2000). Stingless bees outperform honeybees as polli-
nators of crops of some families, such as Compositae,
Cruciferae and Leguminosae (Makkar et al. 2018). Far
less research has been performed on stingless bees
than on honeybees, and knowledge about the gut
microbiota of stingless bees is still limited. Initially,
research on the gut bacteria of stingless bees mainly
focused on host-related lactic acid bacteria (Leonhardt
and Kaltenpoth 2014). Recently, Kwong et al. (2017)
investigated the gut microflora of 13 species of
stingless bees from neotropical regions and Malay-
sia/Australia and found that the bacterial phylotype
identified as Acetobacter-like appears to be specific to
stingless bees. Exploring microbial communities can
provide insights into gut bacterial symbionts with
potential favourable functionalities in stingless bees.
However, stingless bee resources and their microbial
functions are often ignored in tropical China.
The present study used the 16S rRNA Illumina deep
sequencing technique to characterize the gut microbial
community diversity of three common species of
stingless bees in China, Lepidotrigona terminata,
Lepidotrigona ventralis and Tetragonula pagdeni. In
addition, to understand the dominant bacterial com-
position of stingless bee beebread, we collected
beebread from the T. pagdeni colony to analyse the
microbial community structure.
Antonie van Leeuwenhoek (2021) 114:1293–1305
Materials and methods
Sampling and identification
To analyse the microbiome composition across adult
stingless bees, we collected 9 colonies of L. terminata,
8 colonies of L. ventralis and 21 colonies of T. pagdeni
within a 10 km region in Xishuangbanna Dai
Autonomous Prefecture, Yunnan Province, China, in
mid-May 2019. We placed a clean clear plastic bottle
in the hive entrance, thereby catching worker bees
leaving the nest (Leonhardt and Kaltenpoth 2014).
They were transferred to a 5-mL centrifuge tube and
anaesthetized on ice, and they were stored in 100%
ethanol at - 80 °C until mid-June 2019 (Kwong et al.
2017).
To analyse the bacterial composition of stingless
bee beebread, we collected beebread using sterile
scissors and tweezers from five colonies of T. pagdeni
at the same place and time. These samples were
transferred into sterile 15-ml centrifuge tubes stored
on ice. After arriving at the laboratory, the samples
were stored in a refrigerator at -80 °C. The species of
stingless bees were first identified by morphological
methods (Wu 2000). Then, the mitochondrial 16S
rRNA gene was sequenced following the methods of
Rasmussen and Cameron (2010). We included the
following controls: (1) a negative control for sampling
to ensure that sampling equipment (tubes, swaps, etc.)
was not contaminated and (2) a negative control for
DNA extractions, to ensure the identification of
potential kit contamination (Hornung et al. 2019).
DNA extraction
In a sterile environment, we surface-sterilized the bees
in 75% alcohol for 20 s and then washed them with
sterile water. Finally, the entire gut was removed from
the abdomen of each bee. All samples were homog-
enized with 1 ml of Krebs–Ringer solution in a 3rd
generation variable speed (Catalogue number OSE-
Y50, Beijing, China). The total DNA of the gut
microbiota were extracted from three individual
worker bees of all thirty-eight colonies sampled
(Leonhardt and Kaltenpoth 2014) with an E.Z.N.A.Ò
soil DNA Kit (Omega, USA). The total DNA of the
beebread microbiota were extracted with the same soil
DNA kit from five colonies of the sampled T. pagdeni.
1295
16S rRNA gene amplification and sequencing
The DNA concentration was determined using a
NanoDrop 2000 UV–vis spectrophotometer (Thermo
Scientific, NC, USA), and the variable V3 and V4
regions of the bacterial 16S ribosomal RNA (rRNA)
gene were amplified with forward primer (ACTCC-
TACGGGAGGCAGCAG) and reverse primer
(GGACTACHVGGGTWTCTAAT) (Dong et al.
2020). PCR products were analysed by 2% agarose
gel electrophoresis to verify size and were purified
using an AxyPrep DNA Gel Extraction Kit (Axygen
Biosciences, Union City, CA, USA) and then quan-
tified using QuantiFluor-ST kits (Promega, USA) (Liu
et al. 2018). Purified amplification products were
pooled in equimolar amounts and then paired-end
sequenced (2 9 300 bp) on the Illumina MiSeq
platform of Majorbio Bio-Pharm Technology Co.
Ltd. (Shanghai, China).
Processing of sequencing data
Raw data were trimmed and filtered by Trimmomatic
0.36 and merged by FLASH 1.2.11 (Dong et al. 2020;
Wang et al. 2019) with the following criteria: (i) the
reads were truncated at any site receiving an average
quality score \ 20 over a 50 bp sliding window; (ii)
sequences with overlaps longer than 10 bp were
merged, allowing 2 nucleotide mismatches, and (iii)
reads containing ambiguous bases were removed.
Operational taxonomic unit (OTU) clustering with
97% sequence similarity was performed using
UPARSE 7.0.1090 (Shang et al. 2020). The taxonomy
of OTUs was analysed by the Ribosomal Database
Project (RDP, version 2.11) Classifier algorithm, and a
confidence threshold of 70% was used for comparison
