Hypomagnetic Fields Influence the Developmental Duration, Fecundity and Temperature Stress Resistance of Drosophila melanogaster via Frataxin-Associated Traits

Simple Summary

As a highly conserved and important mitochondrial protein, frataxin plays a key role in iron homeostasis and metabolism, and its deficiency is associated with the Friedreich’s ataxia phenotype, a cardio- and neurodegenerative disease in humans. A hypomagnetic field can lead to various biological effects including increased oxidative stress, abnormal neurological functions, and developmental disorders; yet, its effects as an environmental stressor that exacerbates the inherent metabolic vulnerabilities in organisms, e.g., frataxin-deficient fruit flies, is still unknown. Thus, we used a hypomagnetic field to investigate its biological effects on frataxin-deficient fruit flies, and found that hypomagnetic field exposure could extend developmental duration, increase the fecundity of fruit flies, and accelerate recovery from heat shock in frataxin-deficient flies. Our findings indicate that the hypomagnetic field is adverse for growth and development but in favor of reproduction and temperature stress resistance to some extent in frataxin-deficient fruit flies. This study provides the first evidence of the multidimensional effects of hypomagnetic fields on frataxin-deficient fruit flies and offers inspiration and reference for space hypomagnetic effect exploration and Friedreich’s ataxia therapy.

Abstract

Frataxin is a highly conserved mitochondrial protein that plays a key role in iron homeostasis and metabolism, and its deficiency leads to oxidative stress, mitochondrial dysfunction, and neurodegeneration. Hypomagnetic fields (HMF) can lead to various biological effects including increased oxidative stress, neurological and developmental disorders; yet, their effects acting as environmental stressors that exacerbate the inherent metabolic vulnerabilities in frataxin-deficient Drosophila melanogaster flies are still unknown. In this study, the bio-effects of HMF on growth, development, reproduction, and temperature stress resistance of frataxin-silenced flies were investigated. The results showed that HMF extended egg-to-adult and pupa developmental durations of both the control line of repo-GAL4; tub-GAL80^ts>GFP-RNAi (GFP-RNAi) and frataxin-deficient line of repo-GAL4; tub-GAL80^ts>fh RNAi (fh-RNAi) compared to those reared under a geomagnetic field (GMF). Compared with GMF, HMF significantly increased offspring fecundity in fh-RNAi flies, whereas the change in GFP-RNAi controls was not significant, while showing no significant effects on the adult weight of fh-RNAi flies. The impact of HMF on temperature stress resistance was particularly specific: it enhanced recovery from chill coma in control (GFP-RNAi) flies, while it accelerated recovery from heat shock in frataxin-silenced (fh-RNAi) flies. The mechanisms through which HMF modulate frataxin-associated phenotypes at a fundamental physical level warrant further investigation.

1. Introduction

The geomagnetic field (GMF, with intensity value 25–65 μT at present) can protect organisms from solar wind and other cosmic radiations to keep the Earth hospitable for living on, and is used for orientation and long-distance navigation in many species of animals [1,2,3]. The GMF is closely linked to the growth and development of organisms [4,5,6,7]. Hypomagnetic fields (HMF) can affect plant germination and growth, animal metabolism, development, cognitive behavior, and certain biological effects [8,9,10,11,12]. It was found that exposure to transient hypomagnetic fields resulted in increased sensitivity of first optic ganglion cells in Drosophila retina, delayed sperm aging, and stimulated sperm swimming [13,14]. Drosophila flies cultured in a hypomagnetic environment for more than 10 generations exhibited a loss of learning memory and only recovered it after several generations of exposure in a geomagnetic environment [15]. Furthermore, hypomagnetic fields significantly enhanced the phototaxis of the migratory insect, Sogatella furcifera, and affected the flight ability of S. furcifera in a sex-differentiated manner [16]. The oriental armyworm Mythimna separata was easily disoriented in hypomagnetic fields, while its flight orientation in a deflected geomagnetic field varied regularly with the direction of the magnetic field [17]. So far, HMF can lead to various biological effects including increased oxidative stress, abnormal neurological functions, and developmental disorders [18,19,20,21,22].

