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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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... Recently, several research groups have been investigating other probiotic treatments [28][29][30][31], as reviewed in [26], which are not yet commercially available to beekeepers. One probiotic that is currently being tested is the The BioPatty™, which is made of a mixture of three Lactobacillus strains, including Apilactobacillus kunkeei (a hive-associated bacterium that is often found in the honey bee gut) [29,30]. ...
... Recently, several research groups have been investigating other probiotic treatments [28][29][30][31], as reviewed in [26], which are not yet commercially available to beekeepers. One probiotic that is currently being tested is the The BioPatty™, which is made of a mixture of three Lactobacillus strains, including Apilactobacillus kunkeei (a hive-associated bacterium that is often found in the honey bee gut) [29,30]. The BioPatty™ has been shown to increase the expression of bee immunity genes, reduce infection my P. larvae, and help restore the microbiome following antibiotic perturbation [29]. ...
Antibiotic use in apiculture is often necessary to ensure the survival of honey bee colonies. However, beekeepers are faced with the dilemma of needing to combat bacterial brood infections while knowing that antibiotics also kill beneficial bacteria important for bee health. In recent years, bee probiotics have become increasingly purchased by beekeepers because of product claims like, being able to replenish the microbes lost due to agricultural modifications of honey bees' environment or promote optimal gut health. Unfortunately, these products have little scientific evidence to support their efficacy, and previous lab experiments have refuted some of their claims. Here, we performed hive-level field experiments to test the effectiveness of SuperDFM- HoneyBeeTM, the most commonly purchased honey bee probiotic in the United States, on restoring the honey bee gut microbiome after antibiotic treatment. Our results indicated that bees administered the probiotic after antibiotic treatment did not recover their microbiota faster than bees only given the antibiotic treatment. Moreover, the microorganisms in the probiotic supplement were never found in the guts of the worker bee samples, even during active treatment. These results highlight that more basic research is needed to test the efficacy and outcomes of currently available commercial honey bee probiotic supplements.
... Indeed, the proximity of thousands of genetically close individuals within a hive facilitates the development and dispersal of pathogens (Fries and Camazine, 2001). It is their foraging behavior that brings them into contact with anthropogenic stressors, as well as beekeepers' treatments (Fulton et al., 2019;Xiao et al., 2022), which can also be detrimental in certain circumstances for honey bees (e.g., intestinal dysbiosis, brood toxicity) (Pettis et al., 2004;Thompson et al., 2005;Daisley et al., 2020;Jia et al., 2022). In the context of deteriorating bee health, the spread of new parasites, increasing exposure to pesticides and hive treatments, beekeeping practices, diminishing floral resources, and declining queen quality could be the main factors responsible for this tragedy (Smith et al., 2013;Hristov et al., 2020a). ...
The honeybee is an important species for the agri-food and pharmaceutical industries through bee products and crop pollination services. However, honeybee health is a major concern, because beekeepers in many countries are experiencing significant colony losses. This phenomenon has been linked to the exposure of bees to multiple stresses in their environment. Indeed, several biotic and abiotic stressors interact with bees in a synergistic or antagonistic way. Synergistic stressors often act through a disruption of their defense systems (immune response or detoxification). Antagonistic interactions are most often caused by interactions between biotic stressors or disruptive activation of bee defenses. Honeybees have developed behavioral defense strategies and produce antimicrobial compounds to prevent exposure to various pathogens and chemicals. Expanding our knowledge about these processes could be used to develop strategies to shield bees from exposure. This review aims to describe current knowledge about the exposure of honeybees to multiple stresses and the defense mechanisms they have developed to protect themselves. The effect of multi-stress exposure is mainly due to a disruption of the immune response, detoxification, or an excessive defense response by the bee itself. In addition, bees have developed defenses against stressors, some behavioral, others involving the production of antimicrobials, or exploiting beneficial external factors.
... Sucrose syrup suspensions remain the most popular method based on ease of application, but osmotic stress can induce rapid cell lysis of many bacteria (e.g., >90% reduction in LAB cell viability within 96 h at 30°C [19]) making it a poor delivery vehicle in most instances. In contrast, pollen patty-based methods offer a more suitable system (viable bacteria shown to reach their intended targets of the honey bee intestinal tract [20,21]) while also contributing nutritional benefits that support overall health [22]. A major drawback to either method is that only adult bees directly consume the product, meaning that distribution throughout the brood chamber (where many larval pathogens exist) is primarily reliant on nutrient flux between nurse bees (which directly consume the product) and larvae (which are fed by nurse bees via trophallaxis) in the hive [21]. ...
