Metabolic and Developmental Changes in Insects as Stress-Related Response to Electromagnetic Field Exposure

: (1) Background: The growing ubiquity of electromagnetic ﬁelds (EMF) due to rapid technological progress raises concerns about potential health implications. While laboratory experiments have generated inconclusive ﬁndings about adverse effects, EMFs have demonstrated efﬁcacy in magnetotherapy. Earlier studies indicate that an EMF can trigger stress responses in organisms, the outcomes of which are dependent on the intensity of the EMF. (2) Methods: This study aims to explore the effects of extremely low-frequency EMF (50 Hz, 1 mT, or 7 mT) on metamorphosis and metabolism rates, which are indicators of stress, across different developmental stages of Tenebrio molitor , including adults, pupae, and larvae. (3) Results: Our ﬁndings reveal that exposure to EMF leads to accelerated weight loss, increased adult metabolism, and higher mortality; however, EMF exposure appears to have no impact on sugar levels or the rate and success of metamorphosis. Notably, signiﬁcant changes were only observed under the inﬂuence of a strong EMF (7 mT), while the weaker EMF (1 mT) did not yield statistically signiﬁcant outcomes. (4) Conclusion: The obtained results suggest that an extremely low-frequency EMF can be considered a stressor, with its effects contingent upon the speciﬁc parameters of exposure and the developmental stage of the experimental model.


Introduction
The presence of an electromagnetic field (EMF) has been intertwined with life on Earth since its inception, influencing and shaping alterations within living organisms.With the progress of human civilization and the profound interaction with the natural environment, the equilibrium between natural and artificial EMFs has been disturbed.Extensive research over the past five decades has been devoted to investigating the potential impacts of the expanding presence of artificial EMFs in the environment.The investigation of the EMF exposure influence on biological systems continues to generate significant interest among both scientific communities and the general public [1].The question that emerges is in regard to the potential consequences of increased exposure to EMFs on health.On the contrary, it is worth noting that EMFs have been effectively employed in therapeutic applications [2][3][4].A comprehensive understanding of the effects and consequences of EMF exposure has been essential for assessing and mitigating any potential risks to the environment and human health [5][6][7].Studies examining the effects of EMFs on animals and plants have been conducted using various models, levels of organism organization, as well as various sources of EMF (different levels or frequencies) and experimental protocols.Consequently, it may lead to discrepancies in the results and the inability to draw clear conclusions.Nevertheless, numerous studies indicate that an EMF is a well-known but poorly understood stressor that can lead to stress, anxiety, depression-like behavior, and memory deficits [8][9][10][11].Those stress responses elicited by EMF exposure primarily pertain to the nervous system and predominantly manifest deleterious consequences.However, certain studies have suggested that an EMF of a low magnitude might manifest a neuroprotective impact, as exemplified by the production of neuroprotective proteins like Hsp70 or BDNF, as well as augmentation in the activity of antioxidant enzymes [12][13][14][15][16]. Since the explanations given so far have not sufficiently addressed the potential underlying mechanisms that account for the conflicting outcomes observed, we propose that the nature of the changes induced by EMF is contingent upon the magnetic flux density of the applied EMF.In this paper, we decided to evaluate the impact of a 50 Hz EMF of two intensities, 1 mT and 7 mT.The selection of the magnetic flux density values was made in accordance with the prescribed occupational exposure limits outlined in the guidelines of the European Union Directive 2013/35/EU [17], as well as parameters commonly utilized in magnetotherapy [18].Furthermore, under natural conditions, birds and flying insects can be exposed to EMF of such intensity, e.g., in close proximity to high voltage power lines where exposure levels can be 0.6 mT at 1 m and almost 14 mT at 1 cm from the conductor of 400 kV transmission lines [19].
Given that an EMF elicits stress reactions within the nervous system, our research aims to investigate whether EMF exposure can also impact the rate of metabolism and metamorphosis serving as potential stress indicators.To evaluate these changes, we took advantage of a widely used and well-documented insect model.
