Introduction

Honeybees have long served as a valuable model organism for scientific and physiological investigations. Their role in agriculture has been recognized for millennia, as they play a crucial role in pollination, which is essential for sustaining life, maintaining the global climate, and advancing food production. However, honeybees face numerous challenges, both in terms of diseases and environmental factors, some of which have become increasingly prevalent in recent times. While pinpointing a single cause for colony losses worldwide over a specific period is challenging, it is evident that a combination of environmental and biological factors can compromise honeybee lifespan and health, leading to colony mortality1,2.

One disease that poses a significant threat to honeybee a colony is American foulbrood (AFB) caused by the Gram-positive, spore-forming bacterium Paenibacillus larvae (P. l.). AFB primarily affects immature honeybee larvae, leaving behind highly infectious dried larval remains. This disease spreads horizontally when healthy colonies opportunistically rob weakened infected colonies, necessitating the removal of diseased colonies and contaminated beekeeping material. As a result, AFB infestations lead to substantial economic losses3,4. Compounding the challenge of AFB is the remarkable resistance of P. larvae endospores to most known antibiotics. Consequently, the use of antibiotics for managing American Foulbrood (AFB) not only fails to eliminate the spores but also results in contamination of honeybee products and detrimental impacts on honeybee health and quality5,6. Furthermore, not only antibiotics but also probiotic bacteria or natural extracts have been explored as alternative treatments for foulbrood7. Antibiotic misuse and long-term use have led to the development of resistant strains and residues that further compromise the integrity of beehive goods. Consequently, many nations have implemented bans on the use of antibiotics for AFB treatment and prevention, necessitating the destruction of affected colonies by burning the hives8. Amidst these challenges, bee venom has emerged as a natural product with potential antimicrobial properties. Bee venom, also known as apitoxin, is a combination of acidic and vital fluids found in the abdomen of worker bees. With a pH of 4.5 to 5.5, bee venom is comparable to nettle toxin and can be injected into targets through a honeybee's stinger, delivering approximately 0.1 mg of venom. Although only about one percent of the population is estimated to be allergic to bee stings, the antibacterial characteristics of bee venom have been attributed to the action of melittin. Melittin exhibits low cell selectivity and aggressively interacts with cell membrane lipids, creating channels that exert antimicrobial effects9. Numerous studies have explored the antibacterial properties of bee venom, particularly against pathogenic bacterial species such as Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, and Escherichia coli. These investigations have revealed varying levels of antibacterial activity depending on the bacterial strain, with bee venom demonstrating effectiveness against several strains, excluding E. coli and Pseudomonas aeruginosa. Furthermore, the antimicrobial activity of bee venom has been demonstrated against various bacterial species, including E. coli and Salmonella spp10.

Given the vulnerabilities of honeybees to diseases like AFB and the potential antimicrobial properties of bee venom, there is a growing interest in exploring the use of bee venom as a natural alternative for managing honeybee diseases and promoting their overall health11. Nanotechnology is one of the most intensively researched topics of modern materials science. Based on basic characteristics such as scale, distribution, and shape, nanoparticles exhibit new or enhanced properties. The development of this technology in the last decade has made it possible to investigate the bactericidal characteristics of nanoparticles12. Several nanoparticles have been employed as antimicrobial agents in a wide range of items, including medical tools and devices, as well as industrial activities including water treatment and food processing13.

Silver nanoparticles (AgNPs) have shown potent antibacterial activity against a wide range of bacterial pathogens. AgNPs can bind to and disrupt the cell membrane, leading to increased permeability and cell death14,15. AgNPs can catalyze the production of ROS, such as superoxide, hydrogen peroxide, and hydroxyl radicals, which damage bacterial DNA, proteins, and lipids16,17. AgNPs can interfere with vital cellular processes by binding to and inhibiting the function of enzymes and proteins, including those involved in energy production and cell division18. AgNPs can release Ag+ ions, which can further enhance the antibacterial activity by interfering with cellular processes and causing additional oxidative stress19,20. The antibacterial efficacy of AgNPs has been demonstrated against both Gram-positive and Gram-negative bacteria, including Escherichia coli14,17, Staphylococcus aureus15, Pseudomonas aeruginosa16,18, and Bacillus subtilis. The antibacterial activity of AgNPs is influenced by several factors, such as size, shape, concentration, and surface functionalization [15, 17, and 20].

How about improving the performance of bee venom through the high activity of the previously mentioned nanoparticles such as silver metal by forming nanocomposite and seeing what can happen for pathogens and the honeybee life span, as an alternative antidote and Vital. This research aims to investigate the potential of bee venom and explore the development of bee venom/nanoparticle nanocomposites to enhance honeybee pathogen resistance and lifespan. By harnessing the unique properties of bee venom and incorporating nanotechnology, we can potentially develop novel strategies to combat honeybee diseases and mitigate the environmental challenges they face, safeguarding their vital role in agriculture and ecological balance.

