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LX3 enhances larval pathogen eradication by antibiotics. Experimental hives were subjected to standard antibiotic treatment with oxytetracycline (OTC) for 2 weeks and then supplemented for 4 weeks with either pollen patties containing LX3 (LX3) or pollen patties containing vehicle (VEH). No treatment control (NTC) hives received no further treatment after OTC. a Schematic diagram outlining the experimental design. b, c Molecular-based quantification of P. larvae in honey bee larvae (whole body) and adults (dissected abdomen) collected just prior to the start of OTC exposure (A.0), and then after 1 (A.1) and 2 (A.2) weeks of exposure. Data are depicted as median ± 95% confidence intervals (Kruskal–Wallis with Dunn’s multiple comparisons) at different time points. Each data point represents either one individual (adults) or three pooled individuals (larvae) sampled equally from a total of n = 6 hives. d, e Molecular-based quantification of P. larvae in larvae (whole body) and adults (dissected whole abdomens) at the start of the supplementation period (S.0; corresponding to 3 days post A.2 time point), and then after 2 (S.2) and 4 (S.4) weeks. Data are depicted as mean ± standard deviation (two-way ANOVA with Sidak’s multiple comparisons) at different time points with each data point representing either one individual (adults) or three pooled individuals (larvae) sampled equally from n = 4 hives per treatment group. f, g Capped brood counts during OTC treatment (n = 6 hives) and subsequent supplementation period (n = 4 hives per treatment group). Data represents the median (line in box), IQR (box), and minimum/maximum (whiskers) of relative change in brood counts normalized by hive. Statistics shown for one-way and two-way ANOVA, respectively, with Sidak’s multiple comparisons for both. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant.
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Widespread antibiotic usage in apiculture contributes substantially to the global dissemination of antimicrobial resistance and has the potential to negatively influence bacterial symbionts of honey bees (Apis mellifera). Here, we show that routine antibiotic administration with oxytetracycline selectively increased tetB (efflux pump resistance gen...
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... Disease prevention and manage the transmission of pathogens within and across apiaries, beekeepers often resort to treating their hives with antibiotics. This practice aims to minimize the spread of infections and protect the health of bee colonies, though it may also contribute to antibiotic residue issues in honey [13]. ...
Laboratory and field studies were carried out to assess the efficacy of the antibiotic tetracycline in preventing and managing American foulbrood (AFB) and European foulbrood (EFB) diseases in honey bees. Experiments with immature worker bees in the laboratory demonstrated that honey bee larvae could endure a broad range of antibiotic doses in their diet. The recommended dose of tetracycline (100 μg/ml) effectively protected young larvae from infection. However, once the dose exceeded this recommended level, it reached a point where it became lethal.
... Our confirmation of immune activation used a relatively broad assessment of antibacterial activity in the bee hemolymph, but it is known that different immune challenges can lead to different host gene expression profiles (67) that could precipitate such differences. The patterns for Gilliamella and Snodgrassella noted in our empirical study can be partially linked to prior findings in correlational analyses examining the influence of probiotic supple mentation on the microbiome and immune system of honeybees (96,97). Positive and negative associations were found between the expression of antimicrobial peptides and the abundances of Gilliamella and Snodgrassella, and several peptides, such as defen sin-2, abaecin, and lysozyme, produced correlations of differing direction or magnitude with the two bacteria (97). ...
... The patterns for Gilliamella and Snodgrassella noted in our empirical study can be partially linked to prior findings in correlational analyses examining the influence of probiotic supple mentation on the microbiome and immune system of honeybees (96,97). Positive and negative associations were found between the expression of antimicrobial peptides and the abundances of Gilliamella and Snodgrassella, and several peptides, such as defen sin-2, abaecin, and lysozyme, produced correlations of differing direction or magnitude with the two bacteria (97). A related study found that increased expression of several antimicrobial peptides was positively correlated with the abundance of Gilliamella, with correlations with Snodgrassella being weaker. ...
