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.