Previously, we investigated the effects of magnetic fields on the phototaxis of the migratory insect, rice brown planthopper Nilaparvata lugens, and found that the expression level of frataxin in N. lugens was downregulated by changed magnetic fields. As a nuclear-encoded mitochondrial protein, frataxin is widespread in prokaryotes and eukaryotes with highly conserved structures. In eukaryotes, it primarily provides biological iron to mitochondria and is involved in the biosynthesis of intracellular iron–sulfur cluster proteins [23,24,25,26]. In yeast, frataxin interacts directly with the co-protein Isu in an iron-dependent manner and facilitates the transport of iron to Isu during the assembly of Fe–S clusters and is therefore considered as a molecular chaperone of iron in mitochondria [27]. In Drosophila, the central role of frataxin is to maintain mitochondrial Fe-S cluster synthesis and iron metabolism homeostasis, and its absence leads to impaired energy metabolism, oxidative stress, and neurodegenerative phenotypes [28,29,30,31,32,33,34]. The knockdown or mutation of dFh (Drosophila frataxin homolog) leads to elevated ROS and triggers apoptosis, especially in neural and muscular tissues [35]. The dFh-deficient Drosophila flies exhibit dyskinesia, shortened lifespans, and neurodegeneration [36,37,38]. Complete deletion of dFh is lethal, suggesting the critical role of frataxin in the early development of Drosophila flies.

Based on the established roles of the above-mentioned factors, a compelling rationale for coupling HMF with frataxin deficiency emerges. Frataxin is crucial for mitochondrial iron homeostasis and Fe–S cluster synthesis, and its deficiency leads to oxidative stress, mitochondrial dysfunction, and neurodegeneration. Separately, HMF exposure is known to induce oxidative stress and cause neurological and developmental abnormalities. We therefore hypothesize that HMF acts as an environmental stressor that exacerbates the inherent metabolic vulnerabilities in frataxin-deficient flies. The mitochondrial dysfunction and heightened oxidative stress from dFh knockdown likely creates a sensitized background, making the flies more susceptible to the additional metabolic perturbation imposed by HMF. In this study, the bio-effects of HMF on growth, development, reproduction and temperature stress resistance of frataxin-silenced Drosophila melanogaster were investigated. The GAL4/UAS system was employed to induce post-transcriptional silencing of Drosophila frataxin gene (fh) through transgenic double-stranded RNA interference (RNAi). It is supposed that exposure to HMF would affect multiple traits of Drosophila melanogaster via its effects on dFh knockdown.

In this study, we chose the pan-glial driver repo-GAL4 instead of pan-neuronal driver elav-GAL4 because glial cells are increasingly recognized as key contributors to Friedreich’s ataxia (FRDA)-related pathology, including mitochondrial dysfunction, iron/ROS dysregulation, and inflammatory/stress signaling [38]. Importantly, repo-GAL4-driven frataxin (fh) RNAi is also a well-established Drosophila FRDA-relevant strategy that produces robust, quantifiable phenotypes (e.g., reduced lifespan, locomotor/brain phenotypes, stress sensitivity), supporting its suitability for mechanism and environmental-modulation studies [34]. Furthermore, pan-neuronal fh knockdown may introduce stronger neurodevelopmental disabilities or viability defects depending on RNAi strength and timing (even with temporal control as GAL80^ts), and in some contexts elav-GAL4-driven neuronal knockdown yielded weaker biological fitness phenotypes than glial knockdown, making it less sensitive for detecting environmental modulation [39]. Therefore, for our aim of sensitively detecting effects of HMF on frataxin-associated phenotypes, we focused on the glia-targeted adult-onset knockdown of fruit flies.

2. Materials and Methods

2.1. Drosophila Lines, Cultivation and Diets

The tub-GAL80^ts/CyO;repo-GAL4/TM6B lines with the Gal4-UAS system for driving RNAi expression were obtained from China Agricultural University. The RNAi lines used in this work are as follows: UAS-fh RNAi (Bloomington Line Center, BDSC 24620, Bloomington, IN, USA), and UAS-GFP-RNAi (Tsing Hua Fly Center, TH00871.S, Beijing, China). Drosophila stocks were standardly maintained for routine feeding at 25 ± 1 °C, with relative humidity 60 ± 5% and 12: 12 h light: dark cycle, whereas all genotypes including parents and progeny carrying tub-GAL80^ts were reared at 18 °C to suppress GAL4 activity during development.

