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Stingless bees and microbial interactions
Gabriela Toninato de Paula1, Cristiano Menezes2,
Mônica Tallarico Pupo1 and Carlos Augusto Rosa3
Stingless bees (Meliponini) are a monophyletic group of
eusocial insects inhabiting tropical and subtropical regions.
These insects represent the most abundant and diversified
group of corbiculate bees. Meliponini mostly rely on
fermentation by symbiont microbes to preserve honey and
transform pollen in stored food. Bee nests harbor diverse
microbiota that includes bacteria, yeasts, filamentous fungi,
and viruses. These microorganisms may interact with the bees
through symbiotic relationships, or they may act as food for the
insects, or produce biomolecules that aid in the
biotransformation of bee products, such as honey and bee
bread. Certain microbial species can also produce
antimicrobial compounds that inhibit opportunistic bee
pathogens.
Addresses
1
Departamento de Ciências Farmacêuticas, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-
903 Ribeirão Preto, SP, Brazil
2
Brazilian Agricultural Research Corporation, Embrapa Meio Ambiente,
Jaguariúna, SP, Brazil
3
Departamento de Microbiologia, ICB, C.P. 486, Universidade Federal
de Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
Corresponding author: Rosa, Carlos Augusto (carlrosa@icb.ufmg.br)
Current Opinion in Insect Science 2021, 44:41–47
This review comes from a themed issue on Ecology
Edited by Rachel Vannette and Robert R Junker
https://doi.org/10.1016/j.cois.2020.11.006
2214-5745/ã 2020 Elsevier Inc. All rights reserved.
Introduction
Meliponini (stingless bees) represents a monophyletic
group of eusocial insects that belongs to a larger group
known as the corbiculate bees (Hymenoptera: Apidae)
that includes honeybees (Apini), bumble bees (Bombini),
and orchid bees (Euglossini) [1]. Meliponini are found in
all tropical and subtropical regions of the world. As their
name suggests, all species of this group share a peculiar
characteristic among bees, specifically the absence of a
functional sting. Although Meliponini encompass a
diverse group with approximately 550 species and 61 gen-
era, a number of features are common to most species
[2,3]. Nearly all Meliponini species live in perennial nests
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that are typically located inside cavities sealed with plant
resins and beeswax, and these species store food over long
periods of time and proliferate by swarming, with transfer
of nest materials from existing (mother) to newly founded
(daughter) colonies [2]. These attributes, in addition to
the high humidity of tropical environments, have favored
the establishment of numerous microbial interactions in
stingless bees throughout their natural history, as we
discuss below.
A major challenge that these bees had to overcome during
their evolution was to develop a strategy by which food,
including nectar and pollen, could be stored for extended
durations. Even in tropical environments, these resources
are not available throughout the year due to seasonal
patterns in plant flowering [4]. When food resources are
scarce, honey bees abandon their nests and migrate to
areas of greater resource abundance. This behavior is rare
in stingless bees, and they have developed strategies to
sustain their colonies during periods of food scarcity [5].
Therefore, to survive during periods of low resource
availability, colonies maximize foraging during the peak
flowering season and store collected resources for later
consumption [4–6]. Some species diminish their foraging
activity and downregulate their brood production to the
minimum level necessary for colony maintenance, and
these changes in brood cell production are primarily
associated with the quantity of stored pollen [5]. Other
species inhabiting the regions with pronounced seasonal
changes decrease or even interrupt their brood production
during adverse climatic conditions, and their reproductive
diapause is triggered by abiotic environmental factors [5].
While Apini use dehydration techniques to preserve
honey, Meliponini mostly rely on fermentation by sym-
biont microbes [7]. Although Meliponini also dehydrate
nectar up to a certain level of humidity, it is extremely
difficult to attain high sugar concentrations in humid
environments, particularly in cavities. Consequently,
sugar concentrations of stingless bee honey generally
range from 60% to 75% (min. 55%; max 86%) [8]. This
makes it a suitable growth medium for many types of
microbes.
Pollen is also fermented following storage, and this pro-
cess is characterized by an increase in moisture and lactic
acid, and also by increased proliferation of microorgan-
isms [9]. It has been speculated that stingless bees prefer
fermented pollen over fresh pollen [9]. This hypothesis
was tested using experiments examining Scaptotrigona aff.
