Most plants produce chemicals that are toxic to at least some animals. Whether or not the toxins are harmful to a particular animal depends on how much they consume and the specific biochemistry that occurs during digestion. The enzymes produced in the gut both by the animal and by the microbes that reside there often help break down toxic substances into less harmful molecules. However, some products of this breakdown can be toxic themselves. While these products can harm the animal, they may also be detrimental to parasites living in the gut, resulting in an overall positive effect.

Almonds and their pollen are consumed by humans and bees without apparent harmful effects. However, almonds contain amygdalin, a molecule that can produce the highly toxic compound hydrogen cyanide upon digestion. Although amygdalin can be toxic to bees in high doses, the amount usually found in almond nectar is not harmful, and indeed, it may protect bees from parasites. Motta et al. wanted to know how amygdalin is digested in the gut of bees, and whether gut microbes have a role in this digestion.

To answer these questions, Motta et al. compared the effects of consuming amygdalin on normal bees and bees lacking gut microbes. Bees without gut microbes broke down amygdalin into a harmless substance called prunasin. However, only bees with gut microbes could further break down prunasin into hydrogen cyanide. Interestingly, the full metabolism of amygdalin had no detectable effect on whether the bees survived for longer times or on which microbes were found in the gut. Motta et al. also found some gut bacteria in bees that can break down amygdalin and release hydrogen cyanide, and identified the enzyme responsible for the process. When the gene encoding this enzyme was inserted into a different species of bacteria, the second species gained the ability to break down amygdalin.

The findings of Motta et al. explain a role of gut microbes in processing amygdalin in bees. In the future, this may be the key to understanding how humans and other creatures process plant toxins. Future work on the relationship between animals and microbes living in their guts could help scientists understand how to manipulate the digestion and processing of toxins, nutrients, or drugs to benefit human health.

Many animals ingest potential toxins along with their food, and these toxins can have complex consequences. Dietary toxins are often deleterious, but they sometimes prove beneficial, by providing protection against natural enemies, including pathogens and parasites (Gowler et al., 2015). Once ingested, enzymatic metabolism of dietary compounds can render them more or less toxic (Mason et al., 2019; Dearing and Weinstein, 2022). The host itself can produce enzymes that degrade toxins. Additionally, gut microbiota members have been shown to contribute to enzymatic degradation of dietary compounds, including toxins.

As generalist foragers, honey bees and bumble bees can be exposed to a wide range of plant secondary metabolites (Irwin et al., 2014), which are usually produced by plants as defenses against pathogens and herbivores (Zaynab et al., 2018). Even when in low concentrations, these metabolites can have a range of effects on bee behavior and health, from negative to neutral to positive, and can be involved in attraction or deterrence (Detzel and Wink, 1993; Hagler and Buchmann, 1993; Stephenson, 1982). Interestingly, some bee species cannot detect naturally occurring concentrations of certain nectar metabolites, such as quinine, nicotine, caffeine, and amygdalin (Tiedeken et al., 2014). This poor acuity may lead to long-term side effects depending on the toxicity of the metabolite.

A plant secondary metabolite to which generalist bees may be chronically exposed is amygdalin, a cyanogenic glycoside found in almonds, apples, cherries, and nectarines (London-Shafir et al., 2003; Barceloux, 2009; Bolarinwa et al., 2015; Lee et al., 2017). Studies on almonds show that amygdalin is present in nectar and pollen (London-Shafir et al., 2003). The western honey bee, Apis mellifera, is the primary pollinator of almonds and likely also encounters amygdalin in other crops. The toxicity of amygdalin derives from its degradation products (Jaszczak-Wilke et al., 2021). Degradation occurs during plant tissue damage, such as chewing by herbivores, since amygdalin is stored in cell vacuoles and the glycoside hydrolases (GHs) involved in degradation are present in the cytoplasm. During degradation, amygdalin is usually first broken down into prunasin and a glucose molecule. Then, prunasin is broken down into another glucose molecule and mandelonitrile, with the latter compound converted into benzaldehyde and hydrogen cyanide. Hydrogen cyanide is a toxic molecule that can lead to acute poisoning in animals (Khandekar and Edelman, 1979; Carter et al., 1980; Newton et al., 1981; Kolesarova et al., 2021; Kovacikova et al., 2019; Salama et al., 2019) as it interferes with the electron transport chain during oxidative phosphorylation (Cooper and Brown, 2008).