with the SILVA 128 16S rRNA database. We
processed and filtered OTU sequences as follows:
(i) removal of Cyanobacteria sequences, and (ii)
sampling depth based on the lowest number of read
sequences among samples (value = 20, 253) (Gong
et al. 2020).
Statistical analyses and comparison of microbial
communities
The software Mothur 1.30.2 was used to calculate the
alpha diversity, including community diversity (Shan-
non, Simpson and PD indexes) and community
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richness (Ace, Chao and Sobs indexes), using different
random sampling (Gong et al. 2020; Schloss et al.
2009), and the Kruskal–Wallis test was used to
compare whether there were significant differences
between the three groups. The Kruskal–Wallis statis-
tical method was used to detect the differences in
species in the microbial communities of the three
groups. The community diversity within and among
the three groups was evaluated by principal coordi-
nates analysis (PCoA, based on binary Jaccard
distance) and partial least squares discriminant anal-
ysis (PLS-DA). The group comparison of PCoA was
determined by permutational multivariate analysis of
variance (PERMANOVA) and analysis of similarities
(ANOSIM) (Dong et al. 2020; Gong et al. 2020).
QIIME 1.9.1 software and the stats package in R were
used for these analyses. The results with P \ 0.05
between groups were considered statistically
significant.
Results
Overview of sequencing data
A total of 2,020,093 high-quality sequences were
generated from 43 samples; there were 55,419
sequences per L. terminata sample (total 498,774),
L. ventralis averaged 52,698 sequences per sample
(total 421,584), T. pagdeni averaged 42,026 sequences
per sample (total 882,550), and beebread averaged
43,437 sequences per sample (total 217,185). The
average L. terminata sequence length was 418 bp, L.
ventralis was 415 bp, T. pagdeni was 419 bp and
beebread was 420 bp. The qualified sequences were
clustered into 1,136 bacterial OTUs at a difference
level of 3%. The OTU% of 43 samples was estimated
by the Good coverage index, and the average bacterial
coverage rate was 0.99849 ± 0.00060, indicating that
the obtained data could cover the microbial species in
the samples well.
Composition of the gut microbiota of stingless
bees
We classified and identified the 16S rRNA gene
sequences collected from stingless bee workers. OTUs
were assigned to the phylum level (Figure S1A), and
most OTUs were identified as Proteobacteria,
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Antonie van Leeuwenhoek (2021) 114:1293–1305
Firmicutes and Actinobacteria; these were the core
phyla shared by the three species of stingless bees. The
relative abundance of Proteobacteria was the highest
in the gut samples, accounting for 66.17%, 88.61%
and 82.46% of L. terminata, L. ventralis and T.
pagdeni samples, respectively; the second most abun-
dant phylum in gut samples was Firmicutes, account-
ing for 27.95% in L. terminata, 6.15% in L. ventralis
and 12.29% in T. pagdeni. Through intergroup com-
parison, the P-values were 0.0064 for Proteobacteria
and 0.0005 for Firmicutes (Figure S1B).
Additionally, we mapped the OTUs to the repre-
sentative bacterial genus (Fig. 1a, b). Out of the top 15
dominant genera of stingless bees (Fig. 1a, Table 1),
the relative abundances of Acetobacter-like (no-
rank_f_Acetobacteraceae), Psychrobacter, Pseu-
domonas and Bifidobacterium were not different
among the three species. However, there were signif-
icant differences in the abundances of other dominant
bacteria among different species of worker bees. In L.
terminata and L. ventralis, Snodgrassella was not
found, but Snodgrassella was found in the T. pagdeni
samples at a relative abundance of 28.86%. The
highest abundance of Lactobacillus was 27.57% in the
L. terminata, followed by 11.39% in T. pagdeni, and
the lowest abundance was 5.54% in L. ventralis. The
content of unclassified_f_Enterobacteriaceae in L.
ventralis reached 8.59%, while the abundances of this
genus in L. terminus and T. pagdeni were only 0.51%
and 0.50%, respectively. Similarly, the relative abun-
dances of Zymobacter and Carnimonas in L. ventralis
were significantly higher than the relative abundances
of the other two neriformis. The content of Acineto-
bacter in the L. terminata and L. ventralis, which are in
the same subgenus (4.67%—3.21%) was significantly
higher than the content of Acinetobacter in the other
genus (0.41%) of bees. Similarly, we used linear
discriminant analysis (LDA) of effect size (LEfSe) to
determine the taxa, at the genus level, that most likely
explained the differences between the samples of the
three stingless bees (Fig. 1c). The results confirmed
the significant enrichment of Lactobacillus and Acine-
tobacter in L. terminata, unclassified_f_Enterobacte-
riaceae, Carnimonas and Zymobacter in L. ventralis,
and Snodgrassella in T. pagdeni. The above results
show that there were significant differences in the
abundances of dominant bacteria in the gut of the three
stingless bee species.
Antonie van Leeuwenhoek (2021) 114:1293–1305 1297