Current Opinion in Insect Science 2021, 44:41–47
42 Ecology
depilis in cages, and it was found that more bees fed on
fermented pollen, and this type of pollen was consumed
at a 2.2-fold higher rate than the fresh pollen [9]. In
contrast, Carrol et al. [10] reported that honey bees
exhibited a marked preference for the consumption of
freshly stored pollen. Foragers of stingless bees use honey
to carry pollen grains on their hind legs. When large
pellets are formed on both legs, they return to the nest
and deposit the collected pollen inside food pots that are
closed once they are entirely filled. A food pot is thus, a
perfect environment for microbial proliferation, as it is
warm, humid, and nutritious [11]. It may also be difficult
for stingless bees to avoid extensive interactions with
microbes on the carried pollen. Given their advantages,
these interactions may have been conserved over time.
Another important feature of almost all stingless bees is
their use of plant resins in nest building. While their sister
group (Apini) uses pure wax for brood and food combs,
Meliponini mix resins with wax to produce their primary
building material, known as cerumen, and this material is
used in all structures of the nest, including brood cells and
food pots [2,12]. Plant resins are well-known for their
potent antimicrobial properties [13]. This strategy may
have evolved in response to pressure from opportunistic
microbes that could quickly exploit the favorable condi-
tions present inside stingless bee nests. Although little is
known regarding the properties of stingless bee propolis,
examples from other social insects suggest that there is a
great potential of revealing interesting findings regarding
this issue in stingless bees. For example, wood ants
(Formica paralugubris) collect pieces of solidified resin
from coniferous trees that they then mix with their nest
material, and this strongly improves the survival of adults
and larvae exposed to pathogens [14]. In honeybees (Apis
mellifera), the collection and in-hive use of plant resins is
an important mechanism of social immunity, which pro-
tects the colony from pathogens [15] and stabilizes the
honeybee microbiome [16]. Future studies examining
this subject should include species from Malaysia, known
as ‘crystal bees’ (Figure 1), that almost exclusively use
resins to build their nests and yield extreme levels of
fermentation in stored foods [17].
Finally, the reproductive system of stingless bees, where
the daughter colonies are connected to their mothers for
many days or even weeks, are believed to have favored
the stabilization of beneficial relationships between bees
and microbes across generations [18]. As workers trans-
port building materials and stored food from the mother
to the daughter colony during the swarming process, it is
highly likely that the microbes present in the mother
colony would be transported to the newly founded daugh-
ter colony. In this manner, any new beneficial interaction
that emerged would be replicated in the next generation.
This is also an interesting topic for future research, as it
remains unclear if Meliponini bees actively transfer their
Current Opinion in Insect Science 2021, 44:41–47
beneficial microbes vertically in a manner similar to that
of fungus-growing (attine) ants [19], or if they acquire
these microbes from the environment when establishing a
new colony in a manner similar to that of most species of
macrotermitini termites [20].
The microbiota associated with stingless bees
The microbiota associated with stingless bee nests
includes viruses, bacteria, yeasts, and filamentous fungi.
Bacteria and yeasts are metabolically active in bee hives,
and their physiological traits, including enzyme produc-
tion, sugar fermentation, and organic acid production, are
be instrumental in the microbial transformation of pollen
and nectar by initiating biochemical changes that may
provide nutritional benefits to the larvae and adult bees
[8,21,22,24,25]. The beneficial interactions among
stingless bees and viruses or filamentous fungi remain
poorly studied, although these microorganisms are the
opportunistic pathogens that are most likely associated
with disease symptoms in these bees.