Terrestrial invertebrates are often used as bioindicator organisms in ecological and pollution studies.Insect populations are sensitive to their habitat quality, and they can indicate the changing of their habitats with their quantitative and qualitative parameters.[20][21][22].For instance, the yellow mealworm (Tenebrio molitor) has served as a prominent bioindicator in numerous experiments owing to its distinct ability of prolonged survival compared to other insect species when exposed to various stress factors [23].Insect metamorphosis and metabolism represent particularly interesting processes to assess stress conditions due to their inherent sensitivity and responsiveness to environmental perturbations.The intricate physiological and biochemical changes that occur during these developmental stages make them reliable indicators of stress-induced alterations.Furthermore, the modulation of metamorphosis and metabolism in response to stressors provides valuable insights into the adaptive strategies employed by insects to cope with challenging conditions.Consequently, studying these processes contributes to a comprehensive understanding of the impact of stress on insect physiology, which can be extended to broader ecological and environmental contexts.
We already know that EMF exposure (50 Hz, 1-7 mT) has an influence on insects, e.g., inducing stress in cockroaches, increasing motor activity, and delaying response to noxious heat.Locusts exposed to EMF showed reduced kick force and movement, along with altered motor neuron activity.EMF-exposed crickets displayed changes in male calling song patterns and brain amine levels [16,[24][25][26].We assume that EMF exposure might have an impact on the metamorphosis and metabolic processes in insects and the potential effects of EMF exposure may vary depending on the insect's stage of development.
In view of these facts, here we focus on investigating the effects of EM (50 Hz, 1 mT, and 7 mT) on the metamorphosis and metabolism rate (through weight loss, mortality, CO 2 production, and sugar levels in the hemolymph) in adults, pupae, and larval stage of Tenebrio molitor.

Results
The weight of the adults decreased with time in all experimental groups (Figure 1).The group exposed to 7 mT showed a faster weight loss, whereas there were no differences between the control animals and those exposed to 1 mT, as shown by a significant treatment*time interaction in the general linear mixed models (GLMMs, Table S1).The weight of the control group decreased to 76.15% of the initial value on the last day of the experiment.The weight of the exposed group decreased to 76.8% for 1 mT and 63.51% for 7 mT on the last day of the experiment.The survival of imagines (Figure 2) expressed as a time to observe the first dead individual (mean ± SE: 10.3 ± 0.6 days) or 50% (15.7 ± 0.5 days) of dead individuals did not depend on the exposure to EMF (Table S2).However, the time to reach 100% mortality was shorter in the group exposed to 7 mT (19.9 ± 0.9 days) than in those exposed to 1 mT and in the control (23.5 ± 0.9 and 24 ± 0.9 days, respectively), as shown by the general linear models (GLMs, Table S2).Measurements of larval weight (Figure 3) were made for 7 days due to the decreasing number of individuals.Some of the larvae metamorphosed into the pupal stage or died.The weight of individuals in all groups decreased with time but at different rates, as indicated by a significant treatment*time interaction in the GLMM (Table S3).The weight decrease in the control group was the lowest (93% of the initial value measured on the last day of the experiment), whereas that of the larvae exposed to 7 mT was the fastest (88.86%).The weight loss of the group exposed to 1 mT was intermediate and reached 91.74% of the initial value.The weight loss of pupae with time was independent of the The survival of imagines (Figure 2) expressed as a time to observe the first dead individual (mean ± SE: 10.3 ± 0.6 days) or 50% (15.7 ± 0.5 days) of dead individuals did not depend on the exposure to EMF (Table S2).However, the time to reach 100% mortality was shorter in the group exposed to 7 mT (19.9 ± 0.9 days) than in those exposed to 1 mT and in the control (23.5 ± 0.9 and 24 ± 0.9 days, respectively), as shown by the general linear models (GLMs, Table S2).The survival of imagines (Figure 2) expressed as a time to ob dividual (mean ± SE: 10.3 ± 0.6 days) or 50% (15.7 ± 0.5 days) of d depend on the exposure to EMF (Table S2).However, the time t was shorter in the group exposed to 7 mT (19.9 ± 0.9 days) than in and in the control (23.5 ± 0.9 and 24 ± 0.9 days, respectively), as linear models (GLMs, Table S2).Measurements of larval weight (Figure 3) were made for 7 day number of individuals.Some of the larvae metamorphosed into t The