A
ta
na
ermi
t
L.
s
ali
ventr
L.

ni
a gde
T. p
0 20 40 60 80 100
Relative abundance on Genus level (%)
norank_f_Acetobacteraceae Psychrobacter uclassified_f_Enterobacteriaceae Carnimonas Sphingomonas
Lactobacillus Pseudomonas Acinetobacter Escherichia-Shigella unclassified_f_Moraxellaceae
Snodgrassella Bifidobacterium Zymobacter Rosenbergiella Rhodococcus others

B 1 norank_f_Acetobacteraceae
Percent of community abundance on Genus level

Snodgrassella
Lactobacillus
Psychrobacter
0.8 Pseudomonas
Bifidobacterium
uclassified_f_Enterobacteriaceae
0.6
Acinetobacter
Escherichia-Shigella
Zymobacter
Carnimonas
0.4 Rosenbergiella
Sphingomonas
unclassified_f_Moraxellaceae
Rhodococcus
0.2 Bombella
Vibrionimonas
unclassified_f_Acetobacteraceae
Pectinatus
0 others
Tp 1

Tp11
Lv 3
Lv 1

Lv 5
Lv 6

Tp21
Lv 2

Lv 4

Lv 7

Tp19
Tp20
Lv 8

Tp12
Tp 4

Tp 7

Tp15
Tp16
Tp17
Tp18
Lt 2
Lt 3

Tp 5
Lt 4
Lt 5
Lt 6
Lt 7
Lt 9

Tp10
Tp 2

Tp 8
Tp 9
Lt 1

Tp 6
Tp 3
Lt 8

Tp13
Tp14

L. terminata L. ventralis T. pagdeni

C g__Lactobacillus L. terminata
g__Acinetobacter
g__unclassified_f__Enterobacteriaceae L. ventralis
g__Zymobacter
g__Carnimonas T. pagdeni
g__unclassified_f__Halomonadaceae
g__norank_c__Acidobacteria
g__norank_f__Xanthomonadales
g__Vulcaniibacterium
g__Snodgrassella
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
LDA SCORE(log10)

Fig. 1 Gut bacterial composition of stingless bees at the genus different genera, and the length of the column represents the
level. a Average relative abundance of each species. Columns of relative proportion. (C) Histogram of linear discriminant
different colours represent different genera, and the length of the analysis (LDA) values. LDA scores for gut bacteria exceeding
column represents the relative proportion. b Gut bacterial the threshold value of 4 indicate a significant difference between
composition of each colony of stingless bees. A column groups, and histogram length (LDA score) represents the effect
represents a sample, columns of different colours represent size of gut bacteria