To induce adult-onset RNAi, F1 adults of flies were kept at 18 °C until post-eclosion and then shifted to 29 °C to inactivate GAL80^ts and permit GAL4-driven RNAi expression. Standard regular diet (RD): agar 20 g/L sucrose 80 g/L, active dry yeast 5 g/L, calcium chloride dihydrate 1.6 g/L, ferrous sulfate heptahydrate 1.6 g/L, sodium potassium tartrate tetrahydrate 8 g/L, sodium chloride 0.5 g/L, manganese chloride tetrahydrate 0.5 g/L, and nipagin 5.3 mL/L [40].

2.2. Construction of Frataxin-Silenced Drosophila Mutants Using RNAi

To achieve time-controlled silencing of frataxin (fh) (repo-GAL4; tub-GAL80^ts>fh-RNAi) in glial cells, we employed the GAL4/UAS system combined with tub-GAL80^ts. The genetic control was a strain silencing GFP in glial cells (repo-GAL4; tub-GAL80^ts>GFP-RNAi), treated under identical protocols [41,42]. First, hybridization experiments were conducted to generate F1 generations with silenced frataxin (repo-GAL4; tub-GAL80^ts>fh-RNAi, abbreviated as fh-RNAi) (Figure 1A) and silenced GFP (repo-GAL4; tub-GAL80^ts>GFP-RNAi, abbreviated as GFP-RNAi) (Figure 1B) in glial cells. For all crosses involving GAL80^ts, both parents and their developmental-stage offspring were reared at 18 °C to suppress GAL4 activity. To induce target gene silencing, F1 offspring were transferred to 29 °C post-eclosion. Upon reaching adulthood, F1 individuals were sorted for specific phenotypes to establish the silencing model. The target gene was suppressed at the adult stage and papal stage when the magnetic field exposure was applied.

Figure 1.

Drosophila mutants with GFP (A) and (B) frataxin silencing in glial cells.

2.3. Determination of Frataxin Silencing Efficiency and Quantitative PCR of Genes Cat and Hsp26

Total RNA was extracted from the heads of 3- to 8-day-old male Drosophila melanogaster according to Invitrogen’s Trizol reagent manual, and RNA quality was assessed by electrophoresis. RNA samples were treated with an RNase-free DNase set and purified using the NucleoSpin RNA Clean-up XS kit (Macherey-Nagel, Düren, Germany). Premix Ex Taq (TaKaRa Bio Inc., Otsu, Japan) was used with 100 ng cDNA on an ABI 7500 PCR system (Applied Biosystems, Carlsbad, CA, USA). The extracted RNA was reverse-transcribed into cDNA according to the manufacturer (TaKaRa Bio Inc., Otsu, Japan)’s instructions. Based on the concentration measured by UV spectrophotometry, the volume corresponding to 1 µg RNA was calculated, and the appropriate volume of RNA solution was used for reverse transcription.

Based on the CDS region of the obtained full-length gene cDNA sequence, qPCR primers were designed using NCBI online software (Primer-BLAST), with ribosomal protein 49 (Rp49) serving as the internal control gene. Real-time quantitative PCR was performed using an ABI kit to detect target gene expression in the head of flies. Gene expression levels were normalized against the internal control using the quantitative Ct method for relative quantification, with results plotted as relative mRNA expression. Each experiment comprised three independent biological replicates.

In addition, for a better understanding of the mechanism involved in the effects of HMF, we chose two genes of Cat and Hsp26 for quantitative PCR to investigate the ROS and thermal stress associated with HMF exposure. As a key antioxidant enzyme indicative of ROS/oxidative stress status, the Cat is involved in hydrogen peroxide catabolic process, reactive oxygen species metabolic process, and response to oxidative stress in Drosophila [43]. Meanwhile, as a representative gene of the heat shock response/proteostasis pathway that contributes to lifespan determination and the response to cold and heat, the Hsp26 encodes a small heat shock protein that is robustly induced upon heat stress in Drosophila, and its promoter contains heat shock regulatory elements required for heat-inducible transcription [44,45]. Given this in this study, it is speculated that these two genes may be associated with oxidative and thermal stress pathways induced by hypomagnetic exposure and frataxin deficiency. The genes and primer pairs analyzed are as follows (Table 1):

Table 1.