Results

The transmission electron microscopy (TEM) images in Fig. 1a,b depict spherical nanoparticles of Ag NPs with an elevated level of uniformity in size and shape. The particles ranged from 20 to 30 nm, with an average diameter of 24.0 ± 2.3 nm. After the formation of BV/Ag NCs, there was no significant change observed in the size and shape of the Ag NPs, as shown in the TEM micrograph in Fig. 1c,d. The TEM micrograph illustrate that the BV/Ag NCs exhibit a quasi-spherical shape and possess anisotropic characteristics, with an average size of 26.7 ± 1.8 nm. The standalone Ag NPs may exhibit a uniform size distribution and well-defined morphology, such as spherical or near-spherical shapes, as is typical of many chemically synthesized silver nanoparticles. In contrast, the incorporation of bee venom during the synthesis process may lead to changes in the size and morphology of the resulting BV/Ag NCs. The BV components, such as proteins and other biomolecules, can influence the nucleation and growth of the silver nanoparticles, potentially leading to a wider size distribution or the formation of more complex shapes, such as triangular or anisotropic structures.

Figure 1
figure 1

TEM micrograph for Ag NPs (a) and its histogram curve (b). TEM micrograph for BV/Ag NPs (c) and its histogram curve (d).

Full size image

The UV–Vis absorption spectra of the silver nanoparticles, bee venom, and their composites are presented in Fig. 2a. The color change from colorless to greenish yellow indicates the formation of Ag NPs during the reduction process, which can be visually observed21. Ag NPs were synthesized in an aqueous solution through the reduction of silver nitrate using NaBH4, with PVP serving as reducing and stabilizing agents. The absorption spectra of Ag NPs exhibit a surface plasmon resonance peak at 420 nm. Bee venom shows an absorption peak at 250 nm in the UV range, with no absorption peak in the visible range. The absorption peak of Ag NPs red shifts from 425 to 434 nm for BV/Ag NCs, indicating the conjugation of Ag NPs with bee venom. The position of the absorbance peak in the UV–Vis spectrum of the BV/Ag NCs (Fig. 2b) remained unchanged over the 6-month period. The lack of shift in the absorbance peak position indicates the high colloidal stability of the BV/Ag nanocomposites during this extended storage period. This stability observation is an important finding, as it demonstrates the long-term viability and potential for practical applications of the BV/Ag NCs. X-ray diffraction (XRD) analysis of the synthesized silver nanoparticles in Fig. 2c reveals diffraction peaks at angles of 32.5°, 38.3°, 44.4°, 64.6°, and 77.6°, corresponding to crystal planes {122}, {111}, {200}, {220}, and {311} of Ag crystals, respectively22,23,24. These peaks confirm that the synthesized silver nanoparticles are in a nanocrystalline form and possess a crystalline nature. The average estimated particle size of the sample is 25 nm, determined from the full width at half maximum (FWHM). Furthermore, the Fourier-transform infrared (FTIR) spectra (Fig. 2d) display different bond stretches at distinct peaks. The FTIR spectra (Fig. 1d) reveal the presence of various functional groups and chemical bonds in the sample. The peak at 3432 cm–1 corresponds to the N–H stretching vibration, indicating the presence of amine or amide groups. The peak at 2777 cm–1 is assigned to the single aldehyde O–H stretching vibration, suggesting the existence of aldehydes' functional groups. The peak at 2085 cm–1 represents the C≡C stretching vibration, which is characteristic of alkyne groups. Furthermore, the peak at 1637 cm–1 indicates the presence of C=C stretching vibrations, corresponding to alkene or aromatic functional groups. Additionally, the FTIR spectrum shown in Fig. 1e displays a prominent peak around 3440 cm–1, which is assigned to the OH stretching vibration, indicating the presence of hydroxyl groups. The peak at 1041 cm–1 corresponds to the C–N stretching vibration of the amine functional group. The peaks near 677 cm–1 and 651 cm–1 are assigned to the CH out-of-plane bending vibrations in substituted ethylene structures (cis configuration). The FTIR spectrum of the silver nanoparticles (Ag NPs) exhibits prominent peaks at 2927 cm–1, 1631 cm–1, and 1383 cm–1, which suggest the presence of various functional groups and chemical bonds associated with the nanoparticles. In the FTIR spectrum of bee venom (BV), the absorption region of 2950–3450 cm–1 indicates the free vibration of N–H stretching, suggesting the presence of amine or amide groups. The BV FTIR spectrum also shows characteristic amide bands, namely amide I (1651 cm–1) and amide II (1532 cm–1), which are associated with the protein content of the bee venom. Furthermore, the bands at 1112 cm–1 and 1040 cm–1 indicate the presence of non-systematic helical conformations. When the BV and Ag NPs are combined to form BV/Ag NCs, the FTIR spectrum shows some changes. The BV absorbance peak for N–H stretching at 3318 cm–1 is reduced and shifted to 3439 cm–1, suggesting a potential interaction or binding between the BV and the Ag NPs. Additionally, the amide I band in BV at 1658 cm–1 is shifted to 1649 cm–1, further indicating the interaction between the bee venom components and the silver nanoparticles. These FTIR analyses provide valuable insights into the chemical composition and potential interactions between the different components in the sample, which can help in understanding the formation and properties of the BV/Ag NCs.