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Table 1 Relative Genus Mean relative abundance (%) P value

abundances of the top 15
gut bacteria at the genus L. terminata L. ventralis T. pagdeni
level across different
stingless bees norank_f__Acetobacteraceae 36.55 47.70 34.78 0.6407
Lactobacillus 27.57 5.54 11.39 0.0005
Snodgrassella 0.00 0.00 28.86 0.0001
Psychrobacter 10.60 6.46 9.39 0.1998
Pseudomonas 3.35 3.18 4.63 0.1873
Bifidobacterium 3.89 2.89 3.72 0.6543
unclassified_f__Enterobacteriaceae 0.51 8.59 0.50 0.0047
Acinetobacter 4.67 3.21 0.41 0.0171
Zymobacter 1.01 5.06 0.00 0.0026
Carnimonas 1.28 4.59 0.03 0.0000
Escherichia-Shigella 1.15 2.37 1.49 0.0263
Rosenbergiella 0.22 4.19 0.39 0.0471
Sphingomonas 1.68 1.24 0.72 0.0023
unclassified_f__Moraxellaceae 3.42 0.03 0.01 0.3609
Rhodococcus 1.10 0.81 0.63 0.0695

In addition, we found greater variation in the
interspecific community composition in T. pagdeni,
with the presence of two gut ecotypes dominated by
Snodgrassella or Acetobacter-like bacteria. This also
occurred in the two other stingless bee communities
(Fig. 1b). This suggests that the microbial composi-
tion varies considerably between different colonies of
the same stingless bee species.
Bacterial diversity of the stingless bee gut
The ACE index, Chao index, Sobs index, Shannon
index, Simpson index and PD index were used to
evaluate the alpha diversity of the gut microflora of the
three kinds of stingless bees. Through the Kruskal–
Wallis test, we found that there were significant
differences in the Chao, ACE and Sobs indexes among
the three groups of samples (Fig. 2a, b, c), while the
Shannon, Simpson and PD indexes were not signifi-
cantly different (Figure S2). The results showed that
the community richness of the gut bacteria of the three
bee species were different, while the community
diversities were not significantly different. The P-
values of diversity index significance were as follows:
ACE, P = 0.0013; Chao, P = 0.0025; Sobs,
P = 0.0094; Shannon, P = 0.1838; Simpson,
P = 0.1637; and PD, P = 0.6198 (Table S1).
PCoA (based on binary Jaccard distance) and PLS-
DA were used to evaluate the microbial community
diversity within the three species. Through PCoA, we
found that the three species of stingless bees were
divided into three clusters (Fig. 2d), and the host
species could be clearly distinguished by the commu-
nity diversity with statistical significance (P = 0.001,
ANOSIM; P = 0.001, PERMANOVA, 999 permuta-
tions in each test). PLS-DA analysis could also
effectively distinguish host species (Fig. 2e). These
analyses indicated that the gut microbe structures are
distinguishable among the three host species.
Composition of bacteria in beebread
We analysed the bacterial community composition of
beebread for stingless bees at the phylum and genus
levels. We found that the most abundant phylum in
beebread of five colonies of T. pagdeni samples was
Firmicutes (64.64%), followed by Proteobacteria
(21.93%), Bacteroidetes (6.71%) and Actinobacteria
(6.12%) (Fig. 3a). At the genus level, Lactobacillus
was the most abundant bacterial genus, accounting for
39.94% to 92.26% of the total population, followed by
a small population of the genus Carnimonas (1.20% to
15.18%). Lactobacillus, Carnimonas, Escherichia-
Shigella and Acinetobacter were found in all beebread
samples (Fig. 3b).

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Antonie van Leeuwenhoek (2021) 114:1293–1305 1299

A B C 240
** 300 ** **

Chao index of OTU level

Ace index of OTU level

280

Sob index of OTU level

180
200
200

100 100
120

40 0 20
ata ventralis i ata ventralis gden
i ata ventralis gden
i
L.termin L. T. pa
gden L.termin L. T. pa L.termin L. T. pa
Group name Group name Group name

D PCoA on OTU level E PLS-DA on OTU level

L.terminata
16
0.4 L.terminata L. ventralis
L. ventralis 12 T. pagdeni
T. pagdeni

COMP2(6.06%)
8
PC2(6.38%)

