Acaricidal and insecticidal activities of entomopathogenic nematodes combined with rosemary essential oil and bacterium-synthesized silver nanoparticles against camel tick, Hyalomma dromedarii and wax moth, Galleria mellonella

Introduction

Ticks are ectoparasites that feed on vertebrates, resulting in significant blood loss and decreased weight gain in infested livestock (Mukhebi et al., 1999), and they are crucial in the spread of numerous fatal diseases to both livestock and humans (Raoult et al., 2001; Parola, 2004). The camel industry faces a significant challenge from ixodid ticks due to their ability to transmit diseases, as well as their tendency to inflict economic losses in infected animals (Jabbar et al., 2015). However, research revealed that over 20 species of ixodid infest camels (Banaja & Ghandour, 1994), making their active control crucial. The most common species among them are ticks belonging to the genus Hyalomma, which could serve as vectors for Theileria spp., Babesia spp., and Anaplasma spp. (Fard et al., 2012; Barghash & Hafez, 2016; Alanazi et al., 2018).

Hyalomma dromedarii (Acari: Ixodidae) was predominantly tick-infested camels. Mostly, ticks and mites infest camels, but they also affect cattle (Barker & Murrell, 2004). About 95.6% of Sinai camels, 89% of Sudan camels, and 57.13% of Benha and Belbis (Egypt) camels were infested (Ashjaran & Sheybani, 2019; Al-Ghamdi, Aly & Sheshtawi, 2020). Saudi Arabia is one of the nations where the camel population has grown rapidly in recent years. As of 2017, the country had about 500,000 camels, with the largest percentage living in Riyadh Province (FAO, 2019). Saudi Arabia has documented numerous species, including different Hyalomma spp., infesting camels and cattle (Al-Afaleq et al., 2018).

Currently, we use various synthetic acaricides like avermectin, deltamethrin, and cypermethrin (Al Thabiani et al., 2021) to manage H. dromedarii. However, acaricide-resistant tick strains developed, polluted the environment, contaminated meat and dairy products, and the high cost as a result of the ongoing usage of these acaricides made them inaccessible by small-scale farmers (Gupta & Gupta, 2018; Singh, Abhijit & Harkirat, 2019). The existence of several tick species that present diverse antigens also limits vaccination efficiency (de la Fuente & Estrada-Pena, 2019). Consequently, there was a worldwide movement to assess and propose environmentally friendly and efficient agents as a substitute since they were accessible, affordable, and had low levels of toxicity, resistance, and environmental pollution.

Biological acaricides are safe, cost-effective, and biodegradable (Karthi et al., 2020). An integrated tick management program (ITMP) requires a variety of techniques with different modes of action for successful execution. This will optimize effectiveness while lowering the possibility that target pests will develop resistance to the chosen treatments, which may include chemical acaricides (de Leon, 2017; Rodriguez-Vivas, Jonsson & Bhushan, 2018).

The only species in the genus Galleria is the greater wax moth (Hamza & Sayed, 2019), often known as the honeycomb moth (Galleria mellonella L.; Lepidoptera: Pyralidae). The caterpillar larvae feed on the honeycomb found inside bee hives, so they commonly known as wax worms. However, certain traits that G. mellonella possesses make it a suitable choice for researching host-pathogen relationships. It is an excellent, inexpensive, simple, and quick in vivo model to evaluate the pathogenicity of microorganisms, the toxicity of different compounds, and also to study nanoparticle interactions with living systems, and has numerous advantages over other animal models as well (Cutuli et al., 2019). We can run their larvae at the human core body temperature of 37 °C, and they display a response to the innate immune system akin to that of mammals, leading to pathogen elimination through comparable mechanisms (Wojda et al., 2020; Noureldeen et al., 2019; Andrea, Krogfelt & Jenssen, 2019).

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae offer effective substitutes to the acaricides and insecticides currently in use because many investigators have found encouraging results, with some isolates exhibiting acaricidal and insecticidal activities on certain tick and insect species or developmental stages (Monteiro & Prata, 2015; EL Roby, Rezk & Shamseldean, 2018; Alotaibi et al., 2021; Noureldeen et al., 2022). Together with the help of mutualistic bacteria, EPNs kill ticks and other insects. Photorhabdus and Xenorhabdus bacteria and Heterorhabditis and Steinernema nematodes live together in harmony (Gaugler & Kaya, 1990). The only free-living stage, infective juvenile (IJ), enters hosts by spiracles, the mouth, anus, and occasionally the cuticle. They kill the host within 48 h after releasing their bacterial symbionts into the hemocoel (Koppenhofer, 2007).