Figure 2
figure 2

UV/Vis absorption spectra of the prepared Ag NPs, BV/Ag NCs, and BV (a) after one day of preapration and (b) after 6 mnths of preparation. The XRD pattern of the prepared Ag NPs (c), and FTIR spectra of the prepared Ag NPs, BV, and BV/Ag NCs (d).

Full size image

By examining the effect of different treatments and concentrations of BV, Ag NPs, and BV/Ag NCs against Paenibacillus larvae (P. l.) bacterium in vitro, the inhibition zones are shown in Table 1 under dark and light conditions.

Table 1 The inhibition zone of different treatments on P. l. bacterium under dark and light irradiation.
Full size table

Table 1 presents the results obtained from the use of BV (bee venom) and Ag NPs (silver nanoparticles) individually and in combination, compared to the veterinary antibiotic Tylosin, against the P. l. bacterium under dark and light conditions in vitro. All treatments exhibited antibacterial activity against the target pathogen at the concentrations specified in the table, although with some significant variations. BV, despite being used at higher concentrations than Ag NPs (the maximum concentration used was 6.250 mg/mL, which was considered safe for honeybees according to Daniels25), demonstrated inhibition zones against P. l. The diameter of the inhibition zones (IZDs) induced by BV alone were 5.67 mm and 7.00 mm for concentrations of 1.560 mg/mL and 6.250 mg/mL, respectively. On the other hand, Ag NPs alone induced IZDs of 5.33 mm and 6.50 mm for concentrations of 0.015 mg/mL and 0.030 mg/mL, respectively, as shown in Fig. 2a and b. However, when treated with BV/Ag NCs (bee venom/silver nanoparticle nanocomposites) at concentrations of 0.030 mg/mL, 0.022 mg/mL, and 0.015 mg/mL of Ag NPs, along with the minimum inhibitory concentration (MIC) of BV (1.56 mg/mL), a significant difference was observed, resulting in average IZDs of 9.67 mm, 8.00 mm, and 7.00 mm, respectively. The highest inhibition zone in this experiment, equaling 28 mm as the average IZD, was achieved by the Tylosin antibiotic, as shown in Fig. 3.

Figure 3
figure 3

Inhibition zones of P. larvae bacterium in dark by BV (a), Ag NPs (b) BV/Ag NCs (c), and veterinary antibiotic Tylosin (d) under dark conditions.

Full size image

In the light experiment depicted in Fig. 4a and b, the effects of Ag NPs alone and BV/Ag NCs on the P. l. bacterium were investigated. The results showed that all treatments had antibacterial effects against the target microorganism at the specified doses, with improved results compared to the dark conditions. BV/Ag NCs exhibited a significant improvement in the inhibition results, with values of 9.170 mm and 13.670 mm for the minimum and maximum doses (0.015 mg/mL Ag NPs + 1.560 mg/mL BV) and (0.030 mg/mL Ag NPs + 1.560 mg/mL BV), respectively. Individually, Ag NPs inhibited the growth of P. l. bacteria with inhibition diameter means (IDMs) of 6.830 mm and 8.500 mm at concentrations of 0.015 mg/mL and 0.030 mg/mL, respectively.

Figure 4
figure 4

Inhibition zones of P. larvae bacterium by Ag NPs (a) and BV/Ag NCs under light irradiation (b).

Full size image

Various environmental factors, such as temperature, oxygen, food availability, and nutrition, can significantly impact lifespan. Adequate environmental nutrients play a crucial role in the growth and survival of organisms. Insufficient or poor diet can negatively affect health and increase susceptibility to infections. The impact of BV, Ag NPs, and BV/Ag NCs treatments on the lifespan of honeybee workers is presented in Table 2.