Bacterial symbionts and their functions
The major bacterial genera associated with stingless bees
are Lactobacillus, Bacillus, Streptomyces, Clostridium, Staph-
ylococcus, Streptococcus, Enterobacter, Ralstonia, Pantoea,
Pseudomonas, Fructobacillus, Lysinibacillus, and Neisseria
[21,22,23,24]. Kwong et al. [18] reported that the sting-
less bee microbiome appears to be highly variable in
composition, richness, and evenness in comparison to
those of Apis and Bombus. These authors demonstrated
that Gilliamella, Bifidobacterium, Lactobacillus Firm-4, and
Lactobacillus Firm-5 are prevalent in these three bee
groups, suggesting that these taxa comprise the core of
the corbiculate gut microbiome. The phylotype identified
as Acetobacter-like appears to be specific to stingless bees
[18]. Ngalimat et al. [22] reported that bacterial isolates
associated with the Malaysian stingless bee Heterotrigona
itama were capable of utilizing carbohydrates, amino
acids, carboxylic acids, and various derivatives. Certain
isolates of Bacillus species exhibit proteolytic, lipolytic,
and cellulolytic activities. The enzymes produced by
these bacterial isolates may be involved in the breakdown
of carbohydrates and proteins present in nectar and
pollen, which helps to form nest products [22]. These
enzymatic activities may play a key role in improving the
nutritional value of the nest products utilized as food by
the stingless bees. Although several bacterial species
were associated with stingless bee nests, the biological
interactions between them are still unclear.
Bacterial species associated with Meliponini nests have
also been reported as producers of antimicrobial com-
pounds [23,26]. Streptomyces sp. ICBG1323, and Micromo-
nospora sp. ICBG1321, also isolated from Melipona scu-
tellaris, produced polyketides (lobophorins and
anthracyclines) that show variable levels of activity
against the entomopathogen Paenibacillus larvae, the
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 Microorganisms associated with stingless bees de Paula et al. 43

Figure 1

(a) (b)

(c) (d)

(e)
Current Opinion in Insect Science

Crystal bees from Malaysia, Tetrigona binghami (a). These bees have received this name because of the characteristics of their nests, built almost
exclusively with dipterocarp resin (c), including the pollen pots (b) and honey pots (d). Honey fermentation is so extreme that it makes the honey
taste like vinegar (e). (Photos: Cristiano Menezes).

causative agent of the fatal honey bee disease American
foulbrood; however, this pathogen has not previously
been detected in stingless bee samples [23,27]. One
isolate of Paenibacillus polymyxa obtained from the larval
food of M. scutellaris produced (L)-(-)-3-phenyllactic acid
and fusaricidins that were active against entomopatho-
genic fungi and P. larvae. Streptomyces sp. ICBG1318
isolated from M. scutellaris produce two novel cyclic
hexadepsipeptides (meliponamycin A and meliponamy-
cin B) that exhibit strong activity against P. larvae [26].
These results indicate that the antimicrobial compounds
produced by the microorganisms associated with stingless
bees could protect their colonies against opportunistic
pathogens. Several species of lactic acid bacteria and
Bacillus exhibit probiotic characteristics, thus, suggesting
their therapeutic potential [28,29]. However, it would be
interesting to study the bioactive metabolites produced
by these bacteria, as they may benefit bee health.
Yeasts associated with stingless bees and their
functions
The yeast species most frequently isolated from stingless
bees belong to the genus Starmerella. This genus includes
approximately 50 species, most of which have been
described to be associated with bee species and their
floral niches [30,31,32]. Gonçalves et al. [31] suggested
that the genus Starmerella is adapted to the floral niche
and associated insects, and many species exhibit unusual

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44 Ecology
metabolic traits such as fructophily and the production of
sophorolipids. Starmerella species are found primarily in
the neotropics, and most of them seem to be restricted to
certain regions possibly following the geographic distri-
bution of their insect vectors [25,30,32]. Starmerella bom-
bicola, Starmerella batistae, Starmerella riodocensis, and Star-
merella cellae are mostly associated with solitary bees,
while Starmerella meliponinorum, S.tarmerella apicola, Star-
merella neotropicalis, and Starmerella etchellsii are mostly
associated with stingless bees [25,30,32,33]. These latter
species are frequently isolated from pollen, nectar, unripe
and ripe honey, garbage pellets, and adult stingless bees.
Certain species, in association with bacteria, ferment the
nectar collected by bees [25,30,32]. Starmerella species
have also been isolated from honeybees and bumble bees
[34,35]. However, the beneficial effects of these yeasts for
bees remain to be elucidated.