Interestingly, some bees are not deterred by amygdalin concentrations encountered in almond nectar (up to 15 μM) (London-Shafir et al., 2003; Stevenson et al., 2017), and can tolerate concentrations up to 219 μM with no effects on survivorship (Irwin et al., 2014; Lecocq et al., 2018). Bees feeding on particular plants may be exposed to even higher doses of amygdalin; for example, concentrations in almond pollen can reach up to 4 mM (London-Shafir et al., 2003). Exposure to these high doses of amygdalin leads to acute malaise symptoms, including a sharp increase in time spent upside down and abdomen dragging (Hurst et al., 2014), and exposure for several days lowers bee survivorship (Kevan and Ebert, 2005). Lower doses can also result in lower survivorship in lab trials (Ayestaran et al., 2010). Despite the potential for toxicity to bees, colony-level exposure to amygdalin may protect bees against parasites, such as the trypanosomatid Lotmaria passim (Tauber et al., 2020), and may reduce the titer of some pathogenic viruses (Tauber et al., 2020; Palmer-Young et al., 2017). Thus, amygdalin exposure may have both positive and negative effects on bee health depending on dose and infection status.

Despite potential consequences for bee health, the routes of amygdalin metabolism within bees have not been elucidated. The genomes of honey bees and bumble bees have fewer detoxification genes compared to other insects (Berenbaum and Johnson, 2015; Sadd et al., 2015), but they do encode some enzymes that can degrade plant metabolites, such as cytochrome P450 monooxygenases, glutathione transferases, and GHs (Berenbaum and Johnson, 2015; Pontoh and Low, 2002; Rand et al., 2015). For example, honey bees secrete a GH into their mouths from their hypopharyngeal glands that is then transferred to the midgut where it can potentially catalyze the initial breakdown of glycosides (Pontoh and Low, 2002; Ricigliano et al., 2017), such as the conversion of amygdalin into prunasin.

Amygdalin toxicity occurs after ingestion, but not after injection into the hemolymph (Hurst et al., 2014), suggesting that enzymes in the gut achieve the conversion of amygdalin into hydrogen cyanide. The source of these enzymes is unknown. Possibilities include bee GHs (Pontoh and Low, 2002), pollen-derived GHs that bees ingest (Ricigliano et al., 2017), or GHs produced by the bee gut microbiota (Kwong and Moran, 2016; Zheng et al., 2018; Motta et al., 2022a). The latter possibility is suggested by the vast arsenal of GHs produced by the dominant bee gut bacterial species (Zheng et al., 2019; Ellegaard et al., 2019). Interestingly, amygdalin itself does not show antibacterial effects in vitro, and the honey bee gut microbiota appears not to be significantly affected by amygdalin exposure (Tauber et al., 2020).

In this study, we investigated the contributions of honey bees and their microbiota to amygdalin degradation. We found that breakdown to prunasin is achieved by hosts without a microbiota and that further degradation can be performed by specific strains of dominant microbiota species. Using biochemical assays, we characterized a GH secreted by bee-associated Bifidobacterium strains that can degrade amygdalin and prunasin. These findings shed light on how the combined contributions of host and microbiome enable degradation of a dietary plant metabolite.

To investigate amygdalin metabolism by bee gut bacteria, we selected representative strains of four bacterial groups involved in food metabolism in the bee gut: Bifidobacterium, Bombilactobacillus (formerly called Lactobacillus Firm-4), Lactobacillus nr. melliventris (formerly called Lactobacillus Firm-5), and Gilliamella (Figure 1). We cultured these strains in semi-defined media (SDM, Figure 1A) or in nutritionally rich media (MRS or Insectagro, Figure 1B) to assess their susceptibility to amygdalin and their ability to metabolize amygdalin into byproducts, such as prunasin, as analyzed by LC-MS (Figure 1C).

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Different mechanisms of amygdalin degradation by bee gut bacteria

Metabolism of amygdalin by Bifidobacterium strain wkB204 and Bombilactobacillus strain BI-1.1 produces prunasin as a byproduct (Figure 1L–M and Figure 1—figure supplement 1G–H), although prunasin was only detected in wkB204 cultures after providing an excessive amount of amygdalin (Figure 1—figure supplement 1G). This suggests that wkB204 and BI-1.1 encode enzymes to break down the glycosidic bond between the glucose residues in the amygdalin structure, releasing prunasin and one glucose molecule, which can then be used as carbon source by these bacteria. On the other hand, prunasin was not produced by Bombilactobacillus strain BI-2.5 or Gilliamella strain wkB112 (Figure 1M–N) even after adding excess amygdalin (Figure 1—figure supplement 1H–I). Therefore, BI-2.5 and wkB112 seem to metabolize amygdalin in a different way than wkB204 and BI-1.1, probably by breaking down the glycosidic bond that links the two glucose residues to the aglycone, releasing a disaccharide and mandelonitrile into the medium. These mechanisms are corroborated by LC-MS analyses of spent medium taken from these cultures on a daily census (Figure 2). These results suggest that amygdalin breakdown via a prunasin intermediate is limited to wkB204 and BI-1.1.