According to previous research (El-Sadawy, Zayed & El-Shazly, 2008), heterorhabditid strains have a greater influence on H. dromedarii engorged females than do stienernematid strains, with mortality rates ranging from 12–92% for the former and 4-88% for the latter. Furthermore, it was mentioned that G. mellonella, which is utilized as a model to investigate EPNs, is extremely vulnerable to infection by IJs (Ehlers & Shapiro-Ilan, 2005). Research has indicated that specific EPN isolates exhibit a stronger attraction to G. mellonella when compared to other insect species (Koppenhöfer & Fuzy, 2008). According to Bisch et al. (2015), EPNs form a symbiotic association with Enterobacteriaceae bacteria, which may act as an effective biocontrol alternative for G. mellonella.

Nanotechnology, a rapidly developing field, creates biomaterials with precise size and shape. Nanoparticles (NPs) are widely used in numerous disciplines and incorporated into a wide range of commercial products, as well as biomedical and veterinary applications, due to their small size (de Oliveira et al., 2021; Abdel-Ghany et al., 2022). At numerous stages of the parasite’s life cycle, metallic and non-metallic nanoparticles have demonstrated significant potential for quickly causing toxicity and lowering lethal doses (Benelli et al., 2017; Esmaeilnejad et al., 2018). Currently, nanobiotechnology is thought to be a revolutionary approach to tick control. Silver is a valuable and widely available metal, and studies have suggested that its nanoparticles may have anti-tick and anti-insect properties because they are preferable to other nanosized metal particles (Norouzi et al., 2019).

Today, a novel technique for producing nanomaterials is facilitating the development of biotic systems, particularly microorganisms (Saha et al., 2017). Because biological methods are more economical, sustainable, and ecologically benign than chemical and physical processes, they are preferable for synthesizing nanoparticles. As a result, nanotechnology scientists are interested in using prokaryotic organisms and their exudate to produce NPs (Sengani, Grumezescu & Rajeswari, 2017). Numerous microorganisms, including yeasts, actinomycetes, fungi, and bacteria, can create NPs by extracellular or intracellular pathways. Several bacteria, including Rhodopseudomonas capsulate, Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa, Brevibacterium casei, Escherichia coli DH5a, and Stenotrophomonas maltophilia, were used to produce silver nanoparticles (Rajeshkumar et al., 2016). Previous studies have documented the negative effects of the active components of the gram-negative bacteria Proteus mirabilis on the tick Hyalomma marginatum (Hendry & Rechav, 1981). Recently, Proteus mirabilis is considered one of the most important bacteria that have been utilized for synthesizing silver nanoparticles (AgNPs), showing their antioxidant ability in vitro, and being as antimicrobial and disinfectant agents (Elhariry et al., 2018; Yasr & AL-Ramahy, 2022).

As an alternative to pesticides (Abdel-Meguid et al., 2022), botanicals like essential oils (EOs) show promise because they are safe for non-target organisms, break down quickly, have a complex makeup that keeps resistance from developing, and have effects that kill adults, eggs, larvae, and other pests (Baz et al., 2022). Plant products have benefits, but they also have disadvantages, such as inconsistent effectiveness, chemical instability, and little residual activity, often caused by photosensitivity or excessive volatility (George et al., 2014). The literature reveals that researchers have globally examined 356 plant species from 105 families against 31 tick species (Nwanade et al., 2020). In previous studies, essential oils of different plants revealed acaricidal activity against different tick species, such as Lavandula angustifolia, Eucalyptus globulus, Mentha piperita, Artemisia annua, Rosmarinus officinalis, and Thymus vulgaris oils against Rhipicephalus microplus and Hyalomma spp. (Djebir et al., 2019; Valcarcel et al., 2021).

Worldwide, there are no studies that have examined the interaction effects of EPNs with either nanoparticles or plant products against H. dromedarii. Therefore, this study was conducted for the first time to assess the in vitro acaricidal and insecticidal efficacies of locally two EPNs, Heterorhabditis indica and Steinernema sp. integrated with either Proteus mirabilis-synthesized silver nanoparticles or Rosmarinus officinalis essential oil, against the camel tick Hyalomma dromedarii and greater wax moth Galleria mellonella, as well as determine their potentials on biological parameters of H. dromedarii-engorged females.