Several species of the osmophilic yeasts of the genus
Zygosaccharomyces have been found to be associated with
different aspects of honey bee and stingless bee colonies,
including those of Zygosaccharomyces gambellarensis, Zygo-
saccharomyces mellis, and Zygosaccharomyces siamensis
[36,37]. Zygosaccharomyces strains appear to be involved
in the spoilage of food in the hive; however, Z. siamensis
was also recently associated with the honey fermentation
process in Apis cerana [36]. Zygosaccharomyces machadoi was
described in association with garbage pellets of the sting-
less bee Tetragonisca angustula in Brazil [38]. Z. osmophilus
was obtained from different samples of ripe and unripe
honey, and pollen from Scaptotrigona crf. bipunctata and
also from the ripe honey of T. angustula. Honey has been
suggested as the natural habitat of Zygosaccharomyces
osmophilus due to its high abundance in this substrate [39].
Despite the reports of Zygosaccharomyces strains in bee
colonies, including stingless bees, honeybees and bumble
bees [35,36,38,39], little is known about their ecological
functions and their benefits to the host bees. A more
comprehensive description of the molecular mechanisms
involved in nutritional/hormonal symbiosis has been
reported in the interaction between a novel species of
Zygosaccharomyces (without formal description) and the
stingless bee Scaptotrigona depilis (Figure 2). This fungus
grows inside the brood cells of S. depilis, and its ingestion
by the larvae is essential for their survival and metamor-
phosis [40,41]. Metamorphosis in insects is controlled by
a balance between two hormonal systems that include
juvenile hormones and ecdysteroids [42]. Insects
acquire steroidal compounds from their diet as precursors
for the biosynthesis of ecdysteroids, as they do not syn-
thesize these hormones de novo. Zygosaccharomyces sp.
associated with S. depilis was found to accumulate the
steroid ergosterol in lipid droplets in its cytoplasm. In vitro
experiments examining S. depilis larval development
revealed that larvae that were fed food supplied with
ergosterol could complete metamorphosis at a rate similar
Current Opinion in Insect Science 2021, 44:41–47
to that observed when they ingested larval food supplied
with Zygosaccharomyces sp., thus, revealing the molecular
mechanism underlying this symbiotic interaction [41].
The ecdysteroid 24-epi-makisterone A was detected in
the pupae, suggesting its production from ergosterol
during the metamorphic process of S. depilis [41]. The
modulation of Zygosaccharomyces sp. growth in S. depilis
colonies appears to be partially regulated by small mole-
cules produced by two additional microorganisms isolated
from the cerumen, Candida sp. (possibly a novel Starmer-
ella species), and Monascus ruber [40,41]. This Candida sp.
produces the volatiles ethanol and isoamyl alcohol that
stimulate Zygosaccharomyces sp. growth. In contrast, M.
ruber produces lovastatin, an inhibitor of steroid biosyn-
thesis, and monascin. Lovastatin inhibits the growth of
Zygosaccharomyces sp., whereas monascin inhibits Candida
sp. growth [41]. These results, not previously reported in
the literature, demonstrate the intricate relationship
between the microbiota of the brood cell and the stingless
bee S. depilis. The occurrence of similar symbiosis
between Zygosaccharomyces and other species of stingless
bees is a plausible hypothesis that is worthy of further
investigation.
The origin and life cycle of Zygosaccharomyces sp. strains in
bee colonies remain to be described. Interestingly, A.
mellifera queens harbor larger quantities of Zygosacchar-
omyces in the gut and ovaries compared to that in the
workers [43], thus, providing opportunities for similar
investigations in stingless bees. Further studies are
required to better understand the role of Zygosaccharo-
myces in host insects.
Filamentous fungi and their interactions with stingless
bees
Filamentous fungi associated with stingless bees and
their hives typically possess a saprophytic lifestyle. Some
species of these fungi have also been reported as oppor-
tunistic pathogens of many bee species. Barbosa et al. [44]
reported Penicillium and Talaromyces species from honey,
pollen, and the interior of nests of M. scutellaris. Twenty-
one species were identified in Penicillium and six in
Talaromyces, including four novel species of Penicillium
and three of Talaromyces. These authors verified that
these species produced bioactive extrolites; however,
the benefits of such compounds to their hosts were not
determined. Menezes et al. [45] reported, for the first
time, a mutualism between a fungal species, identified as
Monoascus sp., and the bee Scaptotrigona postica. The
authors demonstrated that the fungal mycelia growing
on the provisioned food within the brood cells is essential
for the larval development of this bee. Additionally,
fungal spores could also be utilized as a food resource
by stingless bees, although the nutritional value of these
spores is lower than that of pollen [44]. The beneficial
relationships of these microorganisms with stingless bees
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 Microorganisms associated with stingless bees de Paula et al. 45
Figure 2
(a)
The Brazilian stingless bee Scaptotrigona depilis. (a) Egg in the brood cell; (b) Zygosaccharomyces sp. growth in the borders of the brood cell
(white mass) and larva in the center. (Photos: Cristiano Menezes).
and the species richness of these fungi that are associated
with these insects both remain poorly studied.