0.2
4

0
0
-4

-8
-0.2 -12
-0.2 0 0.2 0.4 -12 -8 -4 0 4 8
PC1(12.55%) COMP1(8.63%)

Fig. 2 Alpha (a, b, c) and beta (d, e) diversity of the gut
microbiota of stingless bees. a ACE index. b Chao index. c Sobs
index. 0.01 \ P B 0.05 is marked as *, and 0.001 \ P B 0.01
is marked as **. d Principal coordinates analysis (PCoA, based
on binary Jaccard distance). e Partial least squares discriminant
analysis (PLS-DA) of stingless bee gut microbiome samples.
Each point represents an individual sample, and clustering of
points means similarity in operational taxonomic units (OTUs)
among the samples. Samples belonging to the same biological
replicate are represented by the same colour

Discussion The dominant microbiota remained in relatively

high abundance in the three stingless bee species and
Microbial diversity among and within bee species may play an important role within stingless bees. At
present, other studies have found certain dominant
We collected L. terminata, L. ventralis and T. pagdeni bacterial members of the stingless bee gut to be critical
in tropical regions of China and then analysed the in host immunity and digestion (Emery et al. 2017;
composition of their gut-dominant bacteria by high- Zheng et al. 2019). For example, Alberoni et al. (2018)
throughput sequencing. We found that there were sprayed Lactobacillus and Bifidobacteria in honeybee
dominant microbiota in the gut of stingless bees (the colonies and subsequently found significantly
set of bacterial species that is present in most members increased brood populations and honey production.
of a host species), represented by members of the Zheng et al. (2019) found that Bifidobacteria were
Acetobacter-like, Snodgrassella, Lactobacillus, Psy- among the major degraders of hemicellulose and
chrobacter, Pseudomonas and Bifidobacterium gen- pectin in the A. mellifera gut. Moreover, Daisley et al.
era; our results are similar to the reports by Leonhardt (2020) found that Lactobacillus attenuates antibiotic-
and Kaltenpoth (2014) and Koch et al. (2013). induced immune and microbiota dysregulation in
honeybees. These results suggest that there may be a