Understanding factors influencing the composition and maintenance of beneficial host-associated microbial communities is central to understanding their ecological, evolutionary, and health consequences for hosts. Host immunity is often implicated as a regulator of these microbiota, but immunity may also play a disruptive role, with responses to infection perturbing beneficial communities. Such effects may be more prominent from innate immune responses, with more rapid-acting and often non-specific components, compared to adaptive responses. We investigated how upregulation of antibacterial immunity in the bumble bee Bombus impatiens affects its core gut microbiota, testing the hypothesis that immunity-induced perturbation impacts the microbiota structure. Freshly emerged adult bees were fed a microbiota inoculum before receiving a non-pathogenic immune stimulation injection. We quantified microbial communities using 16S rRNA amplicon sequencing and targeted quantitative PCR. Coarse community membership shows apparent robustness, but we find that immune stimulation alters the abundance of two core community members, Gilliamella and Snodgrassella. Moreover, a positive association in communities between these bacteria is perturbed following a Gram-negative challenge. The observed changes in the gut microbial community are suggestive of immune response-induced dysbiosis, linking ecological interactions across levels between hosts, their pathogens, and their beneficial gut microbiota. The potential for collateral perturbation of the natural gut microbiota following an innate immune response may contribute to immune costs, shaping the evolutionary optimization of immune investment depending on the ecological context.
IMPORTANCE
Our work demonstrates how innate immunity may influence the host-associated microbiota. While previous work has demonstrated the role of adaptive immunity in regulating the microbiota, we show that stimulation of an innate immune response in bumble bees may disrupt the native gut microbial community by shifting individual abundances of some members and pairwise associations. This work builds upon previous work in bumble bees demonstrating factors determining microbe colonization of hosts and microbiota membership, implicating immune response-induced changes as a factor shaping these important gut communities. While some microbiota members appear unaffected, changes in others and the community overall suggests that collateral perturbation of the native gut microbiota upon an innate immune response may serve as an additional selective pressure that shapes the evolution of host innate immunity.
... As an effort to complement the bee's natural tendency to keep their colonies disease free, many beekeepers (outside of Europe) use antibiotics, which can be immediately effective against certain pathogens, but these medicated treatments are tightly regulated due to concerns about residual accumulation in honey, as well as other offtarget side effects (Lima et al., 2020), including disruption of the natural bee gut microbiota which, paradoxically, can leave colonies more vulnerable to subsequent infection (Daisley et al., 2020;Raymann et al., 2017;Zhang et al., 2022a). Alternative disease management interventions in beekeeping include essential oils (Hybl et al., 2021), RNA interference technologies (Garbian et al., 2012) or variations of transgenerational immune priming (Dickel et al., 2022). ...
... Alberoni et al. (2016) summarize some of these effects, which include nutrient uptake, the production of fatty acids, amino acids and other metabolites, and protection of the host from pathogens and parasites, either by stimulating immune function or by directly inhibiting pathogen growth. Recent research has exploited this coevolved relationship between microbe and host − the holobiont − to demonstrate that strategic manipulation of the worker gut microbiome can help bees recover from dysbiosis (Daisley et al., 2020) and even bolster bee immunity to protect against further gutborne disease (Daisley et al., 2019;Raymann and Moran, 2018). Despite the prospect of microbial therapeutics, the idea of using gut microbe manipulations as a beekeeping tool has received relatively little research attention (Chmiel et al., 2021) and this despite the availability of some reportedly bee-friendly 'probiotic' products (Damico et al., 2023). ...
The holobiont theory of evolution explains how individuals are deeply symbiotic with their gut microbes, such that microbes are adapted to influence host metabolism, immunity and behaviour, as signalled from the gut to the brain. For eusocial taxa like the Western honey bee (Apis mellifera), this brain-gut axis may scale up from the individual to affect entire colonies. Here, we examine how microbial supplementation of honey bee feeds could manipulate the brain-gut axis to affect hygienic and other social behaviours relevant to beekeeping, such as foraging, recruitment (dance language) and defence. To illustrate this concept, we focus on various lactic acid-producing bacteria that can synthesize neurotransmitters such as octopamine, dopamine, serotonin and g-aminobutyric acid, which can influence an individual bee's behavioural cycles and responsiveness to environmental cues. If the behaviour of a worker bee can be deliberately manipulated, and this effect multiplied across many workers, microbial neurotherapeutics could conceivably render colonies more behaviourally responsive to symptoms of disease, or more motivated to forage or possibly less aggressive towards beekeepers. Drawing from the scientific literature, we infer how microbial supplements, such as neurostimulatory or neurosuppressive probiotics, could be applied or even engineered to co-opt the brain-gut axis to bolster colony health or improve performance. The mechanistic link between the gut microbiota and the collective social behaviour of single colonies remains an understudied aspect of honey bee social biology with relevance to apiculture.
... Several animal studies and human clinical trials have demonstrated the protective effect of speci c strains of probiotic Lactobacillus against intestinal diseases, including antibiotic-associated diarrhea. For example, L. acidophilus can regulate antibiotic-induced intestinal ora disturbance and diarrhea in mice and immune and microbiome dysregulation in bees [35,36] . LAB NS8 can improve the damage of antibiotics to intestinal ora and play a key role in maintaining the balance of intestinal ora [37] . ...
In this study, four lactic acid bacteria (LAB) strains demonstrating ciprofloxacin, bile salt, gastric fluid and intestinal fluid tolerance; as well as adhesion ability to Caco-2 and HT-29 cells were used to improve and recover the intestinal flora disorders caused by ciprofloxacin. Among which, Lactobacillus brevis 505 exhibited excellent adhesion ability to two kinds of cells and colonization ability to mouse intestinal. After ciprofloxacin treatment, certain recovery effect on cecum caused by ciprofloxacin in the mice was found during natural recovery (group 5C2), but it was challenging to fully restore the intestinal integrity to the initial level. After L. brevis 505 intervention (group 5C5), the intestinal damage to the colon and ileum caused by ciprofloxacin in mice was significantly alleviated, the recovery effect was better than that of natural recovery. Additionally, L. brevis 505 could effectively regulate INF-γ, sIgA and RegⅢγ increase induced by ciprofloxacin. Shannon and Simpson index of the intestinal flora of mice in 5C5 group were higher than those in other group, the relative abundance of Bifidobacterium and Lactobacillus in the mice in 5C5 group was increased, indicating that LAB can better restore the structure and abundance of intestinal microflora. Consequently, L. brevis 505 shows promise as a probiotic for gut microbiota restoration and rebuilding during antibiotic therapy.
... In the honey bee, LAB plays a protective role against pathogens [58], and supports respiratory requirements in cockroaches [59,60]. Lactobacillus lactis (LX3) enhanced the expression of genes responsible for antimicrobial peptides, improved productivity, and boosted the immune response in adult bees [61]. Therefore, a higher abundance of the LAB in different diet groups can be considered a marker for diet quality. ...
These authors contributed equally to this work. Abstract: Honey bee (Apis mellifera L.) health is crucial for honey bee products and effective pollination , and it is closely associated with gut bacteria. Various factors such as reduced habitat, temperature, disease, and diet affect the health of honey bees by disturbing the homeostasis of the gut microbiota. In this study, high-throughput 16S rRNA gene sequencing was used to analyze the gut microbiota of honey bees subjected to seven diets over 5 days. Lactobacillus dominated the microbiota in all diets. Cage experiments (consumption, head protein content, and vitellogenin gene expression level) were conducted to verify the effect of the diet. Through a heatmap, the Diet2 (probiotic-supplemented) group was clustered together with the Beebread and honey group, showing high consumption (177.50 ± 26.16 mg/bee), moderately higher survival duration (29.00 ± 2.83 days), protein content in the head (312.62 ± 28.71 µg/mL), and diet digestibility (48.41 ± 1.90%). Additionally , we analyzed the correlation between gut microbiota and health-related indicators in honey bees fed each diet. Based on the overall results, we identified that probiotic-supplemented diets increased gut microbiota diversity and positively affected the overall health of individual honey bees.
... Considering that bees feed almost exclusively on syrup after autumn feeding, it is likely that a microbiota disturbed by an antimicrobial (such as Fumidil B ® , used at the end-of-season feeding) will have difficulty returning to a homeostasis state. Overall, significant impairments in the honeybee microbiota have been repeatedly associated with the use of antibiotics such as tetracycline-derived compounds [7,51]. Collectively, these studies highlight that frequently used antibiotics reduce both the abundance and genetic diversity of microbiota strains. ...
Against a backdrop of declining bee colony health, this study aims to gain a better understanding of the impact of an antimicrobial (Fumidil B®, Can-Vet Animal Health Supplies Ltd., Guelph, ON, Canada) and a probiotic (Bactocell®, Lallemand Inc., Montreal, QC, Canada) on bees’ microbiota and the health of their colonies after wintering. Therefore, colonies were orally exposed to these products and their combination before wintering in an environmental room. The results show that the probiotic significantly improved the strength of the colonies in spring by increasing the total number of bees and the number of capped brood cells. This improvement translated into a more resilient structure of the gut microbiota, highlighted by a more connected network of interactions between bacteria. Contrastingly, the antimicrobial treatment led to a breakdown in this network and a significant increase in negative interactions, both being hallmarks of microbiota dysbiosis. Although this treatment did not translate into a measurable colony strength reduction, it may impact the health of individual bees. The combination of these products restored the microbiota close to control, but with mixed results for colony performance. More tests will be needed to validate these results, but the probiotic Bactocell® could be administrated as a food supplement before wintering to improve colony recovery in spring.
... For instance, exposure to antibiotics and agrochemicals disrupts gut bacterial communities and increases mortality under pathogen challenges (Raymann et al., 2017;Motta et al., 2018;Motta & Moran, 2020). Probiotic inoculation partially counteracts the dysbiosis caused by antibiotic treatment and improves resistance to pathogens (Daisley et al., 2020b;Powell et al., 2021). By providing a finescale taxonomy and phylogenetic framework for the gut bacteria of social bees, this database enables improved assessments of the composition, structure and dynamic trends in bee gut microbiota. ...
Honeybees and bumblebees play a crucial role as essential pollinators. The special gut microbiome of social bees is a key factor in determining the overall fitness and health of the host. Although bees harbor relatively simple microbial communities at the genus level, recent studies have unveiled significant genetic divergence and variations in gene content within each bacterial genus. However, a comprehensive and refined genomics-based taxonomic database specific to social bee gut microbiomes remains lacking. Here, we first provided an overview of the current knowledge on the distribution and function of social bee gut bacteria, as well as the factors that influence the gut population dynamics. We then consolidated all available genomes of the gut bacteria of social bees and refined the species-level taxonomy, by constructing a maximum-likelihood core genome phylogeny and calculating genome-wide pairwise average nucleotide identity. On the basis of the refined species taxonomy, we constructed a curated genomic reference database, named the bee gut microbe genome sequence database (BGM-GDb). To evaluate the species-profiling performance of the curated BGM-GDb, we retrieved a series of bee gut metagenomic data and inferred the species-level composition using metage-nomic intra-species diversity analysis system (MIDAS), and then compared the results with those obtained from a prebuilt MIDAS database. We found that compared with the default database, the BGM-GDb excelled in aligned read counts and bacterial richness. Overall, this high-resolution and precise genomic reference database will facilitate research in understanding the gut community structure of social bees.
... environments characterized by an acidic pH and that use carbohydrates as the only or main carbon source to produce lactic acid (Axelsson and Ahrné, 2000;George et al. 2018). Many LAB have been identified as probiotics in insects, as occurs with honeybees (Vásquez et al. 2012), and for their host immune protection (Daisley et al. 2020;Iorizzo et al. 2022). Therefore, it is likely that the function of these LAB in S. acupunctus includes the utilization of sugars and that they mediate gate-keeping activities against pathogens. ...
The agave weevil, Scyphophorus acupunctatus, is a pest of agave. Its larvae cause damage to agaves by boring holes in the plant. Boring requires that the insect consume the constituents of its host plant, which contains sugars and many recalcitrant polymers. It has been hypothesized for many years that the gut bacterial communities of S. acupunctatus play a role in its ability to metabolize agave components. However, studies exploring this insect's gut bacterial communities have yet to be performed. In this work, we used a 16S rRNA gene-based metabarcoding approach to characterize the gut bacterial communities of field-collected agave weevils from different localities in Mexico. We found that external factors, including host plants, have important effects on the structure of the gut bacterial communities of S. acupunctatus. Despite this variability, we found a discrete core bacterial community mainly composed of the genera Prevotella, Pectinatus, Liquorilactobacillus, Secundilactobacillus, Paucilactobacillus, and Pseudomonas. These genera may be necessary for S. acupunctatus as metabolic helpers and/or gatekeepers. Additional studies are needed to fully assess the functionality of the gut bacterial community of this species in terms of its metabolic contribution, which may help to decipher their potential ecological implications. The information we provided here is the first step for guiding further questions.
... Following the recent microbiome revolution 5 , commercial probiotics have been used by beekeepers with the promise of reducing or controlling resident pathogens and restoring or improving the gut microbiome generally, or following antibiotic treatment 6 . Probiotic development and testing in honey bees has been limited [7][8][9] , and there is little empirical evidence supporting the effectiveness of non-native probiotics 10,11 . ...