weight of individuals in all groups decreased with time but a cated by a significant treatment*time interaction in the GLMM (Ta crease in the control group was the lowest (93% of the initial valu Measurements of larval weight (Figure 3) were made for 7 days due to the decreasing number of individuals.Some of the larvae metamorphosed into the pupal stage or died.The weight of individuals in all groups decreased with time but at different rates, as indicated by a significant treatment*time interaction in the GLMM (Table S3).The weight decrease in the control group was the lowest (93% of the initial value measured on the last day of the experiment), whereas that of the larvae exposed to 7 mT was the fastest (88.86%).The weight loss of the group exposed to 1 mT was intermediate and reached 91.74% of the initial value.The weight loss of pupae with time was independent of the exposure to EMF, as shown by the non-significant effect of treatment and its interaction with time in the GLMM (Table S4).

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exposure to EMF, as shown by the non-significant effect of treatment and its interaction with time in the GLMM (Table S4).
(a) (b) The EMF exposure did not significantly affect the time of appearance of the first pupa and imago (Table 1), nor the percentage of successfully metamorphosed pupae and adults (Table 2), as shown by respective GLMs and generalized linear models (Table S5).The EMF exposure did not significantly affect the time of appearance of the first pupa and imago (Table 1), nor the percentage of successfully metamorphosed pupae and adults (Table 2), as shown by respective GLMs and generalized linear models (Table S5).It is worth noting that during pupal metamorphosis into the imago under exposure to 7 mT (rather than 1 mT) began the emergence of developmental abnormalities (27%), which were manifested by the impairment of the first pair of wings-coverts, the lack of walking legs (complete or partial), and the dwarfism of the abdomen.
The effect of EMF exposure on the metabolic rate (measured as CO 2 production, Figure 4) was non-significant in larvae, but adults exposed to EMF exhibited a significantly higher CO 2 release than the control individuals, as indicated by the GLMM (Table S6).The sugar level in the insect haemolymph (Figure 5) did not depend on the EMF exposure (GLMM, Table S7).

Discussion
In this work, we assumed that electromagnetic field (EMF, 50 Hz, 1 mT, an

Discussion
In this work, we assumed that electromagnetic field (EMF, 50 Hz, 1 mT, and 7 mT) exposure functions as a stressor, exerting an influence on the metabolism and metamorphosis rate of mealworms in a manner contingent upon the dosage administered and insect's stage of development.
We found that exposure to the potent EMF (7 mT), as opposed to the weaker one (1 mT), resulted in accelerated weight loss for both adults and larvae, along with elevated mortality rates and increased metabolic activity in adults.Nevertheless, the EMF impact did not affect the rate or success of metamorphosis in pupae and adults, the CO 2 production of larvae, or the sugar content in the heamolymph.Notably, no discernible changes were induced in the pupal stage by the EMF exposure.
The results obtained in the present work indicate that biological systems may react and behave differently under the influence of different parameters of (electro)magnetic exposure.This is similar to what other researchers discovered in studies on both vertebrates and invertebrates.For example, when locusts were exposed to EMFs of 1 mT, 4 mT, and 7 mT, noticeable effects happened only at an EMF exceeding 4 mT.These effects included changes in behavior, physiology, and stress protein levels [16].Interesting outcomes also emerged from the studies on oxidative stress.In animals exposed to an EMF of 1 mT, the levels of markers for oxidative stress and antioxidants were quite similar to the control values.The changes were more pronounced at intensities exceeding 6 mT (e.g., the level of malondialdehyde (MDA) and reactive oxygen species (ROS) were significantly higher, while superoxide dismutase (SOD) activity was decreased) [14,24,27].Additionally, numerous studies suggest that values at or below 1 mT exhibit a stimulating impact of an EMF on brain plasticity processes by the production of protective proteins, e.g., Hsp70 or BDNF, or an increase in the activity of antioxidant enzymes, which could have potential applications in neuroprotection [13,[28][29][30][31].It appears that an EMF of 1 mT could mark the threshold for the appearance of (adverse) biological effects and/or might trigger cellular adaptation aimed at reducing adverse physiological outcomes.However, further, more detailed research is needed concerning a specific EMF-induced effect [14].