Figure 2

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Characterizing an enzyme involved in amygdalin metabolism

After finding that specific strains from different bee gut bacterial species can degrade amygdalin, we focused on honey bee-associated Bifidobacterium strains to investigate the enzyme involved in this metabolism. First, we checked whether the enzyme is secreted or not. Large cultures of wkB204, wkB344, and wkB338 were grown for 5 days (Figure 3A), after which we performed biochemical assays with both spent medium and cell lysate of glucose- and amygdalin-grown cultures.

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As observed in the previous experiment, wkB204 completely degraded amygdalin; we did not detect amygdalin in spent medium (10A sm, Figure 3B) or in cell lysate of amygdalin-grown cultures (10A cl, Figure 3C). To investigate whether the enzyme involved in amygdalin degradation was secreted, we added fresh amygdalin to sterile spent medium (10A sm + 10A) or to sterile cell lysate (10A cl + 10A) originating from amygdalin-grown cultures. After 3 days of incubation, we found full degradation of amygdalin in spent medium (10A sm + 10 A) (Figure 3B), but only slight degradation in cell lysate (10A cl + 10A) (Figure 3C); this was compared to a control sample containing only medium and amygdalin (Fresh 10A). No amygdalin was detected in cell lysates of amygdalin-grown cultures, showing that amygdalin does not enter bacterial cells (10A cl, Figure 3C). Moreover, we detected prunasin in both spent medium and cell lysate of amygdalin-grown cultures supplemented with amygdalin (10A sm + 10A and 10A cl + 10A, respectively) (Figure 3—figure supplement 1).

For comparison, these assays were also performed for wkB344 and wkB338 cultures. Some amygdalin degradation was observed for the spent medium of wkB344 amygdalin-grown cultures, but this degradation was much less than that observed for wkB204 (Figure 3B–C). We also investigated enzyme production and activity in the absence of amygdalin, by adding amygdalin to spent medium and cell lysate from glucose-grown cultures (10G sm + 10A and 10G cl + 10A, Figure 3B–C). None of the strains was able to significantly degrade amygdalin under these conditions.

Since spent medium of wkB204 amygdalin-grown cultures achieved full degradation of amygdalin, we decided to characterize the secreted enzyme involved in the degradation. Spent media from wkB204 cultures, grown with either amygdalin or glucose, were processed to obtain concentrated protein extracts (Figure 4A). Protein profiles were first obtained by SDS-PAGE gel and showed that amygdalin-grown cultures had a distinct secretome when compared to glucose-grown cultures (Figure 4B, Figure 4—source data 1). Then, samples were submitted to proteomics analysis, which confirmed the expression differences, as we found 107 proteins secreted in higher abundance in amygdalin-grown cultures and 131 proteins secreted in higher abundance in glucose-grown cultures (p<0.05, t-test followed by Benjamini-Hochberg procedure to control for false discovery, Figure 4C). Several significantly upregulated proteins in amygdalin-grown cultures are associated with carbohydrate metabolism (Figure 4—source data 2). Interestingly, we detected a highly expressed enzyme belonging to the glycoside hydrolase family 3 (GH3) (WP_254476944) only in amygdalin-grown cultures (Figure 4C), suggesting its involvement in the observed degradation. Other studies have demonstrated that specific bacterial or fungal GH3 enzymes can degrade amygdalin (Gao and Wakarchuk, 2014; Guo et al., 2015; Chang and Zhang, 2012; Li et al., 2018).

Figure 4

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Figure 4—source data 1

SDS-PAGE gel run for cultures of Bifidobacterium strain wkB204.

From left to right, columns represent: (1) PageRuler Plus Prestained Protein Ladder; (2–10) Supernatants of cultures (1–4) grown in the absence of a carbon source, (5–7) in the presence of 10 mM glucose as sole carbon source, or (8–10) in the presence of 10 mM amygdalin as sole carbon source. Each sample (30 μL) was mixed with 5 μL of 6× SDS gel-loading buffer (0.35 M Tris-Cl pH 6.8, 10% w/v SDS, 0.012% w/v bromophenol blue, 30% v/v glycerol, 0.6 mM dithiothreitol), denatured at 100°C for 5 min, then run on a Bolt 4–12% Bis-Tris Plus, 1.0 mm, protein gel at 200 V for 22 min.

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Figure 4—source data 2

Differential protein expression analysis for amygdalin- and glucose-grown cultures of Bifidobacterium strain wkB204.

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GH3 gene expression in Bifidobacterium strains

We used this wkB204 GH3 (WP_254476944) as a query to search a customized database of proteins from bee gut bacteria, including 22 bee-associated Bifidobacterium strains. Ten other Bifidobacterium strains encode a GH3 in their genomes with a high sequence similarity to the wkB204 GH3 (Figure 5—figure supplement 1). Intriguingly, these included a GH3 from wkB344 (WP_121913979), which did not grow in the presence of amygdalin in vitro.