Table 2 The effect of different treatments on the longevity of honeybees.
Full size table

Table 2 shows that three different doses of BV (1.56 mg/mL, 3.12 mg/mL, and 6.25 mg/mL) were fed to bee workers, along with three concentrations of Ag NPs (15 mg/L, 22.5 mg/L, and 30 mg/L), and three concentrations of BV/Ag NCs with 1.56 mg/mL BV for Ag NPs (duplicated three times). The lifespan of bee workers fed with BV was determined to be 41 days, 50.33 days, and 47.67 days for the three concentrations, indicating that feeding with BV at the tested concentrations enhanced the lifespan of bee workers. It was also observed that the concentration of 3.12 mg/mL yielded the optimal lifespan improvement. Bee workers fed with Ag NPs lived for 34.67 days, 32.00 days, and 14.67 days at concentrations of 15 mg/L, 22.5 mg/L, and 30 mg/L, respectively. The concentration of 30 mg/L was found to be fatal for bees, while the lowest concentration (15 mg/L) was the most effective in prolonging bee longevity. The first and second concentrations of the nanocomposites showed significant differences compared to the control, whereas the third concentration resulted in lower longevity compared to the other two nanocomposites but still exhibited more variation than the control. Workers raised in cages and fed on bee venom solution lived longer than those in controlled cages fed on sugar syrup. These findings align with previous research stating that BV supplementation via drinking water had a considerable influence on the initial stages of life, making BV treatment an appealing alternative to antimicrobial growth stimulants. Moreover, this approach could serve as an alternative to regular antibiotic administration26. BV and its components can also impact worker activity and longevity27.

In the Table 3, the results of the best three treatments from the three previous experiments were collected, and it was mentioned that the intake of Ag NPs did not shorten the life of honeybee workers if they were treated with the sugar solution that replaces nectar in the life of the honeybee, whereas the intake of BV/Ag NCs increased its life slightly more than the group treated with Ag NPs only. However, eating BV alone resulted in a significant difference in life length compared to the control, which translates in an improvement in functional performance if the therapies are used at the dosages recommended by the trial results.

Table 3 Life span (days) of worker bees fed with different diets in comparison with control on the longevity of honeybee workers.
Full size table

Discussion

The synthesized silver nanoparticles (Ag NPs) exhibit distinct characteristics when compared to the silver nanoparticles synthesized in the presence of other substances, such as bee venom (BV/Ag NCs) and Ag NPs with Curcumin (Cur/Ag NPs). The incorporation of bee venom during synthesis can lead to the formation of nanoparticles with a wider size distribution and more complex shapes, such as anisotropic structures. The addition of curcumin can result in the synthesis of smaller and more uniform silver nanoparticles compared to the standalone Ag NPs. The bee venom components, such as proteins and peptides, can adsorb or bind to the surface of the Ag NPs, forming a bio-corona that influences the overall surface properties. However, curcumin can function as an effective capping and stabilizing agent, interacting with the surface of the Ag NPs and altering their surface chemistry. The bee venom-derived biomolecules can improve the colloidal stability of the Ag NPs, preventing aggregation and enhancing their long-term stability. The bee venom components can impart additional functionalities, such as antimicrobial, anti-inflammatory, or wound-healing properties, making the BV/Ag NCs more suitable for biomedical applications while curcumin is a well-known bioactive compound with various therapeutic properties, and the Cur/Ag NPs can potentially exhibit enhanced biocompatibility and targeted biological activities28. Moreover, BV/Ag NCs from the results did not show any change in the position of the absorbance in the UV–Vis spectrum that indicates the high stability of the prepared NCs even after 6 months of preparation29.