... In our previous work, we demonstrated that a patty-based hive supplement comprising a three strain LAB consortium (Lactiplantibacillus plantarum Lp39, Lacticaseibacillus rhamnosus GR-1, and Apilactobacillus kunkeei BR-1; herein referred to as LX3) could strongly suppress P. larvae burden in both symptomatic and asymptomatic colonies, as well as induce beneficial effects on adult immune and microbiota systems [20,27]. Based on these multifaceted mechanisms and general improvement of host constitution, LX3 could be expected to increase resistance toward a broad range of infectious diseases. ...
... The dissected hindguts and heads of selected samples were then pooled in biological triplicate and homogenized in TRIzol (Invitrogen) by bead beating, followed by RNA extraction via the manufacturer's instructions. From the remaining TRIzol interphase layer, DNA was extracted using a back-extraction buffer consisting of 4 M guanidine thiocyanate, 50 mM sodium citrate dihydrate, and 1 M Tris base, as previously described [20]. Quality of RNA and DNA was assessed using a microvolume spectrophotometer (DS-11 Spectrophotometer; DeNovix). ...
Managed honey bee ( Apis mellifera ) populations play a crucial role in supporting pollination of food crops but are facing unsustainable colony losses, largely due to rampant disease spread within agricultural environments. While mounting evidence suggests that select lactobacilli strains (some being natural symbionts of honey bees) can protect against multiple infections, there has been limited validation at the field-level and few methods exist for applying viable microorganisms to the hive. Here, we compare how two different delivery systems—standard pollen patty infusion and a novel spray-based formulation—affect supplementation of a three-strain lactobacilli consortium (LX3). Hives in a pathogen-dense region of California are supplemented for 4 weeks and then monitored over a 20-week period for health outcomes. Results show both delivery methods facilitate viable uptake of LX3 in adult bees, although the strains do not colonize long-term. Despite this, LX3 treatments induce transcriptional immune responses leading to sustained decreases in many opportunistic bacterial and fungal pathogens, as well as selective enrichment of core symbionts including Bombilactobacillus , Bifidobacterium , Lactobacillus , and Bartonella spp. These changes are ultimately associated with greater brood production and colony growth relative to vehicle controls, and with no apparent trade-offs in ectoparasitic Varroa mite burdens. Furthermore, spray-LX3 exerts potent activities against Ascosphaera apis (a deadly brood pathogen) likely stemming from in-hive dispersal differences, whereas patty-LX3 promotes synergistic brood development via unique nutritional benefits. These findings provide a foundational basis for spray-based probiotic application in apiculture and collectively highlight the importance of considering delivery method in disease management strategies.
... We showed that genes related to post-translation modifications were over-expressed in the probiotic treatment group, relative to the control and antibiotic treatment groups. Previous studies showed that probiotic diet supplements elicit a proinflammatory response in fish (Nayak, 2010) and honeybees (Daisley et al., 2020), which promotes more effective pathogen clearance and improved disease resistance. In this study, we found that our treatment with probiotics indeed changes the bacterial community composition with increased numbers of potential probiotics taxa (Lactobacillaceae and Bifidobacteriaceae). ...
Differences in gut microbiome composition are linked with health, disease and ultimately host fitness; however, the molecular mechanisms underlying that relationship are not well characterized. Here, we modified the fish gut microbiota using antibiotic and probiotic feed treatments to address the effect of host microbiome on gene expression patterns. Chinook salmon (Oncorhynchus tshawytscha) gut gene expression was evaluated using whole transcriptome sequencing (RNA-Seq) on hindgut mucosa samples from individuals treated with antibiotic, probiotic and control diets to determine differentially expressed (DE) host genes. Fifty DE host genes were selected for further characterization using nanofluidic qPCR chips. We used 16S rRNA gene metabarcoding to characterize the rearing water and host gut microbiome (bacterial) communities. Daily administration of antibiotics and probiotics resulted in significant changes in fish gut and aquatic microbiota as well as more than 100 DE genes in the antibiotic and probiotic treatment fish, relative to healthy controls. Normal microbiota depletion by antibiotics mostly led to downregulation of different aspects of immunity and upregulation of apoptotic process. In the probiotic treatment, genes related to post-translation modification and inflammatory responses were up-regulated relative to controls. Our qPCR results revealed significant effects of treatment (antibiotic and probiotic) on rabep2, aifm3, manf, prmt3 gene transcription. Moreover, we found significant associations between members of Lactobacillaceae and Bifidobacteriaceae with host gene expression patterns. Overall, our analysis showed that the microbiota had significant impacts on many host signalling pathways, specifically targeting immune, developmental and metabolic processes. Our characterization of some of the molecular mechanisms involved in microbiome-host interactions will help develop new strategies for preventing/ treating microbiome disruption-related diseases.
... Six bacterial families were identified as core in C. calcarata. These included prominent bacterial families such as Lactobacillaceae, which has been recognized to play an important role in bee immunity and carbohydrate metabolism (Daisley et al., 2020;Kwong et al., 2017;Lang et al., 2022;Voulgari-Kokota et al., 2019). ...