As anticipated, exposure to EMFs led to enhanced weight loss in both adults and larvae.We link this to the stress response, similar to the reaction of the Gryllus texensis cricket subjected to chronic stress from simulated predator situations [32].Studies suggest that exposure to both EMFs and stress reduces feeding behavior [33,34].In our experiments, we deliberately restricted the insects' access to food.As a result, any observed effects are likely attributable to alterations in their metabolic processes.Exposure to EMFs and stress have the potential to stimulate animals, leading to increased levels of aggression, anxiety, locomotor activity, and the tendency to produce flight behaviors [25,26,32,35,36].The metabolic state of an organism is influenced by its overall level of activity; for instance, a heightened activity corresponds to an increase in the sense of hunger and CO 2 production, thereby facilitating the opportunity to rectify energy deficits [37].Several studies indicate that metabolic changes induced by EMF and static magnetic field (SMF) exposure were evident through an increase in the O 2 uptake or CO 2 production in the exposed animals [38,39].Our findings also indicate that adults exposed to EMFs demonstrated noticeably elevated CO 2 levels; however, the impact of EMF exposure on CO 2 production in larvae was found to be statistically insignificant.The results suggest that adults exhibited the highest susceptibility to EMF exposure.Among the exposed groups, adults experienced the greatest reduction in body weight, followed by larvae.Additionally, adults displayed elevated metabolism rates and an increased mortality rate.Conversely, the pupae did not show any significant alterations in all examined parameters.Clarifying this concept is challenging.In contrast to our findings, studies suggest that the early stages, including pupae and larvae, display a greater susceptibility to stressors such as metals, temperature, and UV radiation [40][41][42].
A large body of animal studies supports the notion that stressful situations reliably produce hyperglycemia via the metabolic effects of increased corticosterone secretion [43,44].Also, exposure to EMF can lead to changes in sugar levels [11,45]; however, our findings revealed no significant alterations in total hemolymph sugar levels.This lack of an effect might be attributed to the dominant influence of severe food deprivation induced stress, which could have overshadowed the impact of EMF exposure.This is also the likely cause for the absence of notable changes in the metamorphosis rate, though many insect studies indicate that an extremely low-frequency EMF (below 100 Hz, 0.5-7 mT) is able to affect longevity and developmental dynamics by shortened developmental time, reduce number of enclosed insects, survivability, as well as weakened oviposition in subsequent generations [46][47][48][49].During the experiments, anomalies were observed in adults that had emerged as a result of the prolonged exposure of larvae and pupae to a 7 mT EMF.Other studies also demonstrate that an extremely low-frequency EMF (<100 Hz) can cause cellular harm and irregularities in the development, e.g., internal gut damage in the adult and larval stage or fluctuations in asymmetry and an increased frequency of abnormal phenotypic traits [46,50,51].Elevated stress levels initially serve an adaptive purpose, as seen in responses like the flight-or-fight reaction.However, these elevated stress levels can transition into a pathological state if they persist for prolonged periods [52].Continuous activation of the stress response over an extended duration (as was the case with the entire developmental cycle exposure to EMF, from the larval stage) triggers physiological transformations leading to the depletion of energetic and molecular reserves, the accumulation of toxic compounds and disruptions in regulatory pathways that may be reflected in the disturbance of transformations and success of metamorphosis [53].