The importance of finding alternatives to antibiotic treatment for honeybees cannot be overstated, as the use of antibiotics in food-producing organisms raises concerns. Improper use of antibiotics can lead to the development of resistant bacteria, posing risks to human health. Additionally, the presence of biological substances in honey, resulting from antibiotic misuse, can affect its quality. Previous studies have explored the antibacterial properties of various honeybee products, including bee venom, propolis, and royal jelly. One of the key components responsible for bee venom's antibacterial effects is melittin, a compound found within it. Melittin plays a significant role in destroying cell membrane permeability by forming pores on the cell wall and disrupting peptides and antimicrobial proteins. This compound has been associated with antibacterial, antiviral, anti-inflammatory effects, as well as the inhibition of cell growth and induction of cell death in cancer cell lines30. These discoveries regarding bee venom's antibacterial properties are supported by a body of research that has explored other honeybee products. Previous studies have indicated that bee venom outperforms propolis and royal jelly in terms of its effectiveness against bacteria and inhibition of bacterial growth. Specifically, bee venom's ability to destroy cell membrane permeability is intricately linked to the presence of melittin. This compound forms pores on the cell wall and disrupts peptides and antimicrobial proteins, leading to the destruction of bacteria31. Melittin has been linked to a variety of biological activities, including antibacterial, antiviral, and anti-inflammatory properties, as well as the inhibition of cell growth and death in multiple cancer cell lines32. The electrostatic interaction between negatively charged bacterial cells and positively charged nanoparticles greatly affects the activity of nanoparticles against bacteria as bactericidal materials33. The mechanism of silver ions' inhibitory impact on microbes is still partially understood. It was discovered that when exposed to Ag+, DNA loses its capacity to replicate and cellular proteins become inactivated. Furthermore, it has been demonstrated that Ag+ attaches to functional groups of proteins, resulting in partial or full protein denaturation. Bacterial cells were harmed by Ag NPs because they attacked their membranes, disrupting their characteristics. Thus, causing leakage of cell components and eventually cell death34. Based on the findings, a combination of bee venom and silver nanoparticles, known as BV/Ag nanocomposite, demonstrates enhanced antimicrobial activity compared to their individual use. In the study conducted by Amini and Shahroudiian35, regarding the antimicrobial activity of silver and gold nanoparticles prepared with curcumin, they determined that nanoparticles of either silver or gold at their concentrations used, which were in micrograms/ml, did not cause any inhibitory effect against all types of microbes tested by the disk diffusion method, while in our presented study, some inhibition of the growth of the tested bacterium, Paenibacillus larvae, occurred when it was treated by the disk diffusion saturated with any of the prepared silver nanoparticles in a size of 24.0 ± 2.3 nm at concentrations 0.015, 0.022,and 0.030 mg/ml, or silver nanoparticles at the same concentrations added to the natural product, bee venom 1.560 mg/ml separately. The success of the nanoparticles in showing their antimicrobial effect with this way here than the other researcher in their mentioned study is due to one or many of the next reasons. The high concentration of silver nanoparticles used, and at the same time was the lowest concentration and safer used to cause harm to the bee workers when mixed with bee venom based on the results of tested materials on the bee’s longevity (Tables 2 and 3). The disk saturated with the material has a specific carrying capacity that it does not exceed, but there may be a positive effect for the length of time the disk was soaked in the tested material, nanoparticles, which in our study presented here was 24 h under cooling conditions. A third reason is that bee venom, in its nature, has a great degree of antimicrobial property and it increased the effect of silver nanoparticles when combined together unlike nanoparticles alone, as it is shown by the numbers in Table 1, this combination between these two substances is perhaps succeeded the method of the saturated discs diffusion for exhibiting its effect. Finally, the different of the tested microbial strains in the two studies, Perhaps the tested bacterium, Paenibacillus larvae, was more sensitive than others, which led to the demonstration of the action of silver nanoparticles against it by the saturated disk method, which is a strong reason in our humble opinion, and this issue still requires further studies and scrutiny in the future.

The nanocomposite exhibits a higher significance against bacteria after photo-stimulation with visible light. However, it is important to note that increasing the concentration of bee venom in the BV/Ag nanocomposite negatively correlates with the value of the inhibition zone. Therefore, it is not advisable to raise the concentration of bee venom in the nanocomposite. These results highlight the potential of bee venom and silver nanoparticles as effective antimicrobial agents for addressing honeybee diseases, both individually and in combination. Further research is necessary to optimize the concentration and application of these treatments to maximize their efficacy in promoting honeybee health.

Antibiotics like oxytetracycline and tylosin have been commonly used to treat AFB, but concerns have risen about antibiotic resistance developing in the Paenibacillus larvae bacteria that causes AFB36,37. Essential oils from plants like thyme, oregano, and clove have shown some antibacterial activity against P. larvae, but their efficacy has been variable38,39. Propolis, a resinous substance collected by honeybees, has demonstrated promising antimicrobial effects against AFB in laboratory studies40. Bacteriophages (viruses that infect bacteria) have been explored as a more targeted approach to killing P. larvae, with some successful results in controlling AFB outbreaks41. However, Silver nanoparticles (Ag NPs) have been investigated in a few prior studies for their antibacterial activity against P. larvae. These studies generally found Ag NPs to be effective at inhibiting the growth of the bacterial pathogen42,43. Other metallic nanoparticles like CdO, PbO, and ZnO have also shown antibacterial potential against AFB in lab experiments44,45. Chitosan nanoparticles, derived from chitin, have exhibited promising results in killing P. larvae spores and vegetative cells46.