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1300
A 100 B 100
Firmicutes
Relative abundance on Genus level (%)
Proteobacteria
80 Bacteroidetes
Actinobacteria
60 others
40
20
0 0
Tp_B
Fig. 3 a The proportion graph of total bacterial phyla among
five beebread samples of T. pagdeni. b Distribution of beebread
bacteria at the genus level among five beebread samples of T.
pagdeni. Distribution of gut bacteria at the phylum and genus
relationship between these two bacterial genera and
the nutrient uptake of stingless bees. Lactobacillus
spp. are important probiotics (Anderson et al. 2011;
Vasquez et al. 2012), and they can secrete bacterio-
static substances (bacteriocins and lactic acid) to
protect their hosts (De Keersmaecker et al. 2006; Li
et al. 2021). Recently, Suphaphimol et al. (2020)
isolated lactic acid bacteria from the gut of L.
terminata that were antagonistic to pathogens of
honeybees (Paenibacillus larvae). Therefore, Lacto-
bacillus also plays a key role in the health of stingless
bees. Snodgrassella has been found to play a crucial
role in metabolism and immunity in other social bees.
Snodgrassella can help bumblebees resist the invasion
of pathogens (Koch and Schmid-Hempel 2011), and it
also leads to significant increases in intracellular
transport, secretion, vesicular transport and cell
movement of the honeybee (Engel et al. 2012).
Snodgrassella is the dominant genus of gut bacteria
in honeybees, bumblebees and stingless bees. There-
fore, it may play the same role in other bees as it does
in stingless bees. However, Pseudomonas found in the
gut of stingless bees is a common pathogen (P.
aeruginosa) (Behzadi et al. 2021), but its role in
stingless bees has not been reported and needs further
study. Kwong et al. (2017) found that Acetobacter-like
bacteria were present mainly in stingless bees, while
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Antonie van Leeuwenhoek (2021) 114:1293–1305
Lactobacilus
Carnimonas
Vibrionimonas
80 Escherichia-Shigella
Acinetobacter
unclassified_f_Acetobacteraceae
Bacteroides
60 Rhodococcus
Propionibacterium
Sphingomonas
40 norank_f_Mtochondria
Psyclrobacter
unclassified_f_Propionibacteriaceae
unclassified_o_Mcrococcales
20 Coynebacterium
Faecalibacterium
others
B1 p_B2 p_B3 p_B4 p_B5
Tp_ T T T T
levels among beebread samples of T. pagdeni (Tp_B). Columns
of different colours represent different phyla and genera, and the
length of the column represents the relative proportion
this genus was not found in other social bees.
However, little has been reported about the role
played by the dominant gut bacteria in stingless bees.
Therefore, the role of the dominant microbiota in
stingless bees remains to be revealed.
While the three stingless bee species shared some
dominant microbiota, the structures of their gut
bacteria differed. These results are concordant across
two different metrics (binary Jaccard distance and
PLS-DA analysis). In addition, there were no differ-
ences in the microbial community diversity (Shannon,
Simpson and PD diversity indexes) between the three
species of stingless bees, but there were significant
differences in community richness (Chao, ACE and
Sobs diversity indexes). Overall, these results suggest
that the relative abundances of gut-dominant bacteria
of different species of stingless bees were different.
Such results were also found in bumblebees (Suenami
et al. 2019), Megalopta (McFrederick et al. 2014) and
A. mellifera (Kwong et al. 2017).
Moreover, we found that the gut bacterial structures
of L. terminus and L. ventralis were more similar to
each other than to that of T. pagdeni (Fig. 2d). Kwong
et al. (2017) postulated that the microbiota of social
corbiculate bees undergoes dynamic evolution and
will change over time. That is, the closer the evolu-
tionary relationship is, the more similar the
Antonie van Leeuwenhoek (2021) 114:1293–1305
composition of the microbial community will be. L.
terminus and L. ventralis are both members of the
genus Lepidotrigona, while T. pagdeni is a member of
the genus Tetragonula. The two genera fall within
different phylogenetic clades that diverged approxi-
mately 65 million years ago (Rasmussen and Cameron
2010). Thus, the similarity in the structures of the L.
terminus and L. ventralis gut bacteria may be the result
of the dynamic evolution of the gut bacteria.
Food can influence the structure of the host’s gut
microbiota (Maes et al. 2016; Powell et al. 2014;
Rothman et al. 2018). For example, Rothman et al.
found that supplemental floral forage can alter the gut
microbiota of A. mellifera (Rothman et al. 2018).
Therefore, collecting nectar/pollen from different
plants may be an important reason for the difference
in gut bacteria abundances among the three species of
stingless bees. According to relevant reports, the
bacterial community in floral nectar is affected by
honeybees that visit the flowers, and honeybees may
acquire bacteria from the nectar (Aizenberg-Gershtein
et al. 2013). Mcfrederick et al. (2017) found that the
same taxa (OTUs) of Lactobacillus in the gut of
Megachilid bees were also found in flowers. In
addition, Lactobacillus, Pseudomonas, Acinetobacter,
Rosenbergiella, Sphingomonas and acetic acid bacte-
ria found in the gut of stingless bees are also found in
flowers (Jojima et al. 2004; Vannette 2020). Interest-
ingly, to ensure coexistence between different species,
stingless bees have evolved several ways of sharing
competing resources (Keppner and Jarau 2016). For
example, different stingless bee species can share
resources within a habitat using different nectar/pollen
