Background
Rhodiola rosea, informally referred to as the ‘golden root’ or ‘arctic root’, is an adaptogenic plant that has been reported to display positive effects on central nervous system activity and cardiovascular function [
1‐
4]. The additional therapeutic effects of
R. rosea, which derive primarily from its root extract, have been outlined in clinical trials for improving mental and physical work capacity during stress, alleviating mental distress, and ameliorating symptoms of depression [
2,
5‐
10]. Although the traditional medicinal uses of
R. rosea derive from Eastern Europe and Asia,
R. rosea products have gained popularity worldwide among athletes as a natural remedy to prevent fatigue and improve performance [
11]. We reported that
R. rosea significantly extended both mean (24%, both sexes) and maximum (16% in males, 31% in females) lifespan of the fruit fly,
Drosophila melanogaster [
12,
13]. The lifespan extension properties of
R. rosea appear to be conserved among model species since the plant has been shown to extend the lifespan of worm and yeast models as well [
14,
15]. The mechanism of lifespan extension with
R. rosea, however, remains to be determined.
Drosophila melanogaster is emerging as an important model to examine the interactions between non-pathogenic microbes within the host. Since
D. melanogaster can be easily manipulated genetically and experimentally, it can serve as a good model to enhance our understanding of animal–microbial symbiosis. Utilizing the
Drosophila model system provides an integrative approach to study the relationship between an herbal extract supplementation and the impact it may have on the gut microbial composition. Another species of the
Rhodiola family,
Rhodiola crenulata, also exhibits multiple pharmacological traits like that of
R. rosea, such as stress protection, neuroprotection, high altitude sickness mitigation, and anti-inflammatory activity [
16‐
19]. Moreover,
R. crenulata has been demonstrated to treat metabolic disorders in rats [
20] and increase intracellular antimicrobial peptide expression while improving gut morphology in fruit flies [
21]. Here we suspect that
R. rosea may act like
R. crenulata in that it may change the microbial composition of
D. melanogaster. Additionally,
R. rosea may mimic numerous other herbal therapies that have been reported to alleviate gastrointestinal and metabolic disorders, which are particularly prevalent in the process of age-related microbial dysbiosis [
22‐
24]. More specifically, the intestinal microbiota is significantly altered during severe age-related physiological ailments, such as obesity, insulin resistance, and general frailty, suggesting that age-related changes in the gut may have an impact on overall healthspan and lifespan [
25‐
27].
The average adult
Drosophila intestine harbors only 5–20 microbial species which primarily belong to the families
Enterobacteriaceae, Acetobacteraceae, and the order
Lactobacillales [
28‐
31]. Of these three strains, the only bacterial order present in considerable amounts in both
Drosophila and mammals is
Lactobacillales [
32‐
34]. When evaluating the microbial differences between fruit flies, it appears that the microbial content of
D. melanogaster, independent of species uniformity, is similar between species that are fed on the same diet [
35]. Conversely, more closely related species that feed on different diets are known to have a contrasting and diverse microbial compositions [
35]. These findings suggest that certain bacterial families, such as
Acetobacteraceae, may favor the low pH and high ethanol conditions present in fermenting fruits, thus influencing the microbiota of flies which favor fruit based diets [
35].
The purpose of this work was to examine whether the anti-aging properties of R. rosea are due to its impact on the microbial composition of the fly gut. To date, there have been no published studies highlighting the impact of anti-aging botanical extracts on the microbial composition of the gut. These results will aim to build support for investigating the effects of botanical extracts on the gut microbiota and how they may help prevent against age-related intestinal diseases.
Discussion
The aim of this study was to determine whether
R. rosea can change the gut microbial community of
D. melanogaster. Our group had previously reported that the root extract of
R. rosea extends the lifespan and improves the healthspan of the fruit fly, but the exact underlying mechanisms of lifespan extension remains unclear [
12,
13,
36]. In this study, we examined the impact of
R. rosea on the microbial dynamics of the fly gut and whether changing the gut microbiome could be beneficial for host longevity. When evaluating the impact of
R. rosea on the fly gut microbiota, we observed sex specific differences between fly groups which could be contributed to a variety of physiological factors. At adult stages, female fruit flies require a greater protein intake needed for egg production, thus consuming more environmental yeast when compared to their male counterparts [
58]. Due to extensive contact with environmental nutrients, the female flies, along with their microbial communities, experience metabolism-related shifts through alteration of host signaling pathways [
59]. Performing 16S rRNA sequencing exclusively on female fruit flies allowed for investigation into the environmental and nutrient microbe-altering effects of
R. rosea and how it influenced the microbial community of the host. Studies involving both sexes and multiple strains of
Drosophila will be required to thoroughly understand the paired effect of
R. rosea and yeast consumption on the host microbiota.
Our results show that while control female Oregon-R fruit flies establish and maintain a consistent microbial composition throughout their lifespan, the
R. rosea supplemented flies maintained a microbial composition which differed in relative abundance of order
Lactobacillales and genus
Acetobacter when compared to control (Figs.
