The Oregon R strain of Drosophila melanogaster was selected as the model organism for our in vivo experimental studies. Fly cultures were maintained in a controlled environment (incubator) at 25 ± 1 °C with a relative humidity of 60 ± 5% and a 12:12 h light–dark cycle. The flies were reared on a standardized diet consisting of agar, cornmeal, dextrose anhydrous, sucrose and yeast extract [Cat No. 1D001-1 L, Himedia Pt. Ltd., India] with the addition of 80% propionic acid or nipagin (methyl-p-hydroxy benzoate) to prevent fungal contamination. These optimized conditions ensured optimal growth, reproduction, and experimental reproducibility. Both the control and treated groups were maintained under identical conditions throughout the study to minimize environmental variables and ensure reliable results.

EMF exposure was performed via a gigahertz transverse electromagnetic (GTEM) cell [GTEM 250A SAE model]. The EMF source was a certified horn-shaped GTEM cell (Figure 1) with a power density of 916 mW, an electric field strength of 44.29 V/m, and an amplitude of −20 dBm. The Oregon R strain of Drosophila melanogaster was exposed to 2.4 GHz HF-EMFs for durations of 10, 20, and 30 min. The experimental groups of Drosophila were meticulously organized based on two key variables: the dosage of ZnO NPs and the duration of exposure to EMF.

ZnO NPs [CAS No. 1314-13-2, >97% Catalog No. 677450-5G, Sigma Chemical Co. Ltd., USA] were used for this study because of their extensive application in various fields. ZnO NPs were spherical with a size of 23 ± 2 nm (Transmission Electron Microscopy) and the mean hydrodynamic size of particle was 110 ± 2 nm in Dulbecco’s Modified Eagle Medium (Dynamic Light Scattering), with a polydispersity index of 0.2 (41). The flies were exposed to ZnO NPs via the ingestion method. ZnO NPs were weighed and dissolved in Milli-Q water to achieve concentrations of 0.1 mM and 0.5 mM, selected based on existing literature and prior lab studies demonstrating their biological relevance, while representing sub-toxic to moderately toxic levels commonly used in nanotoxicology research (41, 42). The solutions were vortexed for homogeneity and sonicated for 30 min at 25 °C, followed by centrifugation at 13,400 rpm for 30 min. The supernatant was collected and then mixed with the Drosophila diet.

Drosophila melanogaster adult flies were exposed to high-frequency EMFs with or without a ZnO NP-containing diet. Eight- to ten-day-old adult flies (10 males and 10 females) were randomly selected and added to each tube on the day of exposure. All the groups were organized in triplicate, and the experiments were carried out three times to ensure significance.

To investigate the combined effects of ZnO NPs and EMF radiation on flies, a total of 12 groups of fly vials (n = 20; 10 males and 10 females per vial) were used. The experimental design consisted of three main groups. Group 1 was tested for only EMF exposure, in which the flies were exposed to EMF alone for 10 min, 20 min and 30 min for 7 days. Group 2, in which the flies were exposed to ZnO NPs at two different concentrations, 0.1 mM and 0.5 mM, without EMF interference. The third group (Group 3) was exposed to the combinations of NPs and EMF, this group was further divided into three subgroups with durations of 10 min, 20 min, and 30 min were used for each concentration (0.1 mM and 0.5 mM). The control groups were kept in separate vials under the same conditions, and all the experimental vials were evaluated under blinded conditions to ensure unbiased results.

To study the effects of ZnO NPs and EMF on lifespan, we employed a longevity assay in Drosophila. Flies were maintained in their standard environment. Twenty newly emerged flies (10 male and 10 female) were reared on diets with or without NPs and further divided into two groups: one with only EMF exposure and the other without EMF exposure. To maintain consistent feeding conditions and prevent larval interference, the flies were transferred to fresh vials every fourth day. Throughout the experiment, the number of dead and live flies was carefully recorded at each transfer. The experiment was conducted for a duration of 60 days. Survival curve was generated from the recorded data.

The crawling assay was used as a behavioral and neurological assessment tool to evaluate larval locomotion, leveraging the well-established correlation between crawling patterns and neuronal function. To facilitate this analysis, a 1.5% agar substrate was prepared in a petri dish, which provided a controlled environment for larval movement. Specifically, third instar larvae were collected for this study, and then the larvae were rinsed with MilliQ to eliminate residual food debris. The larvae were subsequently placed on the agar for 30 s, after which it was allowed to leave distinct trail marks indicative of their locomotor activity. This methodology enables the quantitative analysis of larval crawling behavior by measuring the distance travelled by the larvae (Supplementary Figures S1, S2). This assay was conducted with triple replicates to enable comparisons of crawling behavior across various experimental conditions. The distance travelled by the larvae over 30 s was quantified via a graph paper grid with 0.1 cm² squares, and the data analysis was performed via ImageJ software.

The DCFDA assay was used to measure intracellular ROS and evaluate oxidative stress induced by combined exposure to ZnO NPs and EMF in Drosophila melanogaster. The assay utilized the cell-permeable dye 2,7-dichlorofluorescein diacetate (DCF-DA), which is converted to fluorescent 2,7-dichlorofluorescein upon reacting with hydrogen peroxide. The 2,7-dichlorofluorescein emitted fluorescence detectable by spectroscopy at excitation and emission wavelengths of 495 nm and 529 nm, respectively (43). The third-instar larvae were collected, washed three times with Milli-Q water to remove food residues, and then homogenized in ice-cold 1 X PBS. The homogenate was subsequently centrifuged at 5,600 × g for 10 min, after which the supernatant was treated with 15 μM DCF-DA at an equal ratio. Following a 20-min incubation at 37°C, the fluorescence was measured with excitation at 485 nm and emission at 520 nm. The results are presented as the relative fluorescence intensity normalized to that of the control larvae.

Drosophila melanogaster gustatory preferences and taste perception were investigated using a two-choice taste testing method. For this method, the flies were starved for overnight before being put in a 96-well plate (a taste test chamber) with agar gels that contained either water or sucrose (red dye, acid fuchsin). After a two-hour feeding period, the flies were anaesthetized on ice, and their abdomens were examined under a stereomicroscope for appearance of color, which were then classified as red, blue, mixed, or uncolored.

In this study, flies from two subsequent generations (F1 and F2) were comprehensively examined under a stereo microscope (OLYMPUS Co. Japan) for phenotypic abnormalities caused by NPs and EMF exposure. Following exposure, the wings, eyes, thorax, body color, and other physical features were examined for potential abnormalities. Each treatment was conducted twice to ensure accurate and consistent outcomes. Since the exposure was repeated three times, approximately 300 and 450 flies were screened for phenotypic abnormalities for each treatment, for a total of 1,000 to 1,300 flies per generation.

Statistical analysis of the data was performed via GraphPad Prism software (version 5). All the data are presented as the means ± standard deviations. The analysis included the application of Bonferroni’s multiple comparison test, and the calculation of the arithmetic mean with standard deviation via one-way ANOVA. Two-way ANOVA was used to analyse the survival results. A p value of ≤ 0.05 was used to define significance for all tests.

Survivalship assay, performed on flies exposed to EMF and ZnO NPs independently and in combination between duration of EMF exposure and concentrations of the NPs showed subtle interaction. When flies were exposed solely to EMF, there was an observable, time-dependent increase in survival, as shown in Figure 2B. The survival rate of flies exposed to EMF for 10 min increased significantly from day 14 to day 50 (Figure 2E). This trend was intensified with 20 min of EMF exposure, leading to a prolonged increase in lifespan that lasted from day 14 till end-of-life cycles. A slight but noteworthy increase in survival was also observed in flies exposed to 30 min of EMF, but the effect was more transient, lasting from 32 to 44 days.

Flies exposed to 0.1 mM ZnO NPs alone did not exhibit significant changes in survival (Figure 2A). However, when EMF was combined with 0.1 mM ZnO NPs, the longevity of the flies clearly improved (Figure 2B). Specifically, 20 min of EMF exposure slightly increased survival at 17 and 32 days. A more pronounced survival benefit was observed when EMF exposure was extended to 30 min in combination with 0.1 mM ZnO NPs, with a significant increase in longevity from 14 to 39 days (Figure 2E).

Flies exhibited a marked decline in survival when exposed to 0.5 mM ZnO NPs alone, with survival decreasing steadily from day 7 to day 50 (Figure 2E), indicating toxic effects. However, when EMF was combined with 0.5 mM ZnO NPs, the toxic effects caused by ZnO NPs were suppressed or mitigated to some extent. When combined with 20 min of EMF, 0.5 mM ZnO NPs significantly improved longevity at 14 to 39 days. The 0.5 mM ZnO NPs and EMF treatment for 10 min resulted in a steep decline in survival, which was recorded around day 53. These results imply that the harmful effects of 0.5 mM ZnO NPs might be countered by the protective effects of EMF at 20 min. In conclusion, EMF exposure, particularly at a duration of 20 min, positively influences the longevity of flies, both independently and in combination with ZnO NPs (Figure 2A).

In the F1 progeny, 30 min of EMF exposure caused a slight decline in crawling behavior (Figure 3A), suggesting a potential inhibitory effect on motor activity or neuronal function. However, in the F2 progeny, EMF exposure for 10 min enhanced the crawling ability of the larvae (Figure 3B). When the flies were exposed to the combination of EMF and ZnO NPs, no significant alterations in crawling behavior were detected, indicating that the interaction between EMF and the NPs does not substantially affect motor function.

In the parent flies (F0), no significant changes in oxidative stress levels were observed, neither when exposed to ZnO NPs and EMF individually nor when exposed to their combination. Only a significant induction of oxidative stress was noted when the flies were exposed to a combination of 0.1 mM ZnO NPs and EMF for 20 and 30 min (Figure 4A). Additionally, exposure to 0.5 mM ZnO NPs alone resulted in a significant increase in reactive oxygen species (ROS). Although there were no notable alterations in the parent generation, the flies in the F1 generation showed increased levels of oxidative stress, suggesting a multigenerational effect from parental exposure (Figure 4B). Furthermore, ROS significantly increased with exposure to 0.5 mM ZnO NPs alone.

The two-choice tasting assay was used to assess the effect on sensory abilities caused by co-exposure to ZnO NPs and EMF. The preference index (PI) of parent flies (F0) subjected to EMF for 10, 20, and 30 min showed a gradual deterioration, with the most notable damage occurring at 30 min (Figure 5A). However, in F1 progeny, a significant decline in the PI was observed at 30 min of exposure, with no statistically significant effects at 10 and 20 min, indicating a reduced but still noticeable transmission of the deficit (Figure 5B). In F2 progeny flies, a significant decline was observed at both 20 and 30 min of EMF exposure (Figure 5C), demonstrating a multigenerational effect.

Compared with the individual effects of EMF, the co-exposure of parent flies (F0) to 0.1 mM ZnO NPs and EMF led to a more pronounced decline in PI; however, a marked decline was observed at 10 and 20 min, followed by partial recovery at 30 min, suggesting fluctuating sensitivity across generations (Figure 5B). The negative impact on F1 generation flies was less pronounced than that on F0 generation flies, while F2 progeny flies regained the same results as parent flies (Figure 5C).

In the F0 generation, a significant decline in the PI was observed with 0.5 mM ZnO NP exposure alone, which was further exacerbated when ZnO NPs was combined with EMF, particularly at 20 and 30 min (Figure 5A). In the F1 generation, the combined EMF and ZnO NP treatment also led to significant decline across all exposure durations in comparison with that of the control. However, relative to ZnO NP exposure alone, there was a slight improvement in sensory function under the combined treatment, indicating that F1 flies may exhibit a minor adaptive response that either suppresses or mitigates the effects of ZnO NPs under EMF stress. In the F2 generation, the combine treatment caused a significant decrease in taste perception at 10 and 20 min, with some improvement observed at 30 min compared with that observed after ZnO NP exposure (Figure 5B).

To study the effects of EMF, ZnO NPs, and their co-exposure, flies were carefully examined under a stereomicroscope for visible morphological changes in Drosophila. Flies were screened for subsequent generations: F0 (parent flies), F1, and F2. Phenotypic analysis revealed that exposure to EMF at a frequency of 2.4 GHz led to visible morphological changes in the flies, specifically the crisscross pattern of tergites (Figure 6) and the lack of pigmentation at tergites. These aberrant phenotypes were heritable and passed on to the next generation, indicating that EMF exposure alone induced heritable phenotypic alterations in tergite patterning. When exposed to a combination of 30 min of EMF exposure and 0.1 mM ZnO NPs, F2 progeny flies presented pigmentation deformities in tergites (Supplementary Figure S3).