plant species (Eltz et al. 2001; Leonhardt et al. 2014),
as well as by their preferences for resources at
different heights in the vegetation (Biesmeijer et al.
1999; Nagamitsu and Inoue 1997; Ramalho 2004) and
by preferring rainfall or low light intensity to forage
(Keppner and Jarau 2016). Different plants have
different bacteria (Vannette 2020), and certain com-
ponents of nectar can alter the gut bacteria of bees (Ma
et al. 2020). Therefore, these three species of stingless
bees may have different gut bacteria due to their
visiting of different plants.
Our study also found that within the same species of
stingless bees, there were large differences in the
compositions of the different communities (Fig. 1b).
In particular, in T. pagdeni, the gut bacteria were
dominated by Acetobacter-like or Snodgrassella.
1301
Smith et al. (2017) found that the typical and
maximum homing ranges of T. carbonaria were
333 m and 712 m, respectively. Therefore, the differ-
ences in community composition may also be a result
of the preferred food. Ma et al. found a significant
selected enrichment in the abundance of Snodgras-
sella in the midgut and hindgut of A. mellifera after
feeding on date jujube nectar (Ma et al. 2020). In
addition, different worker bees may have acquired
plant pollen or nectar contaminated pesticides or
heavy metals that can affect the structure of the gut
microbiota (Motta et al. 2020; Rothman et al. 2019),
thus causing differences in the bacteria of different
colonies of stingless bees. Age may be another
important factor in within-species variation in the
gut bacteria of stingless bees. We collected bees
emerging from the entrance of the hive; however,
stingless bees generally fly out of the hive to collect
pollen and nectar between 9 and 30 day after they
emerge due to age (Calderone 1998; Dong et al. 2020),
so the ages of the stingless bees collected may not have
been consistent. Dong et al. (2020, 2021) found that
the different developmental stages of A. mellifera and
A. cerana differed significantly in their compositions
of gut microbes, which changed consistently from
birth to adulthood to the ageing host. In addition, the
influence of genetic differences between different
stingless bee colonies on the colony cannot be ignored.
(McFrederick et al. 2012; Tarpy and Nielsen 2002).
Dominant beebread bacteria of stingless bees
Unlike the gut bacteria of stingless bees, we found a
different core bacterial composition in the beebread of
stingless bees. Lactobacillus, Carnimonas, Escher-
ichia-Shigella and Acinetobacter were the dominant
bacteria in the beebread of T. pagdeni. Lactobacillus
was the most abundant bacterial genus in the beebread
of T. pagdeni, which may be related to the fermenta-
tion of pollen (Ngalimat et al. 2020). Lactic acid
bacteria transform sugar in pollen into water and lactic
acid by lactic fermentation (Audisio et al. 2011), and
this fermentation results in beebread (Detry et al.
2020). This also shows that the beebread samples we
collected were about to mature or had already
matured. In addition, when bees collect and store
pollen, they will mix the pollen with secretions
containing their own beneficial microorganisms
(Detry et al. 2020). Therefore, lactic acid bacteria in
123
1302
beebread may be added by stingless bees when
collecting and storing pollen. Interestingly, we found
the presence of Escherichia-Shigella in the beebread
of five colonies of T. pagdeni, which may relate to the
behaviour of stingless bees in collecting soil or animal
faeces (Veen 2014), resulting in the presence of
Escherichia-Shigella in beebread.
Conclusions
We compared the gut microbiota of three common
stingless bee species in China. The bees a group of
dominant bacteria, including Acetobacter-like, Snod-
grassella, Lactobacillus, Psychrobacter, Pseu-
domonas and Bifidobacterium. Although a dominant
set of microbiota existed in the three stingless bee
species, the structures of the gut bacteria were
different among these three species. In addition, the
abundances of the gut bacteria varied between com-
munities in the same bee species. Food differences
may be a common cause of intra- and interspecific
variation, while age and genetic differences may also
contribute to intra-species variation. Moreover, we
investigated the bacterial composition of beebread
from five colonies of T. pagdeni and found that all
samples of beebread contained Lactobacillus, Carn-
imonas, Escherichia-Shigella and Acinetobacter. The
highest abundance of Lactobacillus may be associated
with beebread fermentation. In brief, our work
provides a basis for exploring the composition and
structure of the gut and beebread bacteria of stingless
bees.
Acknowledgements We are very grateful to the Professor Xu
Huanli of China Agricultural University for providing the
morphological identification of the stingless bees.
Authors’ contributions Q-HT, Z-XD and JG designed the
study; Z-WW, C-HM and JG provided samples; Q-HT, Y-FC
and ZC performed research; Q-HT, Y-FC, S-QL and J-XW
analysed data; C-HM and JG contributed new methods or
models; Q-HT, Y-FC, Z-WW and JG wrote the paper; JG
received the funding.
Funding This study was supported by the Natural Science
Foundation of China (31660695).
Availability of data and material The raw data were
deposited in the NCBI Short Read Archive (SRA) database
with the sample accession number of SRR13249848-
SRR13249885 and SRR13263054-SRR13263058.
123
Antonie van Leeuwenhoek (2021) 114:1293–1305
Declarations
Conflict of interest There are no conflict of interests.
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