Identification of fungi isolated from commercial bumblebee colonies

Introduction

Pollinator-associated microbes are generally understudied unless they have been associated with the honey bee, Apis melliera (Yordanova et al., 2022). Since the 1990s growing commercialisation and international trade of other bee groups such as bumblebees (Bombus spp.) and some solitary bee species (e.g., Osmia cornifrons) has led to the need to understand what microbes are present in, and potentially globally transported with, these commercially reared bees (Graystock et al., 2016a; Graystock et al., 2016b; Graystock et al., 2020; Hedtke et al., 2015; LeCroy et al., 2022). In recent years the application of molecular screening and 16s sequencing has helped explore microbial diversity and movement patterns in commercially traded bees, but focus has largely remained on protists and bacteria (Huang et al., 2015; Milbrath et al., 2021). There remains a lack of appreciation of which fungal groups are present in commercially traded populations of bees, and even less of an idea of the risk they may pose to their hosts and to other bees.

Although there is a dearth of research describing bee-associated fungi, what evidence for we do have suggests that they can have a range of parasitic, mutualistic, or commensal relationships with the bees with which they associate (Evison & Jensen, 2018). The pathogenic fungi of honey bees are the best known because they associate with the most utilised agricultural bee species and their impact may be the most significant to human interests (Yordanova et al., 2022). Ascosphaera apis, for example, parasitises the developing larvae of A. mellifera (and some solitary bee species), killing the larvae leaving a chalky cadaver covered in transmissible and highly persistent spores, causing a disease in honey bees known as chalkbrood (Aronstein & Murray, 2010; Evison, 2015; LeCroy et al., 2022). Ascosphaera spp., along with other known fungal parasites pose a threat to bee health by impacting larval survival and leading to decline in reproductive success and (in the case of honey bees) collapse of entire colonies (Evison & Jensen, 2018; Lopes et al., 2015; Ravoet et al., 2014; Vojvodic et al., 2012).

Positive or neutral relationships between bees and fungi are likely to be common, because many bee-associated fungi can persist within the nesting materials and can sometimes grow on food provisions or frass rather than grow directly within the bee, but a general lack of understanding of the nature of their interactions make characterising mutualistic or commensal relationships difficult. An example of possible beneficial roles of fungi can be found with honey bees. There is strong evidence that mycelium of multiple polypore fungal species has a refractory effect on viruses such as deformed wing virus and Lake Sinai virus (Stamets et al., 2018). Several stingless bee species also gain significant benefits from fungi such as Scaptotigona depilis, Tetragona clavipes and Melipona flavolineata which cultivate a Zygosaccharomyces fungi that, at least in the case of S. depilis bees, must be consumed by larvae to initiate pupation (Menezes et al., 2015; Paludo et al., 2018; Vit, Pedro & Roubik, 2013). If the Zygosaccharomyces fungi also benefit from this relationship—via dispersal and the provision of habitat—then perhaps these are examples of mutualism, but this is not yet known. In addition, some fungal species could include roles aiding digestion and parasite defence (Stefanini, 2018). There is a clear need to improve our understanding of the species and interaction diversity of fungi and bees, only then can we start to build a comprehensive understanding of the potential risks or benefits fungi pose to bee health.

It is estimated that globally over two million bumblebee colonies are purchased annually for agriculture (Graystock et al., 2016a; Graystock et al., 2016b). While these colonies presumably contain favourable substrates for harbouring or cultivating fungi, they are often transported internationally with little to no appreciation of what they might contain (Dharampal et al., 2020). The adult bumblebees in commercial nests, and the pollen they are fed, have occasionally been screened for certain pathogenic fungi, namely Nosema spp. (now recategorized as Vairimorpha) and Ascosphaera spp., which have been detected (Graystock et al., 2013a; Graystock et al., 2013b). However, in certain countries such as the UK, such screenings are not a prerequisite for trade or distribution and are only performed at the supplier’s discretion (Cordes et al., 2012; Dharampal et al., 2020; Graystock et al., 2016a; Graystock et al., 2016b; Graystock et al., 2013a; Graystock et al., 2013b; Graystock, Goulson & Hughes, 2015; McIvor & Malone, 1995). International trade of bumblebee colonies for agricultural use is known to have facilitated the spread of parasites and viruses between managed and wild pollinators (Colla et al., 2006; Goka et al., 2000; Graystock et al., 2016a; Graystock et al., 2016b; Graystock, Goulson & Hughes, 2015; Graystock et al., 2020; Sachman-Ruiz, Narváez-Padilla & Reynaud, 2015). The focus on honey bee parasites has, however, left us unaware of the wider variety of microbes, specifically fungi that may be present in commercial bumblebee colonies.