The genes and primer pairs analyzed in this study.

Gene	Forward Primer	Reverse Primer
Frataxin	5′-GTCACAGTCCGTGGACTTCC-3′	5′-CAAAATCGAACGTTTCAACCG-3′
GFP	5′-ACGTAAACGGCCACAAGTTC-3′	5′-TGCTCAGGTAGTGGTTGTCG-3′
CAT	5′-GACCTGCAAGTTCCCCAGTT-3′	5′-GGTGACATCGAAGGGGTTGT-3′
HSP26	5′-CGACTCCATCTTGGTCGAGG-3′	5′-GTAGCCATCGGGAACCTTGT-3′
RP49 #	5′-CCAAGCACTTCATCCGCCACC-3′	5′-GCGGGTGCGCTTGTTCGATCC-3′

^# Internal reference.

2.4. Magnetic Field Generation System and Insect Exposures

The experiment was conducted in a laboratory in Beijing (39°59′14″ N, 116°19′21″ E), where the local geomagnetic field (GMF) has an intensity of 52,487 ± 841 nT, a declination of 5.30 ± 0.59°, and an inclination of 56°29′ ± 1.02°. To create a hypomagnetic field (HMF), we employed three sets of Helmholtz coils, each powered independently to generate an artificial field that precisely opposed and offset each vector component of the local GMF. This setup produced a spherical hypomagnetic environment of 300 × 300 × 300 mm³ with an average residual intensity of <500 nT (Figure 2). The external diameter of the HMF system is 1200 mm, and insects are placed on the wooden table in middle of Helmholtz coils during experiments. A fluxgate magnetometer (Honor Top model 191A, Qingdao Zhongyu Huantai Magnetoelectric Technology Co., Ltd., Qingdao, China. sensitivity ±1 nT) was used to calibrate and verify the HMF intensity twice daily, both before and after experimental sessions.

Figure 2.

The Helmholtz coil system used to generate hypomagnetic fields. The system consists of three independent coil pairs and each pair of coils are individually powered. The external diameter of apparatus is 1200 mm. The effective area of HMF is generated in the center space with 300 × 300 × 300 mm³. Insects are placed on the wooden table in middle of Helmholtz coil.

In preliminary experiments, we used female flies for qPCR and other assays, and the results showed similar trends to those of male flies. Therefore, to minimize variability introduced by sex-specific physiology and to keep the assay design consistent across molecular and behavioral endpoints, we only used male flies for the related experiments. In this study, 3- to 8-day-old male Drosophila flies from two silenced strains: repo-GAL4; tub-GAL80^ts>GFP-RNAi and repo-GAL4; tub-GAL80^ts>fh-RNAi were used. The flies were housed in groups of 20 per tube, with three replicate groups per condition. Each experimental cycle consisted of a 72 h exposure to either the HMF or the normal GMF, under a light-dark regimen of 12 h of light followed by 12 h of darkness.

2.5. Measurements of Developmental Duration, Adult Weight and Fecundity

Four experimental groups were established: GMF and HMF controls (repo-GAL4; tub-GAL80^ts>GFP-RNAi), and GMF and HMF experimental groups (repo-GAL4; tub-GAL80^ts>fh-RNAi). The Helmholtz coil system was used to generate HMF for the exposure groups, while the controls were maintained in a sham coil of GMF with the same environmental conditions. After three days of consecutive exposure, the parental flies were removed, and the eggs developed under their respective conditions until eclosion. The developmental duration of egg-to-adult and pupae were recorded every 8 h, and the number of pupae and offspring flies per life cycle were counted for each vial. Furthermore, the body weight of six 3- to 8-day-old virgin flies from each group was measured and segregated by sex and line. All experiments were performed in triplicate.

