Native Bees Research Articles

Distance models reveal biases associated with passive trapping methods for measuring wild bee abundance

IntroductionThere is overwhelming evidence of declines in native bee populations and therefore a need for increased monitoring to track these declines and assist in conservation and restoration efforts. Bees can be sampled non-lethally through visual surveys (e.g., distance transects) or lethally through active (e.g., hand netting) or passive (e.g., traps that lure insects from afar) methods. These lethal methods suffer from imperfect detection that is difficult to account for and can confound inferences about habitat characteristics. Additionally, evidence suggests that lethal sampling methods can even invert habitat quality patterns such that high-quality sites yield fewer individuals and low-quality sites yield more individuals.MethodsTo study potential biases associated with imperfect detection, we used hierarchical density estimation with visual surveys to estimate density of bees within 40 young forest patches across Pennsylvania, USA. We surveyed bee communities non-lethally using visual surveys and lethally using blue-vane traps and bee bowls every two weeks between May and September 2019. We collected data on blooming flowers, vegetation structure, and weather during times of survey.ResultsWe found that bee densities estimated from distance transects had a positive relationship with floral resource availability. In contrast, abundance measured via bee bowls and blue-vane traps had no relationship, or sometimes even negative trends with habitat quality, including floral resource availability. Raw bee counts within 2-m of the transect always correlated with modeled densities, showing that some methods do not share the biases of attractive traps.DiscussionOur study demonstrates that failing to account for imperfect detection can impact the interpretation of pollinator surveys and adds to a growing body of literature that acknowledges the value of distance sampling for insects like bees to better understand species’ habitat needs and to monitor populations for conservation.

Bioactives of Melipona rufiventris Propolis: Exploring its Antimicrobial, Anti-Inflammatory, and Antioxidant Activities.

This study explores the potential of propolis, a resinous substance produced by bees, from Melipona rufiventris species. With its composition encompassing resin, wax, pollen, and soil, propolis holds historical significance in traditional medicine within tropical regions. This research is driven by the scarcity of information surrounding M. rufiventris propolis, prompting an investigation into its chemical constituents, in vivo toxicity, and antimicrobial, antioxidant, and anti-inflammatory properties. This exploration could potentially uncover novel applications for this natural product, bolstering both meliponiculture practices and the preservation of native bee populations. The propolis was sampled in Cabo Verde-MG and underwent ethanolic extraction to yield an extract (EEP) for analysis. Chemical assessments (Folin-Ciocalteau, and UHPLC-HRMS) revealed the presence of polyphenols, including flavonoids. The EEP demonstrated higher antimicrobial activity against Gram-positive bacteria and exhibited efficacy against multiresistant strains isolated from complex wounds. Synergistic interactions with commercial antibiotics were also observed. Furthermore, anti-inflammatory evaluations showcased the EEP's potential in reducing NF-kB activation and TNF-α release at non-toxic concentrations. Despite these promising biological activities, the EEP exhibited no antiproliferative effects and demonstrated safety in both the MTS assay and the G. mellonella model. Collectively, these findings highlight the M. rufiventris propolis extract as a valuable reservoir of bioactive compounds with multifaceted potential.

Development of a multiplex real-time quantitative reverse-transcription polymerase chain reaction for the detection of four bee viruses

Viruses in the families Dicistroviridae and Iflaviridae are among the main threats to western honey bees (Apis mellifera) and native bee species. Polymerase chain reaction (PCR) is the gold standard for pathogen detection in bees. However, high throughput screening for bee virus infections in singleplex PCR reactions is cumbersome and limited by the high quantities of sample RNA required. Thus, the development of a sensitive and specific multiplex PCR detection method for screening for multiple viruses simultaneously is necessary. Here, we report the development of a one-step multiplex reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay to detect four viruses commonly encountered in pollinator species. The optimized multiplex RT-qPCR protocol described in this study allows simultaneous detection of two dicistroviruses (Israeli acute paralysis virus and Black queen cell virus) and two iflaviruses (Sacbrood virus and Deformed wing virus) with high efficiency and specificity comparable to singleplex detection assays. This assay provides a broad range of detection and quantification, and the results of virus quantification in this study are similar to those performed in other studies using singleplex detection assays. This method will be particularly useful for data generation from small-bodied insect species that yield low amounts of RNA.