To determine why this GH3 does not enable wkB344 to use amygdalin as a carbon source, we investigated whether this enzyme is expressed in cultures, and used wkB204 and wkB338 cultures as controls for presence and absence of GH3 activity, respectively (Figure 5A). In the presence of glucose as the sole carbon source, strains wkB204 and wkB344, but not wkB338, express the GH3 gene (Figure 5B). When cultivated in the presence of amygdalin as the sole carbon source, only wkB204 shows elevated expression of GH3 transcripts (Figure 5B), which correlates with the ability of this strain to degrade amygdalin in vitro. No elevation in expression was evident for wkB344 (Figure 5B), and the levels of GH3 produced by wkB344 in glucose-grown cultures did not result in observable amygdalin degradation when incubated in 10 mM amygdalin (Figure 3C).

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Figure 5—source data 1

dbCAN meta server results for Bifidobacterium strains wkB204, wkB344, and wkB338.

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The wkB204 GH3 (WP_254476944), which is overexpressed in amygdalin-grown cultures, is encoded in an operon containing four other genes: a major facilitator superfamily transporter (WP_254476943), a glycoside hydrolase family 30 (WP_254477160), and a beta-galactosidase (WP_254477161) (Figure 5C). These were also overexpressed in the presence of amygdalin, based on proteomics data (Figure 5C). The wkB344 GH3 (WP_121913979) is also encoded in an operon, but a beta-galactosidase (WP_121914045) is the only other gene in the operon (Figure 5C), as predicted by the operon-mapper webserver (Taboada et al., 2018).

According to the dbCAN meta server for automated CAZyme annotation, the genomes of these three Bifidobacterium strains encode multiple GH3s: wkB204 encodes 10 distinct GH3s, while wkB344 and wkB338 encode 5 distinct GH3s each (Figure 5—source data 1; Yin et al., 2012; Zhang et al., 2018). Based on the NCBI inference database and amino acid similarity to other annotated GH3s, these three strains have some GH3s highly similar in amino acid sequence and probably similar in function (Figure 5D and Table 1), as noted for wkB204-GH3 (WP_254476944) and wkB344-GH3 (WP_121913979) (Figure 5E).

Table 1

Strain	GH3 loci number	Protein ID (NCBI RefSeq)	Inference (NCBI RefSeq)
wkB204	10	WP_254476932a	WP_007147852
		WP_254476944b	WP_015021504
		WP_254477003	WP_003842825
		WP_254477019	–
		WP_254477308c	WP_015022086
		WP_254477316	–
		WP_254477624d	WP_016461981
		WP_254477626e	WP_004221005
		WP_254478126	–
		WP_254478363	–
wkB344	5	WP_121913968a	WP_007147852
		WP_121913979b	WP_015021504
		WP_121914233c	WP_015022086
		WP_121914846d	WP_016461981
		WP_121914847	WP_004221005
wkB338	5	WP_121912678c	WP_015022086c
		WP_121912768	WP_003838412
		WP_121912769e	WP_004221005e
		WP_121913257	WP_003839235
		WP_121913288	WP_015450023

Bifidobacterium strains also degrade prunasin

To investigate whether Bifidobacterium strains can also degrade prunasin, we performed an additional in vitro experiment in which Bifidobacterium strains wkB204, wkB344, and wkB338 were grown in 10 mM glucose in SDM in the presence of 0.1 mM prunasin (Figure 6A). Under these conditions, all strains grew in the presence of prunasin (Figure 6B) and degraded it (Figure 6C). For comparison, we also checked growth in the presence of 0.1 mM amygdalin (Figure 6D) and found that not only wkB204, but also wkB344 degraded amygdalin (Figure 6E). The lack of growth and, consequently, of degradation observed before for this strain is probably due to the much higher concentration of amygdalin provided in previous cultures (10 or 100 mM).

Figure 6

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Escherichia coli expressing the GH3 enzyme produces prunasin

To confirm the ability of the Bifidobacterium GH3 enzyme to degrade amygdalin and/or prunasin, we cloned and expressed the GH3 gene from Bifidobacterium strains wkB204 (WP_254476944) or wkB344 (WP_121913979) in E. coli (Figure 7A–B). Cell lysates of transformed E. coli expressing GH3 were incubated in the presence of 0.1 mM amygdalin or 0.1 mM prunasin (Figure 7C). After 5 days of incubation, we observed amygdalin degradation (Figure 7D) followed by prunasin production (Figure 7E) for E. coli cell lysates expressing either wkB204-GH3 or wkB344-GH3, but not for E. coli transformed with an empty plasmid, indicating that both enzymes can degrade amygdalin into prunasin. When the cell lysates were incubated in the presence of prunasin, only a small amount of prunasin was degraded (Figure 7F), suggesting that this enzyme, under the tested conditions, still can degrade prunasin, but to a lesser extent. These findings show that this Bifidobacterium-related GH3 enzyme can degrade amygdalin into prunasin, and potentially prunasin into mandelonitrile, and may be responsible for the degradation patterns observed for Bifidobacterium strain wkB204 when cultured in the presence of amygdalin.