2.6. Chill Coma and Heat Shock Recovery Assays

For the chill coma recovery assay, twenty 3- to 7-day-old male flies from each line were transferred without anesthesia to empty vials and maintained at 4 °C for one hour. Following this, flies were returned to room temperature, and the recovery time—defined as the duration from coma to the ability to stand upright for more than two seconds—was recorded. For heat shock recovery assay, twenty 3- to 8-day-old male flies of each genotype were placed in individual glass tubes (24 × 15 mm). The tubes were submerged in a 39 °C water bath until all flies entered a heat-induced coma. They were then transferred to room temperature, and the recovery time was recorded using the same criterion.

2.7. Data Analysis

All data were analyzed using SPSS 20.0 (IBM Inc., Armonk, NY, USA). One-way ANOVA with Welch’s t-test for post hoc comparisons was used to analyze the relative transcript levels of Drosophila frataxin in repo-GAL4; tub-GAL80^ts>GFP-RNAi and repo-GAL4; tub-GAL80^ts>fh-RNAi lines. A two-way ANOVA was used to assess the effect of magnetic fields (HMF vs. GMF) on the following: egg-to-adult developmental duration, pupa developmental duration, and fecundity. Sample sizes were as follows: for egg-to-adult developmental duration, 89 (HMF) and 32 (GMF) eggs for the GFP-RNAi control, and 101 (HMF) and 31 (GMF) for the frataxin-knockdown; for pupal developmental duration, 58 (HMF) and 32 (GMF) larvae for the control, and 92 (HMF) and 30 (GMF) for the frataxin-knockdown; for fecundity, 15 for the frataxin-knockdown and 15 for the control (a pair of female and male adults/vial) with magnetic field condition. Adult body weight was analyzed using a two-way ANOVA, with magnetic field and sex as factors (n = 6 per sex per group). Recovery time from chill coma or heat shock was jointly analyzed using a three-way ANOVA with magnetic field (GMF vs. HMF), genotype (GFP-RNAi vs. frataxin RNAi), and stress (heat vs. chill) as factors. For all ANOVAs, when significant main effects or interactions were found (p < 0.05), Tukey’s post hoc test was used for further pairwise comparisons (p < 0.05). Time to recovery was analyzed as a survival outcome (not yet recovered) using the Kaplan–Meier method that compared the four MF × genotype groups with a k-sample log-rank test; pairwise contrasts used log-rank with Holm adjustment. One-way ANOVA with Tukey’s post hoc test was used to analyze the relative transcript levels of Cat and Hsp26 in repo-GAL4; tub-GAL80^ts>fh-RNAi lines between HMF and GMF.

3. Results

3.1. Frataxin Silencing Efficiency Under GMF

One-way ANOVA indicated that fh gene expression was significantly decreased in fh-RNAi flies (F = 54.12, p = 0.0018) (Figure 3). The silencing efficiency of the UAS-fh-RNAi line is approximately 75% (Figure 3).

Figure 3.

Quantification of frataxin transcripts under GMF. QRT-PCRs were performed on whole RNA extracts of 3- to 8-day-old adult heads (3 samples of 30 heads per genotype). Significant differences are shown at * p < 0.05 (one-way ANOVA with Welch’s t-test performed for each group). The columns represent averages with vertical bars indicating SE.

3.2. Hypomagnetic Field Exposure Extended Egg-to-Adult Developmental Duration in GFP-RNAi and fh-RNAi Flies

HMF significantly increased the egg-to-adult developmental duration of GFP-RNAi (p < 0.001) and fh-RNAi flies (p < 0.001) (Figure 4). Compared with GMF, HMF significantly extended the egg-to-adult developmental duration of GFP-RNAi and fh-RNAi by a mean value of 22.69% and 22.13%, respectively (p < 0.05). A two-way ANOVA with genotype (repo-GAL4;tub-GAL80^ts>GFP-RNAi vs. repo-GAL4;tub-GAL80^ts>fh-RNAi) and field (GMF vs. HMF) showed a significant main effect of field (F = 115.51, p < 0.001), but no significant main effect of genotype (F = 1.41, p = 0.235 > 0.05) and no genotype × field interaction (F = 0.045, p = 0.832 > 0.05).

Figure 4.