As urbanization continues to increase, it is expected that two-thirds of the human population will reside in cities by 2050. Urbanization fragments and degrades natural landscapes, threatening wildlife including economically important species such as bees. In this study, we employ whole genome sequencing to characterize the population genetics, metagenome and microbiome, and environmental stressors of a common wild bee, Ceratina calcarata. Population genomic analyses revealed the presence of low genetic diversity and elevated levels of inbreeding. Through analyses of isolation by distance, resistance, and environment across urban landscapes, we found that green spaces including shrubs and scrub were the most optimal pathways for bee dispersal, and conservation efforts should focus on preserving these land traits to maintain high connectivity across sites for wild bees. Metagenomic analyses revealed landscape sites exhibiting urban heat island effects, such as high temperatures and development but low precipitation and green space, had the highest taxa alpha diversity across all domains even when isolating for potential pathogens. Notably, the integration of population and metagenomic data showed that reduced connectivity in urban areas is not only correlated with lower relatedness among individuals but is also associated with increased pathogen diversity, exposing vulnerable urban bees to more pathogens. Overall, our combined population and metagenomic approach found significant environmental variation in bee microbiomes and nutritional resources even in the absence of genetic differentiation, as well as enabled the potential early detection of stressors to bee health.
... Also, a modulated intestinal permeability and reduced femoral trabecular bone volume were observed [67]. Subsequently in an Apis mellifera model, mixture of three immunostimulatory Lactobacillus strains mitigates both head and gut antibiotic-associated microbiota dysbiosis in adult bees by suppressing larval pathogen loads to near-undetectable levels [68]. Despite multiple reports noting that cocktail or single Lactobacillus consumption improved and recovered the gut microbiota composition toward pre-treatment state while some studied had no obvious results in alteration of the diversity of gut microbial community [84,85], the potential mechanism of gut microbiota restoration by Lactobacillus in microbial niches remains in need of discussion and exploration. ...
A balanced gut microbiota and their metabolites are necessary for the maintenance of the host’s health. The antibiotic-induced dysbiosis can cause the disturbance of the microbial community, influence the immune homeostasis and induce susceptibility to metabolic- or immune-mediated disorders and diseases. The Lactobacillus and their metabolites or components affect the function of the host’s immune system and result in microbiota-mediated restoration. Recent data have indicated that, by altering the composition and functions of gut microbiota, antibiotic exposure can also lead to a number of specific pathologies, hence, understanding the potential mechanisms of the interactions between gut microbiota dysbiosis and immunological homeostasis is very important. The Lactobacillus strategies for detecting the associations between the restoration of the relatively imbalanced microbiome and gut diseases are provided in this discussion. In this review, we discuss the recently discovered connections between microbial communities and metabolites in the Lactobacillus treatment of β-lactam antibiotic-induced dysbiosis, and establish the relationship between commensal bacteria and host immunity under this imbalanced homeostasis of the gut microbiota.
... For example, beneficial gut bacteria in bumble bees reduce the infection rate and presence of the gut parasite Crithidia bombi [32]. Stress tolerance, disease resistance, and stronger immunity to agrochemicals and parasites are associated with the presence of certain microbiota in honey bees [15]. ...
... Bacteria and fungi have been previously reported to be associated with bee health [11,15,20,39]. Osmia lignaria females are known to encounter > 200 flowers per foraging bout from multiple plant species, with several foraging bouts per day [7]. ...
Among insects, symbionts such as bacteria and fungi can be linked to their physiology and immature development, and in some cases are part of a defense system against parasites and diseases. Current bacterial and fungal symbiont associations in solitary bees are understudied, especially in the Pacific Northwest region of the USA. We collected pollen provisions from the native spring-foraging solitary bee, Osmia lignaria Say, across two distinct foraging periods over 2 years at 22 sites along an urban-to-rural gradient in western Washington. We then used next-generation sequencing to identify bacterial and fungi within pollen provisions and assessed the effect of their richness and diversity on O. lignaria larval development success and adult emergence. We detected a significantly positive relationship between bacterial diversity in pollen with O. lignaria larval developmental success, and higher bacterial richness and diversity during the later foraging period. Fungal generic richness and diversity decreased with increasing plant richness. Although neither was associated with O. lignaria developmental success, we did detect Ascosphaera spp. known to be pathogenic to O. lignaria and other bee species. Neither bacterial or fungal richness or diversity was affected by site type when classified as urban or rural. This study provides new information on bacterial and fungal symbionts present in pollen provisions of a native solitary bee when foraging across urban and rural areas of the Pacific Northwest.
... They favor the production of AMP and regulate the immune system. They play a crucial role in the detoxification of ingested toxins and in resistance against pathogens via effect on the immune system, AMP, and biomass effect [52,56,58,[65][66][67]. Due to its close contact with gut epithelium, the microbiota conveys information to the host. ...
Climate change, loss of plant biodiversity, burdens caused by new pathogens, predators, and toxins due to human disturbance and activity are significant causes of the loss of bee colonies and wild bees. The aim of this review is to highlight some possible strategies that could help develop bee resilience in facing their changing environments. Scientists underline the importance of the links between nutrition, microbiota, and immune and neuroendocrine stress resistance of bees. Nutrition with special care for plant-derived molecules may play a major role in bee colony health. Studies have highlighted the importance of pollen, essential oils, plant resins, and leaves or fungi as sources of fundamental nutrients for the development and longevity of a honeybee colony. The microbiota is also considered as a key factor in bee physiology and a cornerstone between nutrition, metabolism, growth, health, and pathogen resistance. Another stressor is the varroa mite parasite. This parasite is a major concern for beekeepers and needs specific strategies to reduce its severe impact on honeybees. Here we discuss how helping bees to thrive, especially through changing environments, is of great concern for beekeepers and scientists.
... Facilitated by social transmission between workers, changes in the microbiome could theoretically quickly go to fixation in a population. Indeed, antibiotic resistance genes have accumulated in bacterial symbionts in managed honey bee colonies, demonstrating long-term impacts with unknown consequences (Tian et al., 2012;Ludvigsen et al., 2017;Daisley et al., 2020;Piva et al., 2020). Considering the social-vertical transfer of the microbiome between worker generations in honey bee colonies with the fact that chemicals including antibiotics accumulate and persist in the hive environment over longer periods (Martel et al., 2006), the damage on the bee microbiome could theoretically go beyond one individual's health affecting a whole population. ...
Microbiomes can enhance the health, fitness and even evolutionary potential of their hosts. Many organisms propagate favorable microbiomes fully or partially via vertical transmission. In the long term, such co-propagation can lead to the evolution of specialized microbiomes and functional interdependencies with the host. However, microbiomes are vulnerable to environmental stressors, particularly anthropogenic disturbance such as antibiotics, resulting in dysbiosis. In cases where microbiome transmission occurs, a disrupted microbiome may then become a contagious pathology causing harm to the host across generations. We tested this hypothesis using the specialized socially transmitted gut microbiome of honey bees as a model system. By experimentally passaging tetracycline-treated microbiomes across worker ‘generations’ we found that an environmentally acquired dysbiotic phenotype is heritable. As expected, the antibiotic treatment disrupted the microbiome, eliminating several common and functionally important taxa and strains. When transmitted, the dysbiotic microbiome harmed the host in subsequent generations. Particularly, naïve bees receiving antibiotic-altered microbiomes died at higher rates when challenged with further antibiotic stress. Bees with inherited dysbiotic microbiomes showed alterations in gene expression linked to metabolism and immunity, among other pathways, suggesting effects on host physiology. These results indicate that there is a possibility that sublethal exposure to chemical stressors, such as antibiotics, may cause long-lasting changes to functional host-microbiome relationships, possibly weakening the host’s progeny in the face of future ecological challenges. Future studies under natural conditions would be important to examine the extent to which negative microbiome-mediated phenotypes could indeed be heritable and what role this may play in the ongoing loss of biodiversity.
... Specific gut symbionts, such as S. alvi, A. kunkeei, Frischella perrara, and L. apis, have been confirmed to induce honeybee innate immune response. They upregulate the Toll and Imd pathway, leading to AMPs expression (Emery et al., 2017;Daisley et al., 2020b;Lang et al., 2022). Considering that the Core-6 consisted of microbes that were able to induce the immune response, the whole gut microbiota balance composition could be more important for regulating the immune system. ...
The gut microbiota of honeybees is highly diverse at the strain level and essential to the proper function and development of the host. Interactions between the host and its gut microbiota, such as specific microbes regulating the innate immune system, protect the host against pathogen infections. However, little is known about the capacity of these strains deposited in one colony to inhibit pathogens. In this study, we assembled a defined microbial community based on phylogeny analysis, the ‘Core-20’ community, consisting of 20 strains isolated from the honeybee intestine. The Core-20 community could trigger the upregulation of immune gene expressions and reduce Hafnia alvei prevalence, indicating immune priming underlies the microbial protective effect. Functions related to carbohydrate utilization and the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS systems) are represented in genomic analysis of the defined community, which might be involved in manipulating immune responses. Additionally, we found that the defined Core-20 community is able to colonize the honeybee gut stably through passages. In conclusion, our findings highlight that the synthetic gut microbiota could offer protection by regulating the host immune system, suggesting that the strain collection can yield insights into host-microbiota interactions and provide solutions to protect honeybees from pathogen infections.