1b and
2b). These changes, with respect to supplementation of
R. rosea, are likely to vary between
Drosophila strains, with additional factors influencing the microbiota such as the nutritional composition and sex [
35,
60‐
62]. Male flies in our study displayed no significant changes in
L. plantarum,
A. pomorum, and the 16S rDNA gene when supplemented with
R. rosea (p > 0.05, Unpaired Welch’s t test) (Additional file
1: Figure S1a–c). CFU tests revealed that male flies displayed significant decrease in CFU counts at early ages of their lifespan (p value = 0.0096 for MRS, p value = 0.0367 for nutrient), but no difference was observed at the later stages of their lifespan, where flies experience an increased bacterial load (Additional file
1: Figure S1d).
Our 16S rRNA amplicon sequencing identified the differences in diversity between control and
R. rosea fed flies. In 10 days old flies, control flies had an average Operational Taxonomic Unit (OTU) count of 18.17, while
R. rosea fed flies had a count of 16.14 (Additional file
2: Table S1). In 40 days old flies, control flies had a OTU count of 22.33, while
R. rosea fed flies had a count of 17.7 (Additional file
2: Table S1). Although we observe a decrease in bacterial diversity in
R. rosea fed flies, the total abundance of bacteria increase, as indicated through 16S rDNA qRT-PCR analysis (Fig.
3a). Although our results suggest that the
R. rosea induced gut microbiome changes are age-dependent, to fully comprehend the time point where
R. rosea begins to induce such changes, additional time points (i.e. time of eclosion) need to be evaluated. Furthermore, since several samples in our study missed certain bacterial genera (
Lactococcus,
Enterococcus), additional samples at various time points need to be evaluated to determine which microorganisms are natively present in the gut in comparison to which are acquired from the environment. To limit the impact on external inputs from contributing to the bacterial load within the
Drosophila gut, many studies have utilized germ-free flies as a model to test the effect of individual bacteria on host physiology [
63,
64]. Utilizing the gnotobiotic model will allow us to control the influence of environmental factors to discern how
R. rosea directly affects individual bacterial species inside the host.
The most notable observations in this study resulted from the ability of
R. rosea to increase the ratio of genus
Acetobacter and decrease the order
Lactobacillales at both the early and later stages of the fly lifespan (Figs.
1c, d and
2c, d). Observations were taken at the order level due to the presence of unidentified reads that belong to the families and genera under the
Lactobacillales order. When comparing the 16S rRNA sequencing data with the 16S rDNA qRT-PCR reads, we noticed that although the genus
Acetobacter was increased in 10 days old flies that were fed
R. rosea, the species
A. pomorum was significantly decreased in these flies (Figs.
1b and
3b). This contrast is possible due to the presence of other commensal species belonging to the genus
Acetobacter, such as
Acetobacter pasteurianus,
Acetobacter aceti, and
Acetobacter tropicalis [
65]. We observed an opposite trend between treatment groups when comparing between the 16S rDNA amplification and CFU counts. A decrease in CFUs corresponded with an increase in 16S rDNA expression, indicating that
R. rosea fed flies experience a lower culturable bacterial load but more overall bacteria (Figs.
3a and
4a). Interestingly enough, both MRS and nutrient agar displayed parallel decreases in bacterial load when
R. rosea fed flies were plated, demonstrating the similarities between the bacteria that are culturable when utilizing the non-selective nature of the nutrient media. A significant decrease in the CFUs with 10 days old flies fed
R. rosea on MRS also suggests that
Lactobacillus is responsible for variation in culturable bacteria at the earlier stages of the fly lifespan (Fig.
4a).
Previous reports demonstrated the impact of an altered diet such as changes in the sugar versus protein composition in the fly media affects the
Acetobacter to
Lactobacillus ratio in flies [
52,
66]. Since a change in the diet impacts host physiology, the effects of the diet on the microbial community suggest that the health of the host is a major determinant for shaping the gut microbial population in flies [
67]. Although studies have reported that the commensal bacterial load fluctuates throughout the
Drosophila lifespan, we observed the dominance of
Acetobacter throughout all stages of the fly life in both control and
R. rosea fed flies [
52]. Since
Acetobacter species thrive under fully aerobic conditions and
Lactobacillus species are incapable of thriving in a ubiquitously oxygenated environment, we propose the possibility that the gut oxygen tension experiences a shift towards aerobic conditions after supplementation of
R. rosea, thus promoting the growth of
Acetobacter [
32,
68]. This is particularly more likely in older flies who consume more oxygen and produce a more severe physiological response to conventional oxygen intake when compared to their younger counterparts [
69]. In addition to
R. rosea playing a role in changing gut oxygen tension, we suspect the extract may further modify immune system function in the
Drosophila gut. An altered gut microbial composition, as a result of the supplementation of
R. rosea, may contribute towards limiting age-related dysplastic changes by positively modulating the process of mis-differentiation in intestinal stem cells (ISCs) and their progeny, leading to the improvement in intestinal function and subsequently benefitting the health of the host. Because epithelial barrier dysfunction is strongly associated with fly aging and mortality, we believe
R. rosea may attenuate this process at the later stages of the fly life [
70]. In summary, evaluating the impact of anti-aging botanical extracts, such as
R. rosea, on the gut microbiome using
D. melanogaster as a model system may provide a platform to understand the interactions between the microbiome, lifespan, and healthspan.
Authors’ contributions
Conceived and designed the experiments: KEL, EAS, SES, MJ. Performed the experiments: KEL, DK. Analyzed the data: KEL, DK, EAS, SES, MJ. Wrote the manuscript: KEL, DK, MJ. All authors read and approved the final manuscript.