Figure 7

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Host and symbionts contribute to amygdalin degradation

We also investigated amygdalin degradation in vivo. To that end, we performed experiments with bees lacking a microbiota (microbiota-deprived or MD), colonized with a conventional microbiota (CV), or monocolonized with Bifidobacterium strains wkB204 or wkB344 (Figure 8A). Bees were hand-fed 5 μL of 1 mM amygdalin in sucrose syrup, or only sucrose syrup. Amygdalin was detected in different compartments of the bee body, including the midgut, the hindgut and the body carcass without the gut of MD, CV, and monocolonized bees (Figure 8B). In the hindgut samples, amygdalin was detected for MD and wkB344-monocolonized bees but not for CV and wkB204-monocolonized bees (Figure 8B). Total amygdalin concentration was significantly lower in CV bees and wkB204-monocolonized bees when compared to control bees not treated with amygdalin but spiked with 5 μL of 1 mM amygdalin during the extraction protocol (Figure 8C). Interestingly, prunasin was only detected in the midgut and hindgut of MD bees (Figure 8D–E).

Figure 8

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These findings demonstrate the role of the microbiota in amygdalin degradation, as amygdalin concentration is reduced in CV bees and prunasin does not accumulate in the guts of CV or monocolonized bees. These findings also show that bees themselves can degrade amygdalin, but that this degradation is partial, since prunasin accumulates in the guts of MD bees. Therefore, the presence of the microbiota contributes to continued amygdalin and prunasin degradation in the bee gut.

Honey bees tolerate typical environmental concentrations of amygdalin

Honey bees exposed to concentrations of amygdalin, ranging from 0.01 to 1 mM, did not exhibit increased mortality rates or dysbiosis (Figure 9A–B). We did not find any significant changes in gut microbial composition (Figure 9C–D) or abundance (Figure 9E) of amygdalin-treated bees when compared to untreated bees, which is consistent with other studies (Tauber et al., 2020). Moreover, amygdalin did not affect mortality rates of MD bees (Figure 9—figure supplement 1). The concentrations used in the in vivo experiments are below the concentrations detected in almond pollen (~4 mM), and the lower concentration resembles what has been detected in almond nectar (~0.01 mM) (London-Shafir et al., 2003).

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Dietary compounds can be metabolized not only by host enzymes but also by the gut microbiota, potentially resulting in increased or decreased toxicity (Koppel et al., 2017). Once consumed by bees, amygdalin is broken down in the bee gut, but whether this degradation is via host or pollen-derived GHs (Pontoh and Low, 2002; Ricigliano et al., 2017) or through activity of the microbiota, has been unclear. We found that members of the bee gut microbiota can degrade amygdalin and its intermediate prunasin in vitro. Specific strains of Bifidobacterium, Bombilactobacillus, and Gilliamella isolated from A. mellifera, B. impatiens, and A. cerana, respectively, were able to degrade amygdalin in vitro. In some cases, the pathway led to the production of the non-toxic intermediate prunasin; in others it did not. While the host alone can degrade amygdalin to prunasin, enzymes secreted by gut bacteria are required for the further degradation of prunasin, and the release of toxic hydrogen cyanide. Our findings for honey bees parallel those for the metabolism of amygdalin in rats, in which the molecule is degraded to produce toxic hydrogen cyanide only in the presence of the gut microbiota, but not when microbiota is absent or when injected (Carter et al., 1980).