Viruses associated with stingless bees and their
functions
Viruses that infect stingless bees have also been poorly
studied. Viruses found within stingless bees include acute
bee paralysis virus (ABPV), deformed wing virus (DWV),
and black queen cell virus (BQCV) [46–48]. Caesar et al.
[46] characterized the Melipona quadrifasciata virome
using high-throughput sequencing in an attempt to iden-
tify potentially pathogenic viruses. A total of 40 621 reads
were mapped to viral contigs of the metagenomes of
unhealthy bees, while only 11 reads were mapped to
contigs identified as viruses of healthy bees. The families
Dicistroviridae, Parvoviridae, and Circoviridae were pres-
ent in the largest copy numbers in the unhealthy bees.
These authors call attention to the presence of a diversity
of as yet unknown bee viruses in stingless bees. Using
PCR analyses, Guimaraes-Cestaro et al. [47] reported the
presence of viruses in samples from ten nests of stingless
bees (Nannotrigona testaceicornis, T. angustula, and Tetra-
gona elongata). ABPV was the most prevalent, followed by
DWV and BQCV. Teixeira et al. [48] studied the diseased
colonies of Melipona spp. in two Brazilian states, and the
authors did not detect viruses in any of the colonies
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(b)
Current Opinion in Insect Science
sampled. These studies focused on the occurrence of
viruses responsible for diseases associated with stingless
bee colonies. However, other viruses with beneficial
properties may be associated with these bees. Bonilla-
Rosso et al. [49] studied the phages associated with the
gut microbiome of A. mellifera using metagenomic
sequencing. The majority of the phages found in the
bee guts in this study were considered unclassified and
represented novel viruses. Similar findings could be
obtained for the gut microbiome of stingless bees.
Conclusion
The microbiota associated with stingless bees confers
advantages to these insects by promoting the microbial
transformation of stored food. The fermentation capabili-
ties, associated with the production of enzymes, organic
acids, antimicrobial substances, and steroids of yeasts and
bacteria isolated from stingless bee products and their
nests suggest that these microorganisms are essential to
the life cycle of these insects. Bacteria, yeasts, and spores
of filamentous fungi may also be considered food supplies
for larvae and adults. These microorganisms represent a
source of concentrated proteins and vitamins that are
essential for insects. The interaction between the yeast
Zygosaccharomyces sp. and the stingless bee S. depilis sug-
gest a nutritional/hormonal symbiosis that is partially
Current Opinion in Insect Science 2021, 44:41–47
46 Ecology
controlled by small molecules produced by Candida sp.
and M. ruber, and highlights the importance of studies
related to the relationship between stingless bees and
their associated microbiota. Zygosaccharomyces and Star-
merella species are frequently isolated from stingless bee
nests, and it is possible that other bee species exhibit
similar symbioses.
Conflict of interest statement
Nothing declared.
CRediT authorship contribution statement
Gabriela Toninato de Paula: Conceptualization, Writing -
original draft. Cristiano Menezes: Conceptualization,
Writing - review & editing. Mônica Tallarico Pupo:
Conceptualization, Writing - review & editing, Supervi-
sion. Carlos Augusto Rosa: Conceptualization, Writing -
original draft, Writing - review & editing.
Acknowledgements
MTP was funded by São Paulo State Foundation (FAPESP) [grants #2013/
50954-0 (MTP) and #2018/03650-0 (GTP)], Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico – Brasil (CNPq) [grant #303792/
2018-2 (MTP]), and Coordenação de Aperfeiçoamento de Pessoal de Nı́vel
Superior (CAPES) – Finance Code 001. CAR was funded by Conselho
Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq – Brazil,
process numbers 407415/2013-1, 0457499/2014-1 and 435040/2018-9), and
Fundação do Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG,
process numbers APQ-01525-14, APQ-01477-13, and APQ-02552-15).
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