Honey consumer's perception: are brazilian consumers familiar with stingless bee honey?

Stingless bees have a fundamental important role in the economies and traditions of South America indigenous cultures. Are mainly responsible for the pollination of several plant species. This way, the aim was to identify if Brazilian consumers know stingless bee honey and its sensorial characteristics, what is the consumption of this, as well as to generate information to assist in the development of the productive chain of the specie. A closed structured questionnaire with 20 questions was used and the participants were invited via social media. Data were collected in November 2020. The statistical analyses were performed related to the description, characterization of the participants and frequencies. In addition to these,, multivariate analyzes, for word association and questionnaire responses were performed. It is clear that most participants have some confusion between the sensory and physical characteristics of honey from native stingless bees and Apis mellifera. This lack of knowledge of the product can cause problems both in the handling of honey for sale and for consumption. Another interference would be linked to the taste, when we highlight the acidity and less sweetness of this pot-honey, and the consumer can often deduce that the product is damaged or not from a good origin. There is little consumer awareness of the benefits and qualities of stingless honey. Many consumers are still unaware of the differences between breeding sites and characteristics of native stingless bees. Still, a large part of these associates the quality of this honey with the honey of Apis Mellifera.

Fire favours the native bee community in a semi‐arid ecosystem

Abstract Fire is a common and frequent disturbance in Mediterranean ecosystems, affecting the diversity and dynamics of native bee communities by modifying the distribution and availability of the resources that bees require to forage and nest. Previous studies have found changes in bee diversity depending on the time since the last fire, with increases in species diversity in the early stages postburn. Using a chronosequence of plots with different times since the last fire in the Mediterranean‐climate chaparral of Baja California, Mexico, we estimated changes in overall abundance, richness, diversity and species composition of the native bee community. Bee abundance and richness were higher at recently burned plots and gradually decreased, but Hill–Shannon and Hill–Simpson diversities did not change over the chronosequences. Nearby plots (in the same locality) of different stages were more similar in composition than distant plots (in different localities) of a similar stage, suggesting a spatial correlation. Plant species richness increased after fires. Bare soil was also greater on recently burned patches and may facilitate nesting resources. The number of bees was positively correlated to plant richness. Our results highlight the importance of fire as a natural disturbance structuring the bee community in the Mediterranean chaparral of Baja California. Furthermore, our results are relevant for future conservation plans since fire management (i.e., prescribed fires) could improve conditions for sustaining greater bee richness and abundance.

Melissopalynology in Brazilian seacoast mangrove areas, a mini review

Along the Brazilian coast, there are several types of mangroves with a variable predominance of trees of the genera Avicennia and Rizophora, as well as of the Laguncularia racemosa species. Mangrove beekeeping is a potential alternative source of income for many coastal communities in Brazil. Research activities on melissopalynology were initiated on Apis mellifera bees mainly by Barth and Luz in Rio de Janeiro, and with native bees by Kerr et al. in Maranhão States during the last century. Pollen analysis of honey, bee pollen and propolis were presented covering the Brazilian macroregions of the south, southeast, northeast and north. Bees showed preferential interest in Laguncularia racemosa in the southeast region. Studies with native bees are underway in an urban mangrove area of Rio de Janeiro. Laguncularia pollen occurred in reduced proportions in both honey and bee pollen. Avicennia predominated in the northeast region samples. Two samples of honey obtained from indigenous bees in the State of Espírito Santo, one sample from Maranhão and one sample of honey from Apis mellifera from Pernambuco, showed both Laguncularia and Avicennia pollen. All mangrove areas studied clearly showed the importance of Laguncularia racemosa. Studies of propolis highlighted the importance of vegetation along the inner edges of mangroves, such as Dalbergia ecastaphyllum, Schinus terebinthifolia and Symphonia globulifera.

Acclimatization and Foraging of Native Brazilian Stingless Bees in Arenas with Covering Materials of Different Spectral Properties.