Materials and Methods

Results

Characterization of P. mirabilis bacterium-synthesized AgNPs

This work described the biosynthesis of AgNPs by the P. mirabilis bacterium. Upon incubating the bacterial filtrate with silver nitrate at room temperature, we observed a visual color change from bright yellow to dark brown, while AgNO₃alone did not exhibit any color change (Fig. 1A). AgNPs were found to be spherical and monodispersed, with an average diameter ranging from 5 to 45 nm, according to the TEM image and size distribution (Figs. 1B, 1C). Figure 1D shows the UV-vis spectrum of AgNP biosynthesis. Consequently, we discovered the largest surface plasmon peak of the colloidal solution’s AgNPs at 320 nm.

Nematicidal activity of EO or AgNPs against EPNs

Figure 2 summarizes the mortality rates of Steinernema sp. and H. indica dauer juveniles exposed to R. officinalis EO and biosynthesized AgNPs treatments at different concentrations and exposure periods. The results revealed significant differences (P < 0.05) between all concentrations examined against both EPN species at various exposure durations. All concentrations of EO or AgNPs were considered harmless to the two species of EPNs, as the treatments demonstrated less than 10% mortality. Long exposure time and high concentration of the EO (60 mg/mL) caused low mortality of both EPN species, which valued 5% for Steinernema sp. and 3.2% for H. indica (Figs. 2A, 2B). At the highest concentration of AgNPs (4 mg/L), however, Steinernema sp. died 9.8% of the time and H. indica juveniles died 4% of the time after 96 h of exposure (Figs. 2C, 2D).

Discussion

This study investigates, for the first time, the combined effects of endogenous entomopathogenic nematodes with either rosemary EO or P. mirabilis-AgNPs against H. dromedarii and G. mellonella. The results showed that applying EPNs alone was lethal to both pests; however, combining EPNs with EO or AgNPs sequentially improved the level of lethality in a synergistic or additive mode. All concentrations of EO and AgNPs were safe for the two species of EPNs that were tested (H. indica and Steinernema sp.), according to the conventional mortality bioassay test used in this study. Notably, Steinernema sp. exhibited significantly higher sensitivity to both compounds compared to H. indica, and AgNPs proved to be more toxic to both EPNs than EO. The findings of the (Barua et al., 2020) study align with these results, confirming the toxicity of rosemary EO to plant parasitic nematodes. While it doesn’t kill slug parasitic and entomopathogenic nematodes, clove, thyme, garlic, and cinnamon oil also harm beneficial nematodes. Our results suggest that rosemary EO, which has demonstrated effectiveness against harmful nematodes without harming beneficial nematodes, is suitable for use in pest management. This study highlights several significant concerns about the application of rosemary essential oil in IPM programs, which also utilize nematode biological control agents. Heterorhabditis and Steinernema species share similar basic biological and physiological traits with plant parasitic nematodes, and they even inhabit similar soil ecosystems (Kenney & Eleftherianos, 2016). In spite of this, Uludamar (2023) found that rosemary oil at 10,000 ppm killed more than 92% of root-knot nematode, Meloidogyne chitwoodi second stage juveniles 24 h after exposure, while our study only found low rates of EPN mortality. The outer layer of EPNs and plant parasitic nematodes has lipids that dissolve in oils. This means that EOs might be able to get through this layer and affect how the organisms work (Stadler & Buteler, 2009). However, unlike most plant parasitic nematodes, EPN IJs have a double cuticle, which would give them an additional defense against these EOs. In line with our study, Kucharska et al. (2011) found that high concentrations (5 ppm) of AgNPs killed 99.6% of H. bacteriophora and 96% of S. feltiae on the fifth day of the experiment. Lower concentrations (0.5 ppm) killed only 4% of H. bacteriophora but 85% of S. feltiae.