It is known that exposure to EMFs can induce morphological and physiological changes in stress-related systems [11,54].The current knowledge shows that biogenic amines (BA), such as dopamine (DA), octopamine (OA), serotonin (5-HT), and tyramine (TA), act as neurotransmitters, neuromodulators, or neurohormones and are involved in the non-specific stress response and their levels change in various insect species under unfavorable conditions.They modulate behaviors and peripheral and sensory organs, enabling the insect to respond correctly to external stimuli [36,[54][55][56][57]. DA and 5-HT are involved in regulating locomotion and play a role in modulating arousal, feeding behavior, aggression, and learning processes.OA is the most frequent insect amine and, along with its precursor, TA, serves as the invertebrate equivalent to the vertebrate adrenergic neurotransmitters [58][59][60].As a "stress hormone", OA has a role in controlling various behaviors, such as flight and aggression, and is responsible for the mobilization of energy that prepares insects for higher metabolic activity related to the stress response and is useful during the recovery period.In the periphery, OA increases heart rate and may modulate ventilation and stimulate mobilization from muscles [58,[61][62][63][64].The activation of energy through the influence of BA occurs due to the triggering of glycogen conversion into trehalose and glucose and the oxidation of trehalose, along with the release of lipids from the fat body [65,66].The elevated levels of BA also intensify the process of energy mobilization by stimulating the release of adipokinetic hormones (AKHs), which are the insect counterpart to the mammalian glucagon.They have a neuromodulatory role and mobilize internal fuel reserves and stimulate insect locomotor activity.AKHs also have a metabolic role and control the release of carbohydrates, lipids, and the amino acid proline from the body fat to the heamolymph during locomotion and flight locomotion [62,67,68].Earlier studies have demonstrated the impact of both an SMF and EMF on BA concentrations in crickets, cockroaches, and fruit flies, as well as on the activity of neurosecretory neurons in gypsy moth caterpillars and yellow mealworm pupae [25,26,[69][70][71].EMF exposure resulted in a decrease in the frequency of spontaneously generated action potentials by the dorsal unpaired median (DUM) neurons of the cockroach Periplaneta americana [72].DUM neurons are believed to provide the majority of OA released into the hemolymph, and their pacemaker electrical activity is modulated by OA [73].The observed decrease in the neuronal activity caused by the EM field was similar to the effects caused by the stimulation of their octopaminergic receptors by the application of octopamine.However, the exact mechanism(s) underlying the EMF-induced changes in excitability is unclear, but it could be mediated by a change in the kinetic of ion channels.It can be elucidated that by acting on cell membrane surface charges, the EMF causes a change in the oscillation pattern of the membrane potential and/or acts as a destabilizing agent of complex protein-to-protein interactions [74,75].Among the various mechanisms proposed in the literature on the biological impacts of an EMF, the one that stands out as the most consistent involves the influx of calcium through voltage-dependent calcium channels.In addition to being a determinant of the electrophysiological characteristics of many cells, it has the capacity to trigger intracellular processes, such as enzyme activation, and can initiate responses at the genomic level.An EMF can cause an increase in the intracellular calcium current by increasing the mean open time [76,77] or increasing the number of calcium channels [78,79], or it may be the effect of a larger current flowing through a single channel [79].The elevated calcium entry leads to an increase in the intracellular Ca 2+ concentration that can initiate a number of physiological processes, including the activation of Ca 2+ -dependent outward K + currents, which regulate the firing frequency [77,80,81].All of the above indicate that biogenic amines may be a potential area to elucidate the underlying mechanisms of EMF-induced stress reactions, including changes in insect metabolism and mortality.It is important to note that impaired biogenic amine signaling has been associated with the development of several neural diseases in humans, including Parkinson's disease, schizophrenia, and depression [82,83].