The use of Meliponini for crop pollination in protected environments is practically non-existent. One of the reasons is the difficulty of acclimatizing Meliponini to the temperature and light conditions inside greenhouses. We investigated how covering materials used in greenhouses, which filter different intensities of ultraviolet (UV) light, affect the foraging behaviors, flight orientation, attraction to walls and ceilings, and mortality of Scaptotrigona cf. postica (Letreille), Frieseomelitta varia (Lepeletier), and Melipona quadrifasciata (Lepeletier). The experiments were conducted in 5.3 m3 arenas covered with four types of plastic films that do not polarize sunlight, with UV transmittance levels ranging from 0.1 to 54%, compared to a transparent glass control. The temperature inside the arenas varied between treatments, from 27 ± 3°C to 31 ± 2°C. All three species collected resources and returned to the colony, regardless of the covering material. However, the proportion of this behavior, the number of bees attracted to the ceiling and wall, and mortality varied among treatments and/or throughout the confinement days for each species. Melipona quadrifasciata and F. varia acclimatized better to the confined environments than S. cf. postica and showed consistent resource collection behavior throughout the confinement days in all tested materials, except for the one that filtered around 90% of UV. In all three species, the mortality gradually decreased throughout the confinement days. The results indicate that the choice of covering material, considering its optical characteristics, can be crucial to ensure greater effectiveness of the pollination services provided by stingless bees in protected systems.

Floral-Devoid Perennial Grass Seed Fields Harbor a Diversity of Native Bees

Floral-Devoid Perennial Grass Seed Fields Harbor a Diversity of Native Bees

An Economic Cost/Benefit Tool to Assess Bee Pollinator Conservation, Pollination Strategies, and Sustainable Policies: A Lowbush Blueberry Case Study

Lowbush blueberry is a mass-flowering plant species complex that grows in both unmanaged wild landscapes and managed agricultural fields in northeastern regions of both the USA and Canada. During pollination, more than 120 native bee species are associated with lowbush blueberry ecosystems in Maine, USA, in addition to three commercially managed bees. Over a 29-year period, we sampled 209 lowbush blueberry fields using quadrat and transect sampling, recording both native bee and honey bee densities, honey bee hive stocking density, and native bees as a proportion of total bees. These data were used to simulate economic uncertainty in pollination. We developed a novel algorithm, the Economic Pollinator Level (EPL), to estimate bee densities that economically warrant pollination investments such as rented hives and planting bee pastures. Statistical modeling indicated both native bee and honey bee activity density predicted proportion fruit set in fields. Honey bee activity density was well predicted by hive stocking density. Proportion fruit set adequately predicted yield. EPL was most sensitive to fruit set/m2/bee and less dependent on berry weight, rented hive stocking density, hive rental cost, lowbush blueberry price, and the annual cost of planting/maintaining pollinator pastures. EPL can be used to sustainably balance economical pollination investments/decisions with bee conservation in lowbush blueberry crops and potentially other pollinator-dependent crops.

Sampling the understory, midstory, and canopy is necessary to fully characterize native bee communities of temperate forests and their dynamic environmental relationships

IntroductionNative bee communities of temperate forests are conventionally sampled from the understory, yet there is growing evidence that bee assemblages in forest canopies are distinct from those in the understory. Therefore, conventional approaches to quantify forest bee–habitat relationships may not comprehensively characterize forest bee communities.MethodsTo examine this, we sampled bees 1–26 m from ground level at 5-m increments at 47 locations in forests located in western Massachusetts, USA. We evaluated bee abundance and species richness responses to a suite of environmental factors measured in the understory with linear and segmented regression comparing four bee sampling strategies: (1) understory sampling only, (2) understory and midstory, (3) understory and canopy, and (4) all strata combined.ResultsWe found that not sampling higher strata underestimated bee abundance and species richness, and linear models had less ability to explain the data when bees of higher strata were included. Among strategies, responses analyzed linearly differed in magnitude due to overall differences in abundance and species richness, but segmented regressions showed relationships with understory characteristics that also differed in slope, which would alter interpretation.DiscussionCollectively, our findings highlight the value of including vertically stratified sampling strategies throughout the flight season to fully characterize native bee and other pollinator communities of forests.