Egg-to-adult duration of repo-GAL4; tub-GAL80^ts>GFP-RNAi and repo-GAL4; tub-GAL80^ts>fh-RNAi lines under GMF and HMF. The columns represent averages with vertical bars indicating SE. Significant differences are set up at *** p < 0.001.

3.3. Hypomagnetic Field Exposure Enhanced Pupal Developmental Duration in GFP-RNAi and fh-RNAi Flies

Exposure to HMF significantly extended the pupal developmental duration in both GFP-RNAi (p < 0.001) and fh-RNAi flies (p < 0.01) compared to GMF. The mean prolongation was 23.15% for GFP-RNAi and 14.70% for fh-RNAi flies (p < 0.05) (Figure 5). A two-way ANOVA for genotype (repo-GAL4;tub-GAL80^ts>GFP-RNAi vs. repo-GAL4;tub-GAL80^ts>fh-RNAi) × magnetic field (GMF vs. HMF) revealed a significant main effect of magnetic field (F = 36.51, p < 0.001), while the main effect of genotype (F = 0.627, p = 0.429) and the genotype × magnetic field interaction (F = 1.32, p = 0.252) were not significant.

Figure 5.

Pupal developmental duration of repo-GAL4; tub-GAL80^ts>GFP-RNAi and repo-GAL4; tub-GAL80^ts>fh-RNAi under GMF and HMF. The columns represent averages with vertical bars indicating SE. Significant differences are set up at ** p < 0.01, *** p < 0.001.

3.4. Hypomagnetic Field Exposure Shows No Significant Effect on Adult Weight of fh-RNAi Flies

For GFP-RNAi flies, we found a significant effect of magnetic fields (F = 4.98, p < 0.05) and a strong effect of sex (F = 201.51, p < 0.001) on adult weight, but no significant interaction between these factors (F = 0.23, p > 0.05) (Figure 6). In contrast, magnetic fields had no significant effect on the weight of fh-RNAi flies (F = 0.13, p > 0.05), although a strong sexual dimorphism was still present (F = 245.45, p < 0.001). Similarly, no significant interaction between magnetic fields and sex was observed in the fh-RNAi group (F = 0.21, p > 0.05).

Figure 6.

Adult weight of repo-GAL4; tub-GAL80^ts>GFP-RNAi (A) and repo-GAL4; tub-GAL80^ts>fh-RNAi flies (B) under GMF and HMF. The columns represent averages with vertical bars indicating SE. Significant differences are set up at * p < 0.05, *** p < 0.001.

3.5. Hypomagnetic Field Exposure Modulated Fecundity in fh-RNAi Flies

Magnetic fields significantly affected the fecundity of fh-RNAi flies. Under HMF, the number of offspring increased by a mean of 10.60% (p = 0.9325, not significant) in control flies (GFP-RNAi) and by 114.52% in fh-RNAi (p < 0.05) flies compared to GMF (Figure 7). A two-way ANOVA revealed a significant main effect of magnetic field (F = 8.31, p < 0.05) on fecundity, but no significant main effect of genotype (F = 2.23, p = 0.17). The genotype × magnetic field interaction showed a non-significant trend (F = 4.20, p = 0.07).

Figure 7.

The fecundities of repo-GAL4; tub-GAL80^ts>GFP-RNAi and repo-GAL4; tub-GAL80^ts>fh-RNAi flies under HMF in contrast to those with GMF. The columns represent averages with vertical bars indicating SE. Significant differences are set up at * p < 0.05. ns = not significant at the 5% level.

3.6. Hypomagnetic Field Modulated the Response of Drosophila Flies to Temperature Stress

During recovery from chill coma stress, flies generally recovered faster in the HMF than in the GMF, and those with frataxin silencing (fh-RNAi) recovered faster than the GFP-RNAi controls. The effect of the genotype was more pronounced under GMF conditions, where the difference was significant, than under HMF, where it was not (Figure 8A). A similar pattern was observed during heat shock recovery, with HMF and fh-RNAi each conferring a faster recovery, and a significant interaction between magnetic field and genotype (Figure 8B).

Figure 8.

Kaplan–Meier curves of chill coma recovery (min) (A) and heat shock recovery (min) (B) across four MF × genotype groups. Groups are distinguished by black/gray line styles (black solid, black long-dash, gray solid, gray short-dash).