Animals
The experimental subjects utilized in this study were adult, pupae, and larval specimens of the Tenebrio molitor L. (Tenebrionidae, Coleoptera) species, commonly known as mealworms.These organisms were obtained from the insectarium facility affiliated with the Department of Animal Physiology and Neurobiology at Nicolaus Copernicus University in Toru ń.The animals were reared at a consistent temperature of 23 ± 1 • C, with a daily lightdark cycle of 12:12.Insects were kept in a transparent container (30.5 cm × 25 cm × 15 cm).The food consisted of a mixture of semolina, flour, and oat flakes.

EMF Exposure
The mealworms were subjected to exposure to an electromagnetic field (EMF) with the domination of magnetic component with magnetic flux density levels of either 1 mT or 7 mT and with a frequency of 50 Hz.EMF generation setup (Figure 6) employed coilmagnetotherapy applicator AS 200 with a diameter of 0.2 m (Elektronika i Elektromedycyna Sp.J.; Otwock, Poland) powered with 230 V 50 Hz through Variac (M10-522-10, MCP, Shanghai, China).The value of magnetic flux density was controlled before each experiment with the gaussmeter (Model GM2, AlphaLab, Inc., Salt Lake City, UT, USA).Insects were placed in the experimental plastic container (8 cm diameter × 3 cm height) in the geometric center of the coil where the EMF was homogeneous (above 90%).
This exposure system and characteristics generated field has been described in detail previously [84,85].The temperature during experiments (for both control and EMF-exposed groups) was monitored using and was set to 24 ± 1 • C. The control group was placed in sham coils with the same experimental conditions (i.e., temperature, light, and humidity), except for the presence of EMF (below 10 µT).
or 7 mT and with a frequency of 50 Hz.EMF generation setup (Figure 6) employed coilmagnetotherapy applicator AS 200 with a diameter of 0.2 m (Elektronika i Elektromedycyna Sp.J.; Otwock, Poland) powered with 230 V 50 Hz through Variac (M10-522-10, MCP, Shanghai, China).The value of magnetic flux density was controlled before each experiment with the gaussmeter (Model GM2, AlphaLab, Inc., Salt Lake City, UT, USA).Insects were placed in the experimental plastic container (8 cm diameter × 3 cm height) in the geometric center of the coil where the EMF was homogeneous (above 90%).The magnetic field distribution inside the coil was numerically simulated using FEMbased COMSOL Multiphysics software.To simulate the magnetic field in the coil, it was first necessary to solve a Helmholtz-type equation for magnetic vector potential A according to the following formula [86]: where ε r and µ r correspond to the relative permittivity and relative permeability, respectively, ε 0 = 8.85•10 −12 F/m and µ 0 = 4π•10 −7 H/m are the electric and magnetic constants, σ (S/m) means an electric conductivity, J (A/m 2 ) is an excitation current density, respectively, and ω = 2πf is an angular frequency of excitation coil current (rad/s) and f (Hz) regular frequency.Next, the vector of magnetic flux density B (T) is obtained as the rotation of magnetic vector potential, namely, B = ∇ × A [86].All constant material parameters employed in numerical computations are summarized in Table 3.To solve the described problem, the magnetic insulation n × A = 0 was assumed on the outer boundaries of the computational area.Moreover, zero magnetic vector potential A = 0 was assumed within the entire computation space at the initial time.It should be noted that the EM field in the coil settles almost immediately after the power is turned on and is considered constant over time.

Experimental Procedures
Observations were carried out on adult insects, pupae, and larvae, which underwent a transformation in the following three groups: control and two exposed to EMF of 1 mT or 7 mT.During the experiments, the insects were kept in a plastic container with a diameter of 8 cm and 3 cm in height.All measurements were carried out at a constant temperature of 23.5 ± 0.5 • C and relative humidity of 50-60%.

Evaluation of Weight Loss, Rate of Metamorphosis, and Mortality
In the first experiment, adult mealworms were selected from breeding cages for similar weights 120-130 mg and placed in plastic containers, 10 individuals per container.The weight of the individuals was measured daily for a period of 14 days.The day of placing the insects in the experimental containers was taken as day "0".Another evaluated parameter was mortality, i.e., the day of appearance of the first dead individual, the day when the mortality reached 50% and 100% in each container.The next experiment was carried out on larvae taken from breeding cages.Larvae with similar weights of 220-230 mg were selected and placed in the experimental container, with 10 individuals in each.Larval weight was assessed every 24 h to determine its decline.The observations were carried out for 7 days (in the longer period, more metamorphoses to the pupa occurred, which disturbed the results).The start day of the experiment was taken as day "0".