Specific members of the bee gut microbiota produce a diverse set of carbohydrate digestive enzymes, including pectin lyases (PLs) and GHs, that help in food processing (Zheng et al., 2019; Zheng et al., 2016; Engel et al., 2012) and detoxification (Berenbaum and Johnson, 2015; Koch et al., 2022). For instance, Gilliamella strains produce PLs and GHs that are involved in the metabolism of pectin and hemicellulose from the pollen cell wall and toxic sugars from nectar or produced during digestion of pectin (Zheng et al., 2016; Engel et al., 2012). Some of these sugars, such as mannose, arabinose, xylose, and galactose, are indigestible for bees and can cause toxicity if accumulated in the gut (Barker, 1977). Bombilactobacillus and Lactobacillus strains also produce enzymes involved in mannose metabolism which potentially contribute to this detoxification mechanism (Zheng et al., 2019; Ellegaard et al., 2019). Interestingly, genomes of Bifidobacterium strains seem to harbor a wider repertoire of GHs than other core members of the bee gut microbiota, but lack PL-related genes (Zheng et al., 2019). These enzymes tend to be substrate-specific and biochemical assays are usually required to verify function. In our study, we identified a specific GH3 in Bifidobacterium strains that contributes to amygdalin and prunasin metabolism in vitro. Other studies have shown that bacterial- or fungal-derived GH3s can degrade amygdalin. For example, the Gram-positive bacterium Cellulomonas fimi encodes a GH3 with activity against β-1,6-linked glycosides (Gao and Wakarchuk, 2014), similar to the linkage found in the structure of amygdalin (Figure 2D). Degradation of amygdalin by GH3s isolated from Rhizomucor miehei (Guo et al., 2015) and Talaromyce leycettanus (Li et al., 2018), or by related extracellular enzymes from Aspergillus niger, has also been observed (Chang and Zhang, 2012). Moreover, different species of mammalian gut-associated Bifidobacterium strains can grow in the presence of amygdalin, potentially due to the production of GH1 or GH3 enzymes (Modrackova et al., 2020).

In our case, heterologous expression of wkB204-GH3 (WP_254476944) or wkB344-GH3 (WP_121913979) using E. coli also led to amygdalin degradation, but to a lesser extent than what was observed for the original host. To accurately quantify the lower degradation rates in transformed E. coli, we used 0.1 mM amygdalin solutions (Figure 7). Potentially, there are other carbohydrate digestive enzymes encoded by Bifidobacterium that contribute to amygdalin or prunasin metabolism (Figure 4C), or the Bifidobacterium host perform specific posttranslational modifications on this enzyme that are not achieved during heterologous expression in E. coli (Adav et al., 2014). Moreover, it seems this GH3 is secreted by Bifidobacterium strain wkB204, but, for cloning purposes, we expressed it in E. coli without a signal sequence for secretion, and, therefore, performed assays with cell lysates, which may not be optimal for characterizing enzyme activity. These features may have masked the potential activity of this GH3, if this is the main enzyme involved in amygdalin degradation by Bifidobacterium strains. Moreover, other hydrolases were identified in the proteomics data (Figure 4C). Although not reported in the literature, some of these could potentially be involved in amygdalin degradation.

As shown in other studies, nectar and pollen metabolites can be chemically transformed when passing through the gut, and this may influence their effects on the host and/or the microbiota (Koch et al., 2019; Vidkjær et al., 2021). To identify the potential contribution of both the host and the microbiota to amygdalin degradation, we performed in vivo experiments in which MD bees and microbiota-colonized bees were exposed to amygdalin for a short period of time. We found that MD bees can degrade amygdalin, but only partially, leading to accumulation of prunasin in gut compartments. In contrast, prunasin accumulation was not observed in microbiota-colonized bees or in bees monocolonized with specific Bifidobacterium strains, and amygdalin degradation was higher in these groups when compared to MD bees. This suggests that members of the microbiota, besides contributing to amygdalin degradation, can also efficiently degrade prunasin and potentially release the final products of amygdalin metabolism, such as hydrogen cyanide. This could potentially increase the side effects of amygdalin byproducts on bees or on the microbiota, since hydrogen cyanide can be toxic to aerobic organisms (Khandekar and Edelman, 1979; Hurst et al., 2014). However, similar to other studies, we did not detect increased mortality rates or changes in microbial community abundance and composition for bees exposed to amygdalin (Lecocq et al., 2018; Tauber et al., 2020), strongly suggesting that field-relevant concentrations of amygdalin are not detrimental to bees. Interestingly, we also found amygdalin in bee carcasses with the gut removed, suggesting that amygdalin is absorbed systemically by bee cells. This has been observed in other studies in which amygdalin was found in the bee hemolymph after oral ingestion (Hurst et al., 2014; Vidkjær et al., 2021).

Studies in other animals have also investigated the roles of the microbiota on amygdalin degradation. Studies in rats, for example, have found higher concentrations of hydrogen cyanide in the blood of microbiota-colonized rats than in MD rats (Carter et al., 1980) or antibiotic-treated rats (Newton et al., 1981) after oral ingestion of amygdalin. However, intravenous administration of amygdalin seems not to lead to hydrogen cyanide formation (Jaswal and Palanivelu, 2018), which could be correlated to the lack of toxicity observed in honey bees after amygdalin injection into the hemolymph (Hurst et al., 2014). These studies suggest that the gut microbiota is a major factor driving amygdalin degradation and hydrogen cyanide release in the guts of animals.