Statistical analysis confirmed significant main effects of MF (F = 55.74, p < 0.001), genotype (F = 72.43, p < 0.001), and stress (F = 324.41, p < 0.001). Significant MF × genotype (F = 7.23, p = 0.0086) and genotype × stress (F = 33.63, p < 0.001) interactions, as well as a significant three-way interaction (MF × genotype × stress) (F = 7.53, p = 0.0073) were observed, indicating that the magnitude of the MF effect depends on both genotype and the type of thermal stress.

3.7. The Relative Cat and Hsp26 Transcript Levels of fh-RNAi Flies Under HMF or GMF Exposure

As mentioned above in Section 2, we chose two genes of Cat and Hsp26 for quantitative PCR to investigate the ROS and thermal stress associated with HMF exposure, as the two genes are supposed to participate in response to oxidative stress and cold and heat shock pathways in Drosophila, respectively [43,44,45]. One-way ANOVA indicated that magnetic fields significantly affected Cat (F = 12.43297, p < 0.01) and Hsp26 (F = 58.7924, p < 0.001) genes expression levels in fh-RNAi flies. Compared with GMF, HMF increased the relative Cat transcript level by 66% in fh-RNAi flies, while it significantly decreased the relative Hsp26 transcript level by 96.8% in fh-RNAi flies (Figure 9).

Figure 9.

Quantification of Cat and Hsp26 transcripts under GMF and HMF. QRT-PCRs were performed on whole RNA extracts of 3- to 8-day-old adult heads (3 samples of 30 heads per genotype), with ribosomal protein 49 (Rp49) serving as the internal control gene. Significant differences are shown at ** p < 0.01, *** p < 0.001 (one-way ANOVA with Welch’s t-test performed for each group). The columns represent averages with vertical bars indicating SE.

4. Discussion

This study provides the first evidence of the multidimensional effects of hypomagnetic fields on glia-specific frataxin-deficient Drosophila melanogaster, offering a framework for exploring HMF interactions with frataxin-associated vulnerabilities. Notably, compared with GMF, while HMF exposure extended the developmental duration of Drosophila flies, which is quite similar with the effects observed on brown planthopper Nilaparvata lugens [7], it increased their recovery after temperature stress, implying that temperature stress resistance depends on frataxin-related pathways, which in turn are modulated by local changes in the magnetic field. Moreover, in contrast with the decreased fertility observed in small planthopper Laodelphax striatellus and brown planthopper N. lugens [19], the present study found increased fecundity in frataxin-deficient flies under HMF exposure, suggesting the role of frataxin in the reproduction of Drosophila flies with changed magnetic fields. It is reported that strong magnetic fields can also prolong the duration of egg-to-adult development [46,47], implying a common primary physical mechanism of magnetic effects on embryonic development.

From above-mentioned effects, it is noteworthy that HMF appears to function through a paradoxical mechanism. Chronic frataxin deficiency causes oxidative stress, yet HMF can induce a state of reductive stress due to an excess of reducing agents like NADPH. In cases of mild frataxin dysfunction, this shift toward a more reduced cellular environment may temporarily disrupt the Fenton reaction cycle, reducing harmful ROS production and offering interim relief. Furthermore, HMF may act as a mild hormetic stressor. Its exposure can trigger an adaptive global response, upregulating protective pathways such as the mitochondrial unfolded protein response and antioxidant defenses. This heightened overall cellular robustness may then partially compensate for the specific vulnerabilities created by low frataxin levels.

As chill coma recovery and heat shock recovery depend on the rapid re-establishment of ionic homeostasis, a process that requires adequate mitochondrial functionality [48,49,50], it has been proposed that loss of mitochondrial frataxin triggers a hypoxia-like, iron starvation process and causally involved ROS production that directly contributes to cellular toxicity [51,52,53,54]. In our study, the Cat transcript was increased and Hsp26 transcript was decreased under HMF exposure, indicating the close associations between ROS and thermal stress and HMF. However, at the lowest level of HMF exposure, frataxin deficiency did not render more toxicity to adult flies as the temperature stress resistance was enhanced rather than decreased. In addition, differences in response to HMF between GFP-RNAi and fh-RNAi flies support the role of frataxin in magnetic field sensitivity [30,55,56,57].

On the other hand, the effects of HMF are supposed to be jointly modulated by genotype and temperature paradigm. During chill coma recovery, a significant MF × genotype interaction was observed: HMF significantly shortened recovery in the GFP background, whereas this contrast was non-significant in the frataxin gene-silenced background. Conversely, during heat recovery, MF and genotype both significantly shortened recovery time independently—HMF and frataxin gene silencing each exerted this effect. While HMF and frataxin silencing both shorten recovery time, the magnitude of the HMF effect is context-dependent: it is large in GFP-RNAi controls during chill coma recovery, more modest in fh-RNAi flies during chill coma, and consistently beneficial during heat shock. Collectively, whether and to what extent HMF accelerates recovery depends on genotype and stress paradigm, reflecting the context-dependent nature of magnetic field effects. Furthermore, it is known that the most widely discussed conceptual model of magnetic biological effects—the radical pair mechanism—predicts only minor relative effects, on the order of fractions of a percent. Therefore, the observed bigger effects in this study, where the measured parameter changes several-fold upon transition from GMF to HMF, are quite rare and worthy of attention. This highlights the absence of GMF, signifying a reliable theoretical explanation for the magnetic effects observed in biology so far.

Finally, we used the pan-glial driver repo-GAL4 rather than the pan-neuronal driver elav-GAL4 because the elav-GAL4 could drive broad expression early in nervous system development, and, due to the fact that fh is a core mitochondrial gene, widespread neuronal impairment during early development may alter circuit formation, baseline activity, feeding, and developmental rate. Therefore, for our aim of sensitively detecting effects of HMF on frataxin-associated phenotypes, we focused on glia-targeted adult-onset knockdown of fruit flies. Nevertheless, pan-neuronal knockdown could be informative and will be addressed in future work to dissect cell-type contributions.

5. Conclusions

In this study, hypomagnetic fields extended egg-to-adult and pupal developmental durations and increased the fecundities of fh-RNAi flies, while showing no significant effects on the adult weight of fh-RNAi flies compared to those reared under GMF. The impact of HMF on temperature stress resistance was particularly specific: it enhanced recovery from chill coma in control (GFP-RNAi) flies, while it accelerated recovery from heat shock in frataxin-silenced (fh-RNAi) flies. Our findings indicated substantial (several-fold) changes observed in key parameters when comparing GMF to HMF conditions, suggesting the necessity of a comprehensive theoretical framework to explain such large-scale biological effects of weak magnetic fields. This observation elevates our work from a purely phenotypic description to a finding with broader implications for the field of biomagnetics, which would eventually frame our findings as a valuable contribution that may stimulate the development of new or refined theoretical models. While our findings establish a clear association between HMF and thermal stress responses, they also generate specific, testable hypotheses regarding the underlying molecular mechanisms. For instance, the observed downregulation of Hsp26 under HMF exposure suggests a potential role for the heat shock pathway in mediating this effect. Future research should employ loss- and gain-of-function models, such as CRISPR-Cas9 gene editing in relevant cell lines, to functionally validate the role of Hsp26 in the frataxin-deficient flies with HMF exposure. Furthermore, subsequent proteomic or epigenomic analyses could elucidate the direct molecular interactions and regulatory changes triggered by HMF, thereby moving beyond correlation toward a mechanistic understanding of the process.

Abbreviations

The following abbreviations are used in this manuscript:

fh	Frataxin
dFh	Drosophila frataxin homolog
GFP	Green fluorescent protein
CAT	Catalase
HSP26	Heat shock protein 26

Author Contributions

Conceptualization, W.P. and H.K.; methodology, H.K. and J.Z.; formal analysis, H.K.; investigation, H.K.; resources, J.Z.; data curation, H.K.; writing—original draft preparation, H.K.; writing—review and editing, W.P. and G.W.; supervision, W.P.; project administration, W.P.; funding acquisition, W.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Funding Statement

This research was funded by the National Natural Science Foundation of China (32271286, 32172414), and the Natural Science Foundation of Jiangsu Province (BK20221510).