The purpose of the next analysis was to answer the question of whether exposure to EMF had an impact on the duration of subsequent development in both the larval and pupal stages of mealworms.First, pupae appearing after metamorphosis from the examined larvae were observed.Each pupa was moved into a separate experimental container just after pupation and further exposed to EMF.The time of emergence of the first pupa was determined.The next step of this part of the experiment was the observation of the adult specimens that emerged from the treated larvae (EMF or control).The time of emergence of the first adult was recorded.The day of emergence of the adult from the pupa was taken as day "1".Insects did not receive food during experiments.The exuvia of the larvae or the remains of pupation were removed.

Metabolism
The rate of metabolism of experimental individuals was estimated by measuring CO 2 production through respirometry.Based on the outcomes we acquired, we opted to confine the subsequent experiments to EMF exposure of 7 mT, as this magnitude was found to induce significant biological effects.The metabolic rate was determined in both adults and larvae immediately after 24 h exposure to 7 mT EMF, along with corresponding control groups.CO 2 production rates were measured in closed glass respirometry chambers using flow-through respirometry (Qubit Systems, Kingston, ON, Canada).A total of the following two channels were connected to the 4-channel gas switcher (G243; Qubit Systems): one empty (baseline) and one experimental containing individual insects.Synthetic air (containing 21% of oxygen in 79% nitrogen and 0% of carbon dioxide; Air Products, Krakow, Poland) was pumped through the system with a high vacuum pump (P652 Vacuum Gas Pump; Qubit Systems).Measurements were carried out in 5 mL glass chambers with a flow rate of 160 mL/min, adjusted with a gas controller and monitor (G248; Qubit Systems).The air was scrubbed of water vapor using a column filled with magnesium perchlorate.Air flew from glass chambers (baseline or experimental, respectively) to infrared CO 2 (S157; Qubit Systems) analyzers.Gas exchange measurements lasted for 80 min.During the time of recording, each 15 min experimental chamber reading was followed by a 5 min recording of the empty chamber (baseline).So, each measurement of an individual insect consisted of four 15 min gas exchange readings and four 5 min baseline readings.Baseline values were used to provide an accurate zero value.Qubit Systems (Kingston, ON, Canada) data analysis software processed carbon dioxide recordings.This software recorded CO 2 levels in ppm and zeroed obtained values using baseline readings.Values were then converted to microliters/hour.Six control and six EMF-exposed insects of each developmental stage (larvae and adults) were tested, with four consecutive measurements taken for each.All measurements were carried out at a constant temperature of 23.5 ± 0.5 • C and relative humidity of 16%.

Total Sugar Content in the Heamolymph
The assessment of the sugar level in the heamolymph was performed on the larvae after 24 h of exposure to EMF of 7 mT in comparison to the control group.We limited the experiments to exposure of 7 mT EMF as the biological effect value.Heamolymph (10 µL) was collected from the insects by cutting the head.In total, 2 µL of collected heamolymph was mixed with 600 µL 70% ethanol and the samples were centrifuged (5 min; 10,000 RCF) to remove precipitated proteins.The 400 µL of supernatant was diluted with distilled water (100 µL) and used for sugar quantification.Phenol-sulfuric method (PSA) was used for the determination of total sugar content according to Dubois et al. [87] and modified by Matsumoto et al. [88].The obtained heamolymph supernatants were mixed with 500 µL of 5% phenol solution and 2500 µL of concentrated sulfuric acid.After 20 min incubation at room temperature, absorbance was measured at 490 nm (Shimadzu UV-VIS 1700 Spectrophotometer).The content of sugar was read from the standard curve for D(+) trehalose.