Obtained data indicated that the combined applications all showed higher mortalities in the host species tested than single applications at the different concentrations and exposure times tested. Previously, Capinera (2008) showed that the toxicological interaction of certain chemicals with EPNs can lead to an increase in the mixture’s final toxicity (synergism), an additive increase in toxicity, or a decrease in toxicity (antagonism). As a result, in the current study, the interaction of the tested EPNs with EO or AgNPs appeared to exhibit a synergistic or additive effect on tick and insect mortality 4 days after treatment. It was the combination of H. indica at half concentration and EO at high concentrations that killed all the larvae and engorged females of H. dromedarii 24 h after treatment (100% mortality rate). On the other hand, the same treatment of H. indica with either a moderate or high concentration of EO killed all the larvae of G. mellonella during the same exposure period. Increasing biological activity against the target organism and lowering participant concentration are the two basic goals of employing synergistic combinations. Furthermore, the higher chemical complexity of combinations reduces the probability of selection for resistance and the subsequent generation of resistant populations (Jyoti et al., 2019).

Interestingly, H. indica IJs alone demonstrated exceptional virulence against all examined host species, surpassing the virulence of Steinernema sp. and other single treatments. It verified more than 91% mortality in H. dromedarii ticks starting on the third day and reaching 100% in just 2 days of G. mellonella exposure. Moreover, the larvae and adults of the tick, as well as the larvae of the wax moth, showed extreme vulnerability to this EPN’s infection, even when exposed in small quantities 4 days after exposure. Because lepidopteran larvae are extremely susceptible to infection by many isolates, this was already predicted. Researchers use this species as a model insect to study EPNs (Dolinski, 2006). According to their behavioral ecology, cruiser EPNs actively seek out insects by recognizing chemical cues released by their hosts, such as uric acid, CO₂, excrement, and intestinal fluid. A specific species of arthropod may attract infectious juveniles more strongly due to the types and quantities of chemical signals they release (Bisch et al., 2015). The greater number of hosts and consequent increase in chemical signal emission are probably the reasons for H. indica’s superior efficacy against H. dromedarii in groups containing larvae or G. mellonella. This might have enhanced the activity of IJs, enabling them to go through the soil more quickly, which would have facilitated and expedited the host’s search, localization, and penetration activities (Monteiro et al., 2020).

We also observed the same trend with Steinernema sp., which ranked second among all separate treatments in terms of virulence effects against tick and insect individuals. Therefore, in the present study, EPNs seemed to be the important factor in tick and insect mortalities when applied in combination with EO or AgNPs. In addition, it was clear from our results that rosemary EO showed higher lethality against all host species than AgNPs. Even though the ixodicidal and insecticidal effects of the AgNPs by themselves were very good in this study, adding EPNs might make them much more effective and even make them more resistant to insects and ticks. The current research revealed a reduction in the toxic effect of nanomaterials evaluated against various stages of H. dromedarii (Benelli et al., 2017). This could be a result of the particles’ composition or the ecological and biological characteristics of ticks, which can change an individual’s susceptibility to nanoparticles relative to others. The P. mirabilis supernatant solution changed color from pale yellow to dark brown upon the addition of AgNO₃. The naked eye could observe this, indicating the bio-reduction of Ag ions and the formation of AgNPs in the reaction solution. These results were in accordance with (Ejidike & Clayton, 2022; Ogunsilea et al., 2024) who reported that the synthesis of AgNPs from Daucus carota and Chromolaena odorata leaf extract was confirmed by a gradual change in color from colorless to darkish brown precipitate. This led to the discovery that the AgNPs in the mixed particles had a big absorption peak centered at 320 nm when the UV spectroscopic study was done. The TEM study showed that the particles were evenly spread out and had a diameter of 5–45 nm. We used P. mirabilis to biosynthesize silver nanoparticles, resulting in particles ranging in size from 35 to 100 nm, in agreement with the previous report by Yasr & AL-Ramahy (2022). Vasyliev et al. (2020) also agreed with the result. They said that the particles’ surface plasmon vibrations are what cause the brown color to be absorbed, and that the shape, size, dielectric medium, and chemical environment around the dispersed nanoparticles in water strongly affect surface plasmon absorption.

In this work, we used GC-MS analysis of R. officinalis essential oil to identify twenty eight phytocompounds significant to science, industry, and biology. There are a lot of different chemicals that make it up, but some of the most important ones are verbenone, 2-(2′,4′-dimethoxyphenyl)-6-methoxy-benzofuran, 2(1H)-phenanthrenone, 4a,9,10,10a-tetrahydro-6-hydroxy-1,1,4a-trimethyl-7-(1-methylethyl)-, (4aStrans)-, demethylsalvicanol, delta.9-tetrahydrocannabivarin, ferruginol, geranyl formate, 2,6-octadiene-1,8-diol, 2,6-dimethyl-, 13-isopropylpodocarpen-12-ol-20-al, caryophyllene oxide, 2,6-dimethylocta-3,5,7-trien-2-ol, and methyleugenol. Other chemicals are present in trace or low amounts. The results were consistent with the findings of Theyyathel et al. (2023), who reported that chemical profiling identified twenty-six chemicals in the methanolic leaf extract of R. officinalis. These substances include ethers, long-chain hydrocarbons, sterols, fatty acid esters, fatty alcohols, terpenes, and vitamin metabolites. Also our results were in harmony with those previous reported by Saleh et al. (2022) in Saudi Arabia, who stated the presence of 18 constituents in rosemary essential oil, representing 99.93% of the total oil content. The major constituents detected in oil were bornyl acetate (26.59%), eucalyptol (17.38%), camphor (10.42%), borneol (9.78%), beta-caryophyllene (7.80%) and α-pinene (3.85%), having strong antimicrobial activity against Staphylococcus aureus. In similar study by Martinez-Velazquez et al. (2011), they recorded that rosemary EO was rich in α-pinene (31.07%), verbenone (15.26%), and 1,8-cineol (14.2%), whereas Mexican oregano essential oil included thymol (24.59%), carvacrol (24.54%), p-cymene (13.6%), and γ-terpinene (7.43%); however, garlic essential oil was rich in diallyl trisulfide (33.57%), diallyl disulfide (30.93%), and methyl allyl trisulfide (11.28%).

With this regard, several bioactivities of R. officinalis EO have been documented, including insecticidal (Waliwitiya, Kennedy & Lowenberger, 2009) and acaricidal activity against two-spotted spider mite, Tetranychus urticae (Miresmailli & Isman, 2006). On the contrary, Laborda et al. (2013) found that rosemary and sage essential oils caused acute contact toxicity against T. urticae, although they showed no insecticidal activity against Ceratitis capitata. Furthermore, rosemary essential oil produced 85% R. microplus larval tick mortality at the higher concentrations (10% and 20%), while Mexican oregano and garlic essential oils had very similar activity, producing high mortality (90–100%) in all tested concentrations (Martinez-Velazquez et al., 2011). In the present study, the acricidal and insecticidal activities of rosemary EO could be attributed to the presence of verbenone, benzofuran, phenanthrenone, and tetrahydrocannabivarin compounds, which have been previously confirmed by other studies (Martinez-Velazquez et al., 2011; Bracalini, Florenzano & Panzavolta, 2024; Park et al., 2019; Díaz et al., 2023). In the same context, Martinez-Velazquez et al. (2011) indicated that the main chemical constituents of rosemary EO, which are probably responsible for acaricidal activity, are α-pinene, verbenone, and 1,8-cineol, compounds. Similarly, GC-MS analyses of R. officinalis and Salvadora persica essential oils showed that the main monoterpenes in both oils were 1,8-cineol, α-pinene, and β-pinene, although in markedly different proportions, suggesting that essential oils exhibit a great potential as substitute methods for controlling Ixodes ricinus ticks (Elmhalli et al., 2019).

Research has recently focused on the pharmacological attributes of ferruginol, a diterpene phenol, which include antimalarial, anticancer, antibacterial, and cardioprotective activities (Lim et al., 2022; Salih et al., 2022). Similar to this study, Fraga et al. (2005) also found that compounds called demethylsalvicanol and ferruginol, which were taken from the hairy roots of Salvia broussonetii, were moderately effective at stopping the Leptinotarsa decemlineata insect from feeding. After oral administration, none of these compounds had antifeedant or negative effects on Spodoptera littoralis ingestion or weight gain. Other chemicals, like Geranyl formate and Caryophyllene oxide, were also found in the essential oil of R. officinalis and in the essential oil of Pelargonium spp. in other studies. These chemicals are known to be toxic to Ae. aegypti larvae and to keep them away (Nararak et al., 2023).

Among all tested host individuals in the present study, G. mellonella larvae demonstrated high susceptibility to all treatments, exhibiting higher mortalities and lower LT₅₀ when exposed to both EPNs incorporated with EO and AgNPs. This suggests that the compatibility of EPNs with EO or AgNPs exhibited stronger insecticidal activity than acaricidal effect. Furthermore, the present study reported the larvicidal and adulticidal effects of compatible EPN treatments with EO and AgNPs on H. dromedarii larvae and engorged females, respectively. Additionally, the larvae responded to the treatments more quickly than the engorged females, who moved slowly during the 2 days following treatment. They laid a normal egg mass and number, slightly lower than the control, but there was a significant variation in hatchability. Furthermore, the engorged females died on the 3^rd day, particularly when exposed to the lower concentration of AgNPs alone; however, the larvae that received the same concentration showed mortality 24 h after treatment. The larger chitinized cuticle area of the engorged females, compared to the larvae’s less chitinized body area, could explain this observation.

Indeed, according to the biological characteristics examined, all of the treatments suggested in this study proved to be compatible with the action of EPNs, either by themselves or in combination, on H. dromedarii engorged females. The parameters showed that the groups treated with H. indica had shorter pre-oviposition, oviposition, and survival periods than the groups treated with Steinernema sp. Also, the values for EW, H%, and NEI% were higher in the Steinernema sp. treatments than in the H. indica treatments. This shows that H. indica is stronger than Steinernema sp., whether they are used alone or together. Our findings reveal that applying EPN H. indica in combination with EO inhibits egg-laying in H. dromedarii females by more than 73%, resulting in only 11.3% of eggs hatching. The infectivity tests in this study confirmed that H. indica is very strong against H. dromedarii. They also showed, for the first time, that these nematodes can kill ticks before they lay their eggs, whether they are used alone or with EO. When engorged females of H. dromedarii were exposed to H. indica, it changed the egg mass weight, but not in a way that was statistically significant when AgNPs were added. In other words, the entomopathogenic nematode infections did not get in the way of the metabolic conversion process or the production of the nutrients needed for egg formation before the eggs were laid. As a result, the best control percentage (95.6%) was obtained from the H. indica + EO treatment; it is not statistically different from the treatment when H. indica alone is present, although the efficacy was lower (89.4%). The results were better than those reported by Saman et al. (2023). They said that using 150 IJ/engorged females of H. indica LPP1 and H. bacteriophora HP88 led to an almost 90% reduction in the number and viability of eggs laid by engorged females of R. microplus. Also, these results are similar to what was reported by Albogami (2024), who said that the two species of Heterorhabditis are among the most dangerous to engorged H. dromedarii females, even more so than EPN from the Steinernema genus.

The results of the current preliminary study indicated that applying EPNs alone or integrated with either EO or AgNPs promise to provide superior efficacy against ticks and insects, but further research targeting other economically important tick or insect species is needed to elucidate their mode of action, side effects, and formulation development to confirm the method’s success in (livestock and apiary).

Conclusion

Researchers all over the world are investigating and introducing novel, safe, and efficient bio-agents as alternatives for chemical pesticides. This trend is influenced by a number of factors, including resistance, toxicity, availability, low cost, low pollution of the environment, and low side effects. It is worth noting that arthropod pests of economic and medical significance can be effectively and economically controlled with EPNs, EO, and bio-synthesized AgNPs. In order to manage H. dromedarii and G. mellonella, our work is the first to document the interaction of EPNs with either EO or AgNPs. Fortunately, the results showed that associating EPNs with EO was more effective than using AgNPs for controlling the two organism species that were being studied. The use of rosemary EO at a sub-lethal concentration that is compatible with EPN appears to be promising for efficiently causing toxicity in tick and insect pests.

We propose that impregnating tick and insect cuticles with R. officinalis EO or P. mirabilis-AgNPs, followed by nematode exposure, shows compatibility for livestock use. This approach, where treated camels are sprayed with EO or AgNPs and then exposed to nematodes in pasture, could allow nematodes to infect ticks post-detachment. Alternatively, combining EPNs with EOs/AgNPs directly on infested camels may enhance tick control and slow resistance development. Although successful EPN applications are simpler pest-host models, similar methods may suppress wax moth populations outside beehives, like in storage by dipping or spraying infested combs. Further research is essential to fully explore EPNs, EOs, and AgNPs for pest control.

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