It is important to note that colonization by the normal microbiota decreases the pH to about 5 in the bee gut (Zheng et al., 2017), and low pH can favor the degradation of amygdalin into prunasin, then prunasin into mandelonitrile, which can undergo spontaneous degradation at acidic pH to give benzaldehyde and hydrogen cyanide (Jaswal and Palanivelu, 2018). Therefore, the presence of the microbiota itself, without the action of GHs, could favor the degradation of amygdalin into prunasin and potentially lead to the production of hydrogen cyanide.

Although bees and their native microbiota seem to tolerate relatively high doses of amygdalin, parasites that commonly inhabit the bee gut may not fare as well. There is increasing evidence that metabolites in nectar and pollen, even those considered toxic in some cases, can improve pollinator health at specific concentrations by controlling or reducing parasite loads (Stevenson et al., 2017; Tauber et al., 2020; Costa et al., 2010; Simone-Finstrom and Spivak, 2012; Stevenson, 2020). Indeed, bees tend to forage on specific plants as a means of reducing colony pathogen loads (Simone-Finstrom and Spivak, 2012). For example, honey bees from hives treated with amygdalin exhibited decreased levels of infection by the parasite L. passim and some viruses (Tauber et al., 2020). In contrast, this does not seem to be the case for Crithidia infection in bumble bees, whose loads are not reduced after amygdalin exposure (Richardson et al., 2015), as the parasite is not susceptible to amygdalin (Palmer-Young et al., 2016). Other plant metabolites, such as the essential oil thymol, can reduce Nosema spore loads in honey bees (Costa et al., 2010; Stevenson, 2020). Therefore, the extent of protection may depend on the exposure level and on the parasite being exposed.

Indeed, some insects, such as bees, ants, flies, and butterflies, can use a wide range of toxic secondary metabolites to medicate themselves in a therapeutic or prophylactic way (de Roode and Hunter, 2019). Honey bees typically collect secondary metabolites in plant resins, nectar, and pollen as prophylactic medication behavior to protect their colonies from parasites (Simone-Finstrom and Spivak, 2012; Erler and Moritz, 2016; Simone-Finstrom et al., 2017). Similarly, Monarch butterflies feed on milkweeds with high concentrations of toxic cardenolides as a therapeutic medication against parasites (Gowler et al., 2015). Monarchs can also use cardenolides as a prophylactic medication by selecting oviposition sites with high concentrations of cardenolides (Lefèvre et al., 2012) or transferring cardenolides to eggs (Sternberg et al., 2015) to reduce parasite growth in their hatching offspring. Some species of fruit flies use transgenerational medication to resist attack from parasitoid wasps (Poyet et al., 2017). Female flies preferentially oviposit in media containing the alkaloid atropine, which reduces infection success of parasitoids, but also reduces fecundity.

More recently, a few studies have brought attention to activities of plant-derived compounds within the host gut, which can be modulated by the host and/or the microbiota, to lead to the final metabolite activity (Koch et al., 2019). The recently described plant metabolite callunene, from heather nectar, can play a prophylactic role in preventing Crithidia infections in bumble bees, especially when parasite cells are present in the crop. However, callunene cannot play a therapeutic role in infected hosts, because host metabolism inactivates it before it reaches the hindgut where Crithidia usually establishes (Koch et al., 2019).

Not only the host, but also the microbiome can play a major role in the metabolism of some nectar metabolites with consequences for activity against parasites. The glycosylation status of a nectar metabolite can directly affect its activity, with the aglycones exhibiting higher activity than the corresponding glycosylated metabolite. This has been observed for the metabolites tiliaside, from linden trees, and unedone, from strawberries (Koch et al., 2022). Tiliaside is a glycoside with in vivo, but not in vitro, activity against Crithidia bombi as it requires deglycosylation by host and/or microbiome enzymes during gut passage to exhibit antiparasitic activity (Koch et al., 2022). Unedone, on the other hand, is not a glycoside and has both in vitro and in vivo activity against C. bombi. Interestingly, unedone is first glycosylated and inactivated in the midgut by bee enzymes, then deglycosylated and reactivated in the hindgut by the microbiome (Koch et al., 2022).

A few other studies have also investigated the roles of the microbiota in the metabolism of other plant metabolites. Kešnerová et al., 2017, demonstrated that honey bees monocolonized with strains of Bifidobacterium, Bombilactobacillus, or Lactobacillus can metabolize flavonoid glycosides. Indeed, genomic analyses have shown that the genomes of strains from these bacterial groups contain a diverse set of carbohydrate processing genes, including GHs that could be involved in the cleavage of sugar residues (Zheng et al., 2019; Ellegaard et al., 2019). We found strain variation for amygdalin metabolism, and such variation likely influences processing of other dietary secondary metabolites. Strain-level variations in the microbiome could, consequently, affect parasite persistence or establishment. Perturbation of the honey bee gut microbiota by pesticides and/or heavy metals (Motta and Moran, 2020b; Rothman et al., 2020; Motta et al., 2022b) could indirectly affect parasite success in the bee gut through changes in the metabolism of secondary metabolites by the microbiome.