Statistical Analysis
To test the effect of EMF on the weight loss of exposed individuals (separately: adults, larvae, and pupae) with time, general linear mixed models (GLMMs) were used, including treatment as a fixed factor (control, 1 mT, 7 mT), time as a covariate (days 1-14), an interaction treatment*time, and experimental replicate as a random factor.The dependent variable in the model was the relative weight of insects on consecutive days, expressed as the percentage of the initial weight (on day 0).If a treatment*time interaction was significant, slopes of weight loss over time were tested separately for each treatment group (to check if the relationship holds for each group).Then, significant slopes were compared with each other to check if they were parallel (i.e., weight loss in both groups had the same rate).Finally, for parallel slopes, intercepts were compared to check if the slopes differed from each other in their vertical position (i.e., the group means were different).
To examine the impact of EMF on the mortality of exposed adults, 1-way general linear models (GLMs) were used, including treatment as a fixed factor (control, 1 mT, 7 mT).The dependent variables in the models were days of the occurrence of particular levels of mortality (the first dead individual, 50% mortality, 100% mortality) in particular replicates.
To assess the influence of EMF on the time (day) to appearance of the first pupa (emerging from larvae) or adult (emerging from pupae), 1-way GLMs were used, with treatment as a fixed factor (control, 1 mT, 7 mT).To test the effect of EMF on metamorphosis success (percentages of successful metamorphoses from larvae to pupae and from larvae to adults), generalized linear models (binomial distribution, log link function) were used, including treatment as a fixed factor (control, 1 mT, 7 mT).
To analyze the effect of EMF on the metabolic rate of exposed larvae and adults, GLMMs were used, including treatment as a fixed between-group factor (control, 7 mT), time as a within-subject factor (4 consecutive measurements of the same individual), treatment*time interaction, individual weight as a covariate to control for its effect on metabolism and individual as a random factor.The dependent variable was the CO 2 production per individual per unit of time.The weight and the dependent variable were log-transformed to linearize potentially allometric relationships between weight and metabolism.
To test the influence of EMF on the sugar content in the haemolymph of exposed adults, a GLMM was used, including treatment as a fixed factor (control, 7 mT) and replicate as a random factor.

Conclusions
It can be concluded that an electromagnetic field (EMF, 50 Hz) has an impact on an insect's metabolism, increasing body mass loss, mortality, and CO 2 production; however, EMF exposure does not seem to affect the process of metamorphosis.Notably, the observed changes were only evident when insects were exposed to a stronger EMF (7 mT) rather than a weaker EMF (1 mT).As a potential explanation for these observed effects, we postulate that EMF exposure acts as a stressor.This idea finds support in previous research indicating alterations in biogenic amine levels due to EMF exposure where ionic conductivities, particularly cellular calcium concentration, may participate.The findings suggest that many of the deleterious consequences associated with exposure to EMFs could be attributed to the prolonged increase in stress hormones.However, the specific mechanisms through which an EMF operates to produce these effects are not yet fully understood.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app13179893/s1,Table S1: General linear mixed model to test the effect of EMF exposure (treatment: control, 1 mT, 7 mT) on weight loss with time (time, days 1-14) in adult mealworms.The model included a random factor: experimental replicate, within which animals were measured on consecutive days.The dependent variable in the model was the relative weight of insects on consecutive days, expressed as the percentage of the initial weight (on day 0); Table S2: General linear models to test the effect of EMF exposure (treatment: control, 1 mT, 7 mT) on adult mealworm mortality.The dependent variables in the models were days of the occurrence of particular levels of mortality in particular replicates; Table S3: General linear mixed model to test the effect of EMF exposure (treatment: control, 1 mT, 7 mT) on weight loss with time (time, days 1-7) in larval mealworms.The model included a random factor: experimental replicate, within which animals were measured on consecutive days.The dependent variable in the model was the relative weight of insects on consecutive days, expressed as the percentage of the initial weight (on day 0); