Discussion
In this study, we provide evidence for differentiation in compositional
profile of honeys produced by stingless bees and honey bees at an
unprecedented spatial scale using advanced analytical and chemometric
methods. Our results highlight that fermentation markers and sugars are
the main compounds explaining the pattern observed between stingless bee
and honey bee honeys, which supports recent studies (Muhammad & Sarbon,
2021; Gadge et al., 2024). The transformation of sugars into acids,
ethanol and a variety of secondary metabolites during the fermentation
process that occurs in SBH is a major driver of increased chemical
composition diversity. These key differences lies in the transformation
of sugars into acetic acid and lactic acid performed by bacteria,
including lactic acid bacteria (LAB) and Bacillus spp. found in
SBH (Menezes et al., 2013; Ngalimat et al., 2020). Our analysis of
chemodiversity show that compared to honey bee honey, SBH had not only a
higher compound richness but also a higher structural diversity of
compounds (i.e functional chemodiversity). The simultaneous presence of
a greater number of compounds with different biochemical properties
fosters synergetic actions among them (Whitehead et al., 2021; Petrén et
al., 2023), presumably conferring significant reported therapeutic
benefits of SBH for both the bee colony and human health (Rao et al.,
2016; Berenbaum & Calla, 2021; Héger et al., 2023). For instance,
organic acids produced during fermentation result in increased
antimicrobial properties of honey (e.g. formic and lactic acid; Raftari
et al., 2012) while coupling with the effects of compounds naturally
present in nectar, including amino acids (e. g. phenylalanine which has
known neuroprotective effects (Zamri et al., 2023)) and organic acids
with metal-chelating antioxidant activity (Viuda-Martos et al., 2008).
Some notable examples include kynurenic acid, a metabolite known for its
anti-inflammatory and antioxidative activity (Turska et al., 2022), that
was only identified in SBH from the Afrotropics (25-198 mg/kg) in
proportions approaching levels of chestnut honey (305 mg/kg in the
chestnut honey sample from Turkey in our dataset), one of the main food
enriched in kynurenic acid (Turski et al., 2016). Neotropical SBH were
associated to methylglyoxal (0-30 mg/kg), a key compound of the medical
grade Manuka (Leptospermum scoparium ) honey bee honey (400+
mg/kg) known worldwide for its exceptionally high levels of
antibacterial activity (Adams et al., 2009). These results show how the
shared use of SBH by bees and microorganisms may increase both their
fitness by protecting the colony against microbial pathogens and toxins
while providing propitious and controlled environment for symbiotic
microorganisms (Menezes et al., 2013; Fernandes et al., 2022), following
theoretical research on extended fitness hypothesis (de Araújo et al.,
2021).
Our study illustrates a significant magnitude of variation in the
compositional profiles of SBH, driven by a range of compounds including
sugars, amino acids and fermentation markers. Unexpectedly, our analyses
did not allow to pinpoint a clear ecological, evolutionary or
geographical driver explaining these differences in chemical properties
among stingless bee honeys. SBH from the Afrotropics tend to be more
similar to honeys from Central and South America than honeys from the
other tropical regions of the Old world, even though global phylogeny
suggests a division of the Meliponinae subfamily into Old and New
World groups (Rasmussen & Cameron, 2010). The non-systematic
composition of SBH highlights its important plasticity but also our lack
of knowledge regarding ecology and evolution of the tribe. Environmental
and evolutionary drivers investigated in this study alone do not explain
all the variations in chemical composition. The environmental variables
were mainly selected according to the available literature on nectar
chemistry (Petanidou, 2007; Parachnowitsch et al., 2019; Nicolson,
2022). The impact of ecological conditions on nectar processing by
stingless bees remains poorly understood, even though some species
perform little or no thermoregulation within the hive (Roubik, 2006;
Ayton et al., 2016) potentially exposing stored nectar to certain levels
of alterations. Available flora and the proportional use of host plants
play a key role in honey composition (Silva et al., 2009; Machado et
al., 2020; Shamsudin et al., 2022) but cannot be included in formal
analysis since the foraging ecology and seasonality of stingless bees in
the Afrotropics and Indo-Malayan region remains largely undocumented
(Aleixo et al., 2017; Bueno et al., 2023). The phylogeny of stingless
bees is also only partially resolved and remains at the genus level,
masking the variations in honey composition that are likely to occur
within the same genus (de Sousa et al., 2016; Ismail et al., 2021). In
addition of microbiota whose role is depicted above, pollen profile
(monofloral vs. polyfloral) (Sousa et al., 2016; Chuah et al.,
2023), maturation stage of the fermentation process acids (Silva et al.,
2022), propolis composition and the management type that varies from
rearing wild colonies to advanced beekeeping methods across the tropics
(Delgado et al., 2020) have demonstrated impacts on honey properties and
should be investigated throughout the tribe. This is of particular
importance as the observed complex interplay of biotic and abiotic
factors shaping honey composition leads to an important phenotypic
variation in properties of SBH that may provide fitness benefits for the
colony as shown for other extended phenotypes (Blamires et al., 2018).
The study of SBH is not only of fundamental interest, but also has
applied purposes. Beekeeping with stingless bees (i.e. meliponiculture)
is promoted as a tool for rural development and sustainable income
generation activity, out of the Maya region where it’s an age-old
tradition and first emerged to meet the medicinal and nutritional needs
of an exploding population (Cortopassi-Laurino et al., 2006; Bahri et
al., 2016; Mustafa et al., 2018; Chemurot et al., 2021; Rasmussen et
al., 2024). The development of meliponiculture is also supported to
prevent destructive honey-hunting practices of stingless bee species,
otherwise severely threatened by agrochemicals (Lima et al., 2016),
pathogens (Fleites-Ayil et al., 2023), climate change (Giannini et al.,
2017; Dalsgaard, 2020) and drastic land use change (Gibbs et al., 2010;
Curtis et al., 2018). One of the main challenges in achieving
economically sustainable meliponiculture is the absence of reliable
market access due to the lack of international standards for SBH.
Although a number of initiatives have been undertaken at national and
regional level (Vit et al., 2004; Malaysian Standards, 2017; de Camargo
et al., 2017), SBH has not been formally acknowledged by the Codex
Alimentarius (Codex Alimentarius Commission, 2019), preventing wider
market access for meliponiculturists and multiplying counterfeits on
sale. In order to establish standards, several studies have investigated
into the species- and genus-specific composition of SBH (Chuttong et
al., 2016; Mokaya et al., 2022; Vit et al., 2022) and the impact of
particular climatic and environmental conditions on its properties
(Sousa et al., 2016; Dezmirean et al., 2017; Lavinas et al., 2023;
Noiset et al., 2024) but relatively few at global scale and on the joint
effect of evolutionary and ecological constraints. Yet, according to our
results, if closely related species or species from the same region tend
to produce similar honeys, this rationale does not extant to more
distant species that can produce comparable or very contrasting honeys
depending on the studied regions. Compositional profile of stingless bee
can also be similar to those produced by honey bees, a pattern observed
in this study and at intercontinental scale (Noiset et al., 2022).
Therefore, precise and unique standards for a species or an
environmentally heterogeneous region (i.e. a country) can ultimately
turn out to be ineffective. By contrast, quality labels associated with
specific local production conditions correspond more effectively to
variations in the chemical composition of SBH. Additionally, compounds
not quantified in this study such as trehalulose are promising markers
to differentiate SBH (Fletcher et al., 2020; Zawawi et al., 2022).
International standards for SBH should remain very broad to include all
the possible compositional profiles, especially as some of the key
compounds that differentiate them from A. mellifera (sugars and
fermentation products) are among those that vary the most within
stingless bee honeys.
Our study confirms the differences in composition between Apis honeys and SBH, while showing the significant variations in SBH
composition across the Tropics due to the many ecological, evolutionary
and human management factors involved. Phenotypic variation in SBH makes
it an ideal model for the study of ecological and evolutionary theory
but it requires more fundamental research on this under-studied tribe to
fully understand the role of honey in bee colony and human health.
Comprehensive characterization of SBH properties in Afrotropics and the
Indo-Malayan region/Australia that takes into account its specificity
and dynamism is a key step for the establishment of scientifically
informed standards to promote sustainable livelihood through
nature-based solutions and the exploitation of non-timber forest
products.