Rapid on‐ground assessment after the 2019–2020 megafires reveals new information on rare and threatened plants in northern New South Wales, Australia

AbstractLack of ecological knowledge is a major challenge for effective conservation of threatened plant species in Australia as disturbance events, such as wildfire, increase in frequency and magnitude. Rapid on‐ground surveys are increasingly important to inform recovery strategies for rare and threatened plants in response to extreme wildfire events, yet resources are rarely available to deploy time‐sensitive surveys. Here, we utilize a rapid on‐ground survey and monitoring program to document basic biological and ecological information for 21 rare and threatened plant species from the New England bioregion in northern New South Wales, which were affected by the catastrophic 2019–2020 megafires. Our results fill an important knowledge gap of ecology, population size, distribution and response to fire for these taxa, document for the first time a species distribution pattern within the Torrington State Conservation Area and reveal previously undocumented plant–insect interactions for nine species, including likely pollination of Persoonia terminalis ssp. terminalis and Monotaxis macrophylla by native bee (Hymenoptera) and fly (Diptera) species. Our findings reveal that two species (Cassinia heleniae and Hakea macrorrhyncha) have scarce distribution and low population sizes, despite neither having a conservation listing and their threat status should be urgently assessed. Simple rapid on‐ground surveys can be more cost effective for delivering long‐term conservation outcomes for rare and threatened taxa and we advocate that future funding and prioritisation processes must support the immediate delivery of such surveys in response to disturbance events.

Effect of protein and lipids in pollen on the developmental success of the native solitary bee Osmia lignaria Say

The health of solitary bees is dependent on the pollen they consume, driving their development from larvae to pupae. Nutrients such as proteins and lipids play an important role in bee development and could be an important key in conservation efforts to support declining populations. We examined the concentration of protein, total lipids, and 13 individual lipid classes in pollen provisions collected from the native solitary nesting bee, Osmia lignaria Say, at 22 sites in urban and rural landscapes in western Washington over three years and during two distinct foraging periods. The results indicate that protein concentrations in pollen did not significantly affect larval development. We detected higher lipid concentrations in the second foraging period, which was also associated with increased larval developmental success. Using Next-Generation sequencing on collected pollen, we also ascertained the plant genera in pollen and observed that plant richness did not differ significantly between the two foraging periods even though levels of lipid concentrations did. Individual lipid classes may play a role in the successful development of O. lignaria, as hexosylceramide and cholesterol ester were positively associated with O. lignaria larval developmental success, which emphasizes the need for additional studies on the role of individual lipid classes on bee development.

Viruses in Brazilian stingless bees

Stingless bees are important pollinators in tropical and subtropical areas. The breeding of native stingless bee species from the Meliponini tribe to obtain honey, pollen, and geopropolis (meliponiculture) is a promising activity in Brazil with economic, social, and environmental impacts. Stingless bees can share food sources with Apis mellifera and other insects that can be vectors of pathogens. Here, we investigated seven stingless bee species from Brazil (Melipona quadrifasciata anthidioides, Melipona scutellaris, Nannotrigona testaceicornis, Partamona helleri, Scaptotrigona xanthotricha, Tetragonisca angustula, and Trigona spinipes) screening for seven of the most common pathogenic viruses (ABPV, IAPV, BQCV, CBPV, SBV, KBV, and DWV) in A. mellifera in summer and winter seasons. All seven stingless bee species studied were positive for ABPV and IAPV (only in M. quadrifasciata anthidioides and T. spinipes), while ABPV was found in samples from summer and winter, IAPV was only found in winter. This is the first study detecting viruses in seven native stingless bee species in Brazil and because of the novelty of this area in Brazil, it is necessary to monitor native stingless bee colonies to investigate the impact of those viruses at a colony and individual level to avoid losses and impact to the native bees.

Exposure of the stingless bee Melipona scutellaris to imidacloprid, pyraclostrobin, and glyphosate, alone and in combination, impair its walking activity and fat body morphology and physiology

The stingless bee Melipona scutellaris performs buzz pollination, effectively pollinating several wild plants and crops with economic relevance. However, most research has focused on honeybees, leaving a significant gap in studies concerning native species, particularly regarding the impacts of pesticide combinations on these pollinators. Thus, this study aimed to evaluate the sublethal effects of imidacloprid (IMD), pyraclostrobin (PYR), and glyphosate (GLY) on the behavior and fat body cell morphology and physiology of M. scutellaris. Foragers were orally exposed to the different pesticides alone and in combination for 48 h. Bees fed with contaminated solution walked less, moved slower, presented morphological changes in the fat body, including vacuolization, altered cell shape and nuclei morphology, and exhibited a higher count of altered oenocytes and trophocytes. In all exposed groups, alone and in combination, the number of cells expressing caspase-3 increased, but the TLR4 number of cells expressing decreased compared to the control groups. The intensity of HSP70 immunolabeling increased compared to the control groups. However, the intensity of the immunolabeling of HSP90 decreased in the IMD, GLY, and I + G (IMD + GLY) groups but increased in I + P-exposed bees (IMD + PYR). Alternatively, exposure to PYR and P + G (PYR + GLY) did not affect the immunolabeling intensity. Our findings demonstrate the hazardous effects and environmental consequences of isolated and combined pesticides on a vital neotropical pollinator. Understanding how pesticides impact the fat body can provide crucial insights into the overall health and survival of native bee populations, which can help develop more environmentally friendly approaches to agricultural practices.

Japanese honey bees (Apis cerana japonica) have swarmed more often over the last two decades

The impacts of temperature increase are a concern for honey bees, which are major pollinators of crops and wild plants. Swarming is the reproductive behavior of honey bees that increases colony numbers. Honey bee colonies sometimes swarm multiple times, with each swarming termed a “swarming event” and a series of these events called a “swarming cycle.” The number of swarming events per swarming cycle varies widely depending on climatic conditions and subspecies, and the recent temperature increase due to global warming might be affecting the number of swarming events per swarming cycle of native honey bees. We clarified long-term changes in the number of swarming events per swarming cycle of Japanese honey bees (Apis cerana japonica) by collecting beekeepers’ swarming logbooks. The survey showed that between 2000 and 2022, Japanese honey bees swarmed 1 to 8 times per swarming cycle. Generalized linear model analysis indicated that year had a significant positive effect (coefficient, 0.03; 95% CI, 0.01–0.04); that is, the number of swarming events per swarming cycle showed a moderate increase over time. In addition, we found that colonies swarmed more often in a cycle when the swarming process began in early spring, especially in March. Considering the notably strong trend in Japan of warmer temperatures in March, the number of swarming events per swarming cycle may be increasing because reproduction is beginning earlier in the year. Further analyses are needed to verify the causal relationship of temperature increase on the number of swarming events per swarming cycle.

Propolis of Tubuna bee (Scaptotrigona bipunctata) from the Meliponini tribe alleviates glycaemic, oxidative, and inflammatory conditions during STZ induced diabetes in Wistar rats also aiding in injury recovery

This study was designed to characterize and describe the in vivo effects of an ethanolic propolis extract (EHAP) from the native Brazilian bee Tubuna (Scaptotrigona bipunctata). Propolis has been recently proposed as an adjuvant during diabetes, mainly because propolis possesses various natural compounds without the elevated sugar quantities found in pure honey. Compounds with biological activity and in vitro antioxidant potential have been detected in propolis from Tubuna. Streptozotocin (STZ) is a well-documented model to induce experimental diabetes in rodents. We have detected potential compounds in Tubuna propolis and observed the effects of daily oral treatment with EHAP in Wistar rats during experimental diabetes. EHAP improved glucose tolerance in STZ animals without significantly changing net and organ weight. STZ increases levels of TBARS and Carbonyl, and reduces levels of -SH, effects partially controlled by EHAP, particularly in serum. EHAP decreases inflammatory mediators TnF-ɑ and IL-1β in diabetic animals and decreases paw swelling during carrageenan induced edema. In conclusion, Tubuna propolis constituents possess interesting biological activities to aid in several diseases, but this still needs further studies in these particular pathologies. This is the first work to characterize Tubuna propolis from Rio Grande do Sul and the first work to describe its effects in an animal model of experimental diabetes or any animal model of disease. Treatment with EHAP shows potential to regulate conditions during STZ induced diabetes, aiding in injury recovery paw edema. Mechanistic studies, other models of long-term diabetes or metabolic syndrome, and a complete characterization of propolis from Tubuna are potential targets for future studies.

An engineered bacterial symbiont allows noninvasive biosensing of the honey bee gut environment.

The honey bee is a powerful model system to probe host-gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to noninvasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular tool kit to genetically modify symbionts and reported for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium Snodgrassella alvi as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report exposure to the molecules in a dose-dependent manner through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or noninvasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host-gut microbiota research.

The dark European honey bee Apis mellifera mellifera in Lithuania: data on mitotype diversity of native bee population

In Europe, the population of the dark European honey bee Apis mellifera mellifera L. has been significantly affected by the introduction and propagation of non-indigenous subspecies of honey bees. For a long time, the status of the native Lithuanian A. m. mellifera bees was unknown. Thus, the aim of this study was to determine the current distribution of the subspecies population in Lithuania and the diversity of its mitotypes. A total of 598 honey bee colonies from traditionally managed apiaries were studied to determine their maternal ancestry using the COI–COII intergenic spacer sequences of mtDNA. The results revealed that 52% of the maternal lines investigated belonged to A. m. mellifera. They represented 20 unique COI–COII intergenic spacer mitotypes, 11 of which were detected for the first time. We hypothesised that a small proportion of ancestral A lineage mitotypes among the dominant M lineage mitotypes could be the relict of the maternal line.

Honey-kombucha beverage with yerba maté infusion: Development, polyphenols profile, and sensory acceptance

Kombucha is a sweetened beverage made by fermenting a symbiotic consortium of bacteria (generally acetic acid bacteria) and yeasts (SCOBY) in black or green tea, with sucrose as source of carbohydrate. Other herbal infusions, such as yerba maté (Ilex paraguarienses), are used to produce kombucha-like beverages. Honey is a good source of sugar and biologically active components for kombucha-like development. The goal was to develop and characterize a fermented beverage from yerba maté infusion using a kombucha culture, with either sucrose or different types of honey from Apis bees and native Brazilian stingless bees. The fermentation process was studied for up to 20 days, monitoring pH, titratable acidity, reducing sugar and soluble protein, total phenolic compounds, chlorogenic acids, caffeine, and rutin. The sensory acceptance of the honey-kombucha-like beverage was also investigated. As fermentation progressed, the pH decreased, titratable acidity increased, and reducing sugars initially decreased due to SCOBY consumption, followed by an increase due to the conversion of non-reducing sugars to reducing sugars. The concentration of chlorogenic acids increased with fermentation, exceeding 300 μg/mL after 15 days. The aroma and flavor of the kombucha beverage received scores around 5–6 on a nine-point hedonic scale. These scores were linked to the high titratable acidity after the 10th day of fermentation and the “vinegar taste” reported by the subjects. To improve sensory acceptance, the fermentation length can be reduced to up to 10 days, resulting in a beverage with a pleasant fruity and sour aftertaste. However, this strategy may compromise the retention of high polyphenols, which is achieved through longer fermentations of around 15–20 days.

Bacterial Strains Isolated from Stingless Bee Workers Inhibit the Growth of Apis mellifera Pathogens.

Apis mellifera bees are an important resource for the local economy of various regions in Argentina and the maintenance of natural ecosystems. In recent years, different alternatives have been investigated to avoid the reduction or loss of colonies caused by pathogens and parasites such as Ascosphaera apis, Aspergillus flavus, and Paenibacillus larvae. We focused on bacterial strains isolated from the intestine of native stingless bees, to elucidate their antagonistic effect on diseases of A. mellifera colonies. For this purpose, worker bees of the species Tetragonisca fiebrigi, Plebeia spp., and Scaptotrigona jujuyensis were captured from the entrance to tree hives and transported to the laboratory, where their intestines were extracted. Twenty bacterial colonies were isolated from the intestines, and those capable of inhibiting enterobacteria in vitro and producing organic acids, proteases, and chitinases were selected. Four genera, Levilactobacillus, Acetobacter, Lactiplantibacillus, and Pantoea, were selected and identified by the molecular marker that codes for the 16S rRNA gene. For inhibition assays, cell suspensions and cell-free suspensions were performed. All treatments showed significant antibacterial effects, in comparison with the controls, against P. larvae and antifungal effects against A. apis and A. flavus. However, the mechanisms by which these bacteria inhibit the growth of these pathogens were not studied.