... Studies sequencing the gut as a whole typically result in libraries biased towards rectum species, but provide less resolution for the ileum biofilm, which often contains an order of magnitude fewer cells 30 . The gut microbiota is disrupted by antibiotic application in the lab and field leading to substantial decreases in Lactobacillius and Bifidobacterium followed by dysbiosis and host susceptibility to disease 6,7,31 . In the United States, honey bees have been exposed to commercially available antibiotics for many decades 32 , and many members of the gut microbiome have acquired antibiotic resistance genes relative to those in honey bees with less recent exposure 33 . ...
... Some of the core gut species alter significantly with task, age and overwintering 18,29,73 , but it is mostly unknown how the microbiota behaves overwinter 69,73 or how various forms of dysbiosis or alternate gut enterotypes affect honey bee health 74,75 . Perhaps replacement by Gilliamella apicola and Commensilabacter (Alpha 2.1) only represents a slightly substandard microbiome although antibiotics have been shown to increase pathogen susceptibility by disrupting the gut microbiome and associated immune physiology 6,7,76 . When sequenced or analyzed as a whole, the healthy honey bee gut microbiome is represented by an even distribution of species 77 . ...
Probiotics are widely used in agriculture including commercial beekeeping, but there is little evidence supporting their effectiveness. Antibiotic treatments can greatly distort the gut microbiome, reducing its protective abilities and facilitating the growth of antibiotic resistant pathogens. Commercial beekeepers regularly apply antibiotics to combat bacterial infections, often followed by an application of non-native probiotics advertised to ease the impact of antibiotic-induced gut dysbiosis. We tested whether probiotics affect the gut microbiome or disease prevalence, or rescue the negative effects of antibiotic induced gut dysbiosis. We found no difference in the gut microbiome or disease markers by probiotic application or antibiotic recovery associated with probiotic treatment. A colony-level application of the antibiotics oxytetracycline and tylosin produced an immediate decrease in gut microbiome size, and over the longer-term, very different and persistent dysbiotic effects on the composition and membership of the hindgut microbiome. Our results demonstrate the lack of probiotic effect or antibiotic rescue, detail the duration and character of dysbiotic states resulting from different antibiotics, and highlight the importance of the gut microbiome for honeybee health.
... Also, the population of lactobacilli in the intestines of bees fed with the treatment of 2 g of probiotic per liter of syrup was significantly higher than other experimental treatments. Lactic Acid Bacteria, Bifidobacteria and Bacillus species, are the major groups which could mitigate the antibiotic-associated microbiota dysbiosis and immune deficits in adult workers(Daisley et al., 2020). The benefits of Bacillus species lie in their ability to produce a number of enzymes and antibiotic-like substances that could assist in the digestion of carbohydrates and suppress the growth of pathogenic or nocuous organisms in honey bees, respectively(Teo et al., 2005;Tootiaie et al., 2021). ...
The purpose of this study was to evaluate the effect of different levels of probiotic on colony functional traits, honey quantitative and qualitative parameters and gut microbial flora in honey bees. A total of 56 hives were randomly allocated to 8 treatments with 7 replicates in the city of Firouzkoh (Iran) for about 60 days. Experimental treatments that were included: control, 2 g antibiotic, 1 g probiotic, 2 g probiotic, 3 g probiotic, 1 g antibiotic + 1 g probiotic, all per liter of syrup for each hive. The results showed that hives fed with treatments 2 g or 3 g probiotic per liter of syrup had higher hive population and honey production than the other treatments (p<0.05). Adding 2 g or 3 g probiotic per liter of syrup increased lactobacillus counts in bees’ intestines compared to the other treatments (p<0.05). Also, supplementation of 1 g or 2 g probiotic and 2 g antibiotic per liter of syrup for each hive decreased Escherichia coli counts in the intestine compared to the other treatments (p<0.05). The adding 1 g or 2 g probiotic per liter of syrup for each hive increased the levels of fructose and glucose in honey compared to the other treatments (p<0.05). The supplementation of 1 g or 2 g probiotic per liter of syrup decreased sucrose in honey compared to the other treatments (p<0.05). The results of the present study suggest that probiotic might be used as a feed additive for increased honey quality and gut microbial flora improvement in honey bees.