Within a typical bumblebee colony there are several different substrates that could culture or harbour fungi. Of particular interest are bumblebee honey, frass, and nest wax associated with eggs and honey cups (we differentiate egg cups and brood here because we are referring to only the wax around the brood, and not the brood itself) (Fig. 1). Though honey from bumblebees is infrequently studied, honey bee honey can exhibit antibiotic properties, and is also known to harbour growth of xerophilic and xerotolerant fungal species, such as Ascosphaera apis, Bettsia alvei and species of Alternaria, Aspergillus and Penicillium (Kačániová et al., 2009; Kačániová et al., 2012; Rodríguez-Andrade et al., 2019; Saksinchai et al., 2012; Snowdon & Cliver, 1996). Bumblebee honey is not capped and is not stored long term, so it has greater similarity to nectar. Nectar has a lesser-known diversity of fungal genera, with the most common being Metschnikowia and Aureobasidium (De Vega, Herrera & Johnson, 2009; Jacquemyn et al., 2013; Mueller, Francis & Vannette, 2023; Pozo, Herrera & Bazaga, 2011). Bumblebee nests (particularly in commercial settings) have frass areas where bee faeces and waste (including dead adults and larvae) accumulate. Across several bee species, frass has been associated with bee pathogenic fungi, such as Ascosphaera (honey bees) and Nosema (honey bees and bumblebees) and plant pathogenic fungi such as Botrytis cinerea and Colletotrichum acutatum (Inglis, Sigler & Goette, 1993; Parish et al., 2019; Wynns, Jensen & Eilenberg, 2013). Bumblebee wax is used to house developing larvae and store honey. Whilst there are no data on fungi found in bumblebee wax, studies linking Zygosaccharomyces sp. (originally Monascus sp.) as an obligate mutualist in stingless bee brood development (Menezes et al., 2015; Paludo et al., 2018), combined with the known antimicrobial properties of honey bee wax (Grange & Davey, 1990), provide an interesting juxtaposition of predicted habitability of this substrate.

Despite well researched information on the presence, functional roles, and impacts of some honey bee fungi, relatively little is known about the fungi associated with bumblebees or their nest substrates. In addition, most of this research has focused on fungal DNA, so the viability of any fungi identified remains unknown (Jacquemyn et al., 2013; Mueller, Francis & Vannette, 2023; Pozo, Herrera & Bazaga, 2011; Wojcik et al., 2008). There is a clear gap in knowledge regarding the community of viable fungal microbes associated with bumblebee nests, which provide multi-component environments for brood development, food storage and debris accumulation (Grabowski & Klein, 2017). Here we survey and ID the fungi present and culturable in commercially reared bumblebee nests, allowing for the first assessment of the potential for the distribution of these fungi within commercially sourced bumblebee nests.

Materials and Methods

Results

Substrate type is associated with different fungal growth

Frass exhibited the lowest incidence of viable fungi, with only 35.7% (six) of the 17 samples containing such fungi. However, this substrate showcased the highest diversity of fungal species. A greater percentage (71.4%) of the honey cup wall and the egg cup wall samples had viable fungi, and 85.7% of honey samples had viable fungi (Fig. 2). Substrate type had a statistically significant effect on the occurrence of viable fungi (X² = 14.134, df = 3, p = 0.0027); there was also significant difference in presence or absence of growth between bumblebee colonies (X² = 27.15, df = 13, p = 0.0119).

Discussion

This study detected 11 different fungal species from four substrates (frass, honey, honey cup wax, and egg cup wax) within commercial colonies of the bumblebee Bombus terrestris audax. This is the first survey of fungal diversity grown from different substrates of commercially reared bumblebees, and provides some interesting insights into how and why such diversity might differ.

Frass is the detritus from bumblebee activity in the nest. It is often a mixture of chewing debris and excrement. The highest fungal diversity was observed in frass, which can be attributed to its role within the colony. Frass has exposure to bee excretion and a number of phoretic mites which all of which may contribute to its high fungal diversity (Osimani et al., 2018; Paniagua Voirol et al., 2018; Pernice, Simpson & Ponton, 2014; Schwarz & Huck, 1997; Weiss, 2006). Frass may hold the highest diversity as well because of the length of time the fungi have to grow and unhomogenized nature of the substrate. There is also the consideration that there is significantly less diversity and abundance of microbes in insects than in (for example) mammals due to the physical barriers between the gut and the rest of the insects’ systems such as the basal lamina (Pernice, Simpson & Ponton, 2014). Almost every phylogenetic grouping found in the colonies was represented in frass (with the exception of Monascus ruber). Candida orthopsilosis and Candida parapsilosis were found in the frass. The isolation of Candida strains on bee surfaces suggests that these yeasts rely on insect vectors for dispersion across flowers but have no negative effect on the bees (Hong et al., 2003). Yeasts are often found in the nectar of flowers visited by bumblebees (Herrera et al., 2009), potentially showing that bumblebees contain yeasts prior to contact with nectar from their preferential flowers, although this could still be an effect of the rearing process. There is also evidence that yeasts use flowers as sites for mass reproduction and insects as platforms for hibernation (Brysch-Herzberg, 2004).

Bumblebee larvae are fed with honey produced by the queen bumblebee and her workers. Honey had the lowest diversity of fungal isolates, which could be attributed to its antimicrobial properties (Aurongzeb & Azim, 2011; Nolan, Harrison & Cox, 2019; Wahdan, 1998). However, there is little evidence to suggest that bumblebee honey, which is significantly closer to nectar in terms of water content than honey bee honey, has similar antimicrobial effects. The isolate found in the bumblebee honey, Aspergillus tubingensis, is a member of the Aspergillus genus which have long been known to be associated with honey bees and includes the pathogen responsible for the highly infectious stonebrood disease in immunocompromised bees (Cheng et al., 2022; Foley et al., 2014). Bumblebee honey is stored in honey cups for future feeding to larvae. The honey cup walls had the second-largest variety of fungal isolates. One interesting fungal isolate was Penicillium crustosum, which causes food spoilage (De Jesus et al., 1983) and has not traditionally been associated with bumblebee species. It is unclear if this fungi spoils the nutritional value of bumblebee honey but if it does, it would be a harmful microbe to colony health even if it does not parasitize the bees themselves.

Bumblebee eggs are placed in small clusters within egg cups where they are nursed and fed by worker bees before being moved to individual cups prior to pupation. The egg cups contained two fungi known to be pathogenic in honey bees but with unknown affects in bumblebees (as of yet—A. tubingensis and A. niger) and one that has been associated with improved nutrition and pupation in stingless bee species (Monascus ruber). Monascus ruber was originally found to be crucial for the survival of a Brazilian stingless bee, Scaptotrigona depilis, by being a food source for its larvae, though this was later corrected by Paludo et al. (2018) to Zygosaccharomyces sp. (Menezes et al., 2015; Paludo et al., 2018), growing inside brood cups over the larval food and being transmitted across generations via recycled contaminated nest materials. Our study identified M. ruber by the same PCR primers (ITS-1 and 4) so it very well could be misidentified similarly—this was later identified in Paludo et al. (2018) with species specific primers. There is also evidence of M. ruber in the brood cups of stingless bees, although it may not serve the same purpose.

All sampling was done from colonies sourced from only one commercial supplier of bumblebees and with no similar data from wild or other suppliers, no comparisons can be made. Nevertheless, the presence of live and culturable fungi confirms commercially imported bumblebee colonies as a source of fungal microorganisms. However, if we are to fully understand the frequency and, indeed, the full diversity of such fungi, more sampling is required. This study provides a snapshot of the fungal profiles within bumble bee colonies ordered from one supplier at one point in time. Suppliers could employ various pollen/facility sterilization techniques and/or rearing methods to reduce or remove the fungi present. Commercial colonies also are not representative of wildtype colonies and likely contain different diversities of microorganisms (Newbold et al., 2015). There is likely spillover of nest fungi between commercial and wild nests, similar to the pathogenic spillover documented in the UK, the US, Ireland, and Japan (Cameron et al., 2016; Graystock et al., 2016a; Graystock et al., 2016b; Graystock, Goulson & Hughes, 2015; Newbold et al., 2015; Niwa et al., 2004), but to fully understand the risks this may or may not pose to wild bumblebees, more research is required. Because the source colonies from this study were isolated and maintained indoors, having never been free-flying in the field, the likely entry points for the fungi found would be either from the founding queens that hibernated in soil or peat materials or through contamination of rearing provisions, such as pollen (David et al., 2015; Dharampal et al., 2020; Mommaerts, Jans & Smagghe, 2010). Although commercial suppliers now often sterilize the pollen that they feed to colonies, the sterilization methods employed have inconsistent effects on fungal spores (Strange et al., 2023), and the overall effectiveness of sterilization against a wide range of fungal spores is unknown (Graystock et al., 2016a; Graystock et al., 2016b).

Conclusions

Though we identify a diversity of fungi from the various substrates of commercial bumblebee colonies, we do not yet know the functional significance of these fungi or the generality of the findings. Further research is needed to explore the interactions between bumblebees and fungi, including the ecological and evolutionary implications of these interactions. This research can inform conservation efforts and help to ensure the continued pollination services provided by commercial and wild bumblebees, which are essential for maintaining ecosystem health and food security.

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