The current study builds on the previous research on Ag NPs but combines them with bee venom for a synergistic antimicrobial effect. The findings indicate that the Ag NPs and bee venom combination was more effective at inhibiting P. larvae growth and reducing spore viability compared to either component alone. This suggests the combination therapy may be a more potent and versatile treatment option for managing AFB compared to previous antidote or single nanoparticle approaches. The study provides promising new insights into leveraging nanomaterials and natural bee-derived compounds for tackling this persistent honeybee disease.

Conclusion

American foulbrood (AFB) poses a significant threat to bee colonies, necessitating effective treatment options beyond antibiotics. Bee venom and silver nanoparticles were evaluated as potential antibacterial agents against AFB. Ag NPs effectively inhibited AFB progression, while the combination of BV and Ag NPs (BV/Ag NCs) extended honeybee lifespans. Oral administration of BV through sugary syrup doubled honeybee lifespans compared to the control group. BV/Ag NCs exhibited enhanced bactericidal efficiency under photo-stimulation with visible light. The study provides insights for the development of safer and more efficient antibacterial agents for honeybee health. BV and Ag NPs show promise as alternative treatments for AFB, without detrimental effects on honeybee lifespan. Future research could explore the application of BV/Ag NCs in beekeeping practices for combating honeybee diseases and promoting their overall well-being.

Material and methods

Preparation of Ag nanoparticles

Using sodium borohydride (NaBH4) and Polyvinyl pyrrolidone (PVP; Mw = 40,000) as reducing and stabilizing agents, respectively, silver nanoparticles (Ag NPs) were synthesized. After 30 min of stirring, 0.2 g of PVP was dissolved in 100 mL of 1 mM silver nitrate, and 40 mL of 2 mM ice-cold NaBH4 was added dropwise until the color changed from colorless to greenish yellow, indicating the production of Ag NPs. The NPS were centrifuge at 6000 rpm and washed with distilled water to remove any access of NaBH4. This high-speed centrifugation step and subsequent washing with distilled water would effectively separate the synthesized Ag NPs from the reaction medium, including any unreacted NaBH4.

Pathogen

The subculture originates from a local strain of bacterium that was previously isolated on a slant of SBA medium from an exemplary American foulbrood (AFB) sample, as depicted in Fig. 5a. The bacterium was identified as Paenibacillus larval bacterium through biochemical API tests conducted by VACSERA47. Over time, this pure isolation has been progressively re-cultured every three months using the method introduced by Alippi et al.48 (Fig. 5b,c).

Figure 5
figure 5

Thread line of decayed larva (distinguished one of AFB symptoms) (a), Isolated colonies of P. l. bacterium on MYPGP medium (b), and Gram + ve rods of P. l. bacterium; Magnify = 400 × (c).

Full size image

Bee venom (BV):

Raw bee venom refers to the soft, fine, and pale white scales of a natural bee product obtained using an electroshock instrument on colonies. The VC-Starter kit's configuration, as illustrated in Fig. 6a–d was modified with the following settings: Timer on 0.5–2 s, Timer off: 3–5 s, Input Voltage: 11.5–13.5 VDC. The collector frames have dimensions of 40 cm by 50 cm. The semi-automatic operation mode involves a temperature range of 5 °C to 40 °C, with a maximum humidity of 95% at 40 °C. The maximum working time is 8 h. The collector consists of a frame connected to the pulse resource, and each collecting treatment lasts for 30 min. During this period, the device automatically activates and sends predefined pulses through the wired networks. When a bee encounters two wires connected by a short circuit, it receives a mild electric shock. This shock induces the bee to sting the glass sheet used for venom collection. After 30 min, the collector frame is removed from the colony, and the bee venom deposited on the glass plate is scraped off using a scraping knife. The collected bee venom is then weighed according to each colony, with a quantity measured for each one49.

Figure 6
figure 6

Scrap off bee venom and the collected bee venom process (a–c) and bee venom collector device (d).

Full size image

The UV–Vis absorption spectrum of the bee venom (Fig. 7a) showed an absorption peak at 266 nm in the UV range, but no absorption peaks were observed in the visible range. This indicates that the bee venom compounds primarily absorb in the UV region of the spectrum. The FTIR spectrum of the bee venom (Fig. 7b) exhibited several characteristic absorption bands. The most intense band was observed at 3327 cm−1, which is attributed to the stretching vibrations of hydroxyl groups from water and carbohydrates (such as glucose, fructose, and trehalose) present in the venom. This broad band likely also overlaps with the N–H stretching vibrations (amide A) of proteins, as bee venom is known to contain a high proportion of protein-based components. The vibration at 2927 cm−1 corresponds to the C-H stretching of methyl and methylene groups from both proteins and lipids. The medium intensity band at 1636 cm−1 was assigned to the C=O and C–N stretching vibrations (amide I) associated with the peptides and enzymes (globular proteins) present in the bee venom. The bands at 1112 cm−1 and 1040 cm-1 indicate the presence of nonstandard helical conformations. An absorption band at 1222 cm−1 was attributed to the PO2- stretching of lipids (phospholipids). The spectral region from 1200 to 900 cm−1 contained numerous vibrations, including a weak band at 1083 cm−1 corresponding to the PO2− symmetric stretching of phospholipids. Additional bands at 1050 cm−1 (C–O–P stretching) and 986 cm−1 (CN asymmetric stretching of (CH3)3N+) further confirm the presence of lipids in the bee venom sample. These FTIR results provide detailed insights into the chemical composition and functional groups present in the bee venom used in the study50,51,52.

Figure 7
figure 7

The UV–Vis absorption spectrum (a) and FTIR spectrum (b) of the produced bee venom.

Full size image

The bee venom was subjected to chemical analysis, the results of which are partially summarized in Table 4.

Table 4 Chemical analysis of the produced bee venom.
Full size table

Bioassay tests

To investigate the antibacterial properties of the propagated materials against the pathogenic bacterium P. larval, 9 cm petri dishes were prepared. The dishes were filled with MYPGP medium which consisted of Muller Hinton Broth 10 g + Yeast Extract 15 g + K2HPO4 3 g + Glucose 2 g + Sodium Pyruvate 1 g + Agar 20 g according to Nordstrom & Fries, 199553. MYPGP medium previously autoclaved at 1 bar and 121 °C for 20 min. Firstly, the medium was cooled to 40 °C, and then 20 mL was poured into each dish. The dishes were then allowed to solidify at room temperature for 24 h. Any contaminated plates were discarded to ensure accuracy. The materials and treatments assessed against P. larvae included five separate groups, with each group consisting of six replicates and evaluated under both dark and light conditions. The treatments were categorized as follows: BV, Ag NPs, BV/Ag NCs, Tylosin veterinary antibiotic (used as a positive control), and sterile distilled water (used as a negative control). To conduct the bioassay, paper discs measuring 4 mm in diameter were created and sterilized. These discs were then assigned to the various treatment groups54, these discs soaked inner samples of the treatment preparations in cooler for along 24 ours. Using a sterile cotton swab, streaks of isolated P. l. bacterial saline were spread on the surface of complete MYPGP dishes, allowing the bacterium to sporulation. The MYPGP dishes were divided into equal groups, with each group containing six replicates. In each group, the appropriate numbers of discs corresponding to a specific treatment were placed on the surface of the medium, independently in each of the six replicates. The plates containing the MYPGP medium were incubated aerobically in the dark at 37 ± 1 °C for 24 h. It is important to note that the treatments involving Ag NPs alone or BV/Ag NCs (2nd or 3rd groups) were repeated under the same conditions, but after exposing them to light and then treating the bacterium. The petri dishes were examined to observe a uniform lawn of bacterial growth, with clear zones indicating inhibition caused by the different treatments. The inhibition zones around the diffusion discs were photographed, measured in millimeters, whereas the inhibition zone diameters were measured by using model of Olympus SZ_6145TR Trinocular Stereo Microscope 0.67 ×–4.5 × provided with micrometric optical lens, for accuracy, the lengths of inhibition diameters that appeared around discs in all 6 replicates in each treatment group were recorded in the micrometric grading with micrometers then converted to millimeters according to the account "1 mm = 1000 µm", finally, these measurements were recorded for analysis55.

Longevity examination

To evaluate the impact of different treatments and their concentrations on the lifespan of honeybees, an experiment was conducted at laboratory on suitable and districted numbers of adult bees inner simulated cages whereas, Sealed brood frames were selected from a healthy colony of Carniolean hybrid honeybee race (Apis mellifera var carnica) from the apiary of the Bee Research Department, Plant Protection Research Institute. These frames were placed in a controlled environment chamber with a constant temperature of 35 °C and a relative humidity of 75%56. One-day-old worker bees were obtained from the frames and placed in cages, with each cage containing fifty bees and fed with the preparations in the same emergence day. The cages made of wood and equipped with a slide able Plexiglas's door with drilled holes, had dimensions of 90 × 80 × 110 mm. There was a feeding hole on the top of each cage, providing an accessible pathway for adding feed (Fig. 8). The feed consisted of either a 50% sucrose solution alone or a 50% sucrose solution mixed with the tested treatments. Both types of solutions were replaced daily. The worker bees' diet comprised a mixture of sucrose solution and the tested materials, soaked for 24 h, with approximately 5 g of the mixture placed in a plastic cap measuring 10 mm × 30 mm. The worker bees' diet was changed and replenished daily57. The experiment consisted of four groups, each group contained on 3 cages of honeybee adult workers (50 workers\cage), the four groups were treated with the following treatments and mentioned concentrations; 1st group fed on sucrose solution 50% mixed with Ag NPs 15, 22.5 or 30 mg\L separately, 2nd group fed on sucrose solution 50% mixed with bee venom 1.53, 3.12 or 6.25 mg\ml separately, 3rd group fed on sucrose solution 50% mixed with pairs of Ag NPs 15 mg\L + bee venom 1.53, 3.12 or 6.25 mg\ml separately, and finally, 4th group fed on only sucrose solution 50% as a control. Each concentration was replicated three times for confirmation. All the cages were kept in an incubator at 32 °C and 65% relative humidity throughout the experimental period. The susceptibility of honeybee workers fed on BV, Ag NPs, BV/Ag NCs, and sugar syrup only (control) was assessed to determine their lifespan using a technique developed by Maggi et al.58. Whereas all cages of each treatment group were monitored, dead workers were counted and removed daily. The longevity of honeybee workers was estimated by constructing life tables following the method of Fukuda and Sakagami59.

Figure 8
figure 8

One replicate of the cages in which performed of the longevity experiment, the replicate shows both cage design and the treatment of honeybee workers by (Ag NPs 15 mg\L and Bee venom 3.12 mg\ml) in sugar feeding putted inner caps on bottom of the cage.

Full size image

When studying the effects of bee venom or other treatments on bee longevity, it is crucial to start with bees that are as healthy and uncompromised as possible. Using newly emerged, one-day-old worker bees helps to establish a baseline of normal, uninfected bee lifespan before any experimental treatments are applied. This approach allows the researchers to more accurately assess the impacts of the bee venom or other factors on bee longevity, without potential confounding effects from pre-existing infections or other health issues. Starting with young, uninfected bees provides a cleaner experimental system to isolate the effects of the specific variables under investigation. Evaluating the longevity of these one-day-old worker bees, even in the absence of bacterial infection, gives important insights into the intrinsic effects of the treatments on normal bee physiology and lifespan. This can provide a stronger foundation to then examine how the treatments may interact with or protect against specific pathogenic challenges.

Adult honey bee worker individuals basically do not show symptoms of the disease under study (American foulbrood) due to the inability of the spores of this disease’s bacterium to transform into the harmful vegetative phase in the midguts of adult worker bees, due to: 1st; The pH level in the intestine of the adult worker differs from that in the intestine of the worker larva in its early stages due to the presence of honey and pollen as main food in the midgut of the adult worker. 2nd; The shape of the tissue structure (histology) of the intestinal walls in whole individuals of worker bees changes and transforms into a thick double wall structure surrounded from the inside by a peritrophic membrane. In the end, this somewhat complex tissue structure prevents the germination of Paenibacillus larvae bacterium and prevents their penetration of the intestine wall into the haemolymph cavity where the nutritional medium is typical for this bacterium to multiply, so that the cycles of replication begin, causing infection, and then the appearance of disease symptoms, similar to what happens in the guts with a simple histological composition of bee larvae in their early ages, and their dependence only on royal jelly appropriate for their food to carry out their action in causing infection. Accordingly, the adults of worker bees only carry the spores of pathogen in their guts, if they are present in an infected bee colony, and then they transmit these spores to the healthy bee larvae in the royal jelly that they secrete for them. Because bee venom, silver nanoparticles and their mixture were tested against the bacterial pathogen initially in the artificial medium, MYPGP in order to determine firstly the extent of their ability to inhibit pathogenic bacterium, and secondly to determine the minimal concentration of them that is inhibitory to bacterium in preparation for using these treatments and these concentrations against the disease in the field within the infected bee colonies in other studies in the future.

Based on the above, it was necessary and logical test of the safety of using these treatments and their effective concentrations discovered in the laboratory, starting with samples of adult bees to find out which of these treatments are harmful, meaning whether they are inherently lethal to the adult bees that will feed the larvae infected with the disease or not. As well as determining which of these concentrations would be harmful to adult bees, represented by measuring the average longevity of adult worker bees considering the presence of these treatments in the nutrition provided to the bees? Therefore, it was necessary to start with adult worker bees, one day old, to obtain an accurate calculation of the ages of the bees as nurses’ bees who are responsible for feeding the larvae that infected with the disease and appear the symptoms, and then their ages as a swarm bee.

Statistical analysis

The obtained results from all treatments were analyzed using MSTAT-C version 1.41, employing a randomized complete block design (ANOVA). GraphPad Prism version 3.03 for Windows software was used for data analysis60. Duncan's multiple range test was employed to compare the means of all treatments at a significance level of 0.0561.