Interactions between gut microbiomes and pathogens are widespread and have repeatedly been shown for the bee gut microbiota (Raymann and Moran, 2018). In honey bees, the core microbiota contributes to protection against RNA viruses (Dosch et al., 2021), pathogenic bacterial infections (Steele et al., 2021; Lang et al., 2022), and the microsporidian parasite Nosema (Wu et al., 2020). Further, infection by Nosema, for example, can lead to microbiome dysbiosis (Paris et al., 2020; Rubanov et al., 2019; Huang et al., 2018). Although microbiota-conferred protection seems to be common in bees, the underlying mechanisms are generally unknown. They may involve the host immune system (Horak et al., 2020; Kwong et al., 2017a) and/or direct microbial interactions within the gut (Steele et al., 2017). Our study on amygdalin shows that microbial metabolism of dietary components may be one mechanism through which microbiota members impact hosts or co-resident microbes.

Our results show the relevance of the microbiota for the metabolism of plant toxins, using amygdalin as an example of a toxin that is jointly metabolized by the host and the microbiota. The consequences for bees of the full metabolism of amygdalin are not yet clear-cut but point toward the beneficial to neutral spectrum of host-microbe interactions. The metabolism of most field-relevant concentrations of amygdalin does not affect bee survival rates or microbiota composition and does not deter bees (Tiedeken et al., 2014). In some instances, dietary amygdalin can reduce parasite and viral loads (Tauber et al., 2020; Palmer-Young et al., 2017). Future work could investigate the amounts of hydrogen cyanide released during in vitro experiments with amygdalin-degrading bacteria or during in vivo experiments with microbiota-colonized and microbiota-deprived bees. The full impact of amygdalin on bee health could be assessed using experiments testing whether hydrogen cyanide released into the gut may protect bees infected by specific parasites. Such experimental studies could better elucidate how gut microbial communities metabolize plant metabolites and how this metabolism affects host fitness.

Bacterial strains are available by request from the Moran Lab. The complete genome sequence of strain BI-2.5 has been deposited at DDBJ/ENA/GenBank under the accession CP031513. The genome assemblies for strains BI-1.1, LV-8.1, BI-4G, L5-31, OCC3 and wkB204 have been deposited at DDBJ/ENA/GenBank under the accessions QOCR00000000, QOCS00000000, QOCU00000000, QOCT00000000, QOCV00000000 and JAFMNU020000000, respectively. 16S rRNA amplicon sequencing data are available at NCBI BioProject PRJNA865802.

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   2. Kwong WK
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   ID JAFMNU000000000.2. Bifidobacterium asteroides strain wkB204, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/JAFMNU000000000.2
2. 1. Motta EVS
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   (2022) NCBI BioProject

   ID PRJNA865802. Cooperative host-microbe metabolism of a plant toxin in bees.

   https://www.ncbi.nlm.nih.gov/bioproject/PRJNA865802/

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   ID QOCV00000000. Lactobacillus bombicola strain OCC3, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/QOCV00000000
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   ID QOCU00000000. Lactobacillus bombicola strain BI-4G, whole genome shotgun sequencing project.

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   ID QOCS00000000. Bombilactobacillus bombi strain LV-8.1, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/QOCS00000000
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   ID QOCR00000000. Bombilactobacillus bombi strain BI-1.1, whole genome shotgun sequencing project.

   https://www.ncbi.nlm.nih.gov/nuccore/QOCR00000000
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Author details

1. Erick VS Motta

   Department of Integrative Biology, The University of Texas at Austin, Austin, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   erickvsm@utexas.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9360-4353

2. Alejandra Gage

   Department of Integrative Biology, The University of Texas at Austin, Austin, United States

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Thomas E Smith

   Department of Integrative Biology, The University of Texas at Austin, Austin, United States

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Kristin J Blake

   Mass Spectrometry Facility, Department of Chemistry, The University of Texas at Austin, Austin, United States

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Waldan K Kwong

   Instituto Gulbenkian de Ciência, Oeiras, Portugal

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Ian M Riddington

   Mass Spectrometry Facility, Department of Chemistry, The University of Texas at Austin, Austin, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

7. Nancy Moran

   Department of Integrative Biology, The University of Texas at Austin, Austin, United States

   Contribution

   Resources, Supervision, Funding acquisition, Writing - review and editing

   For correspondence

   nancy.moran@austin.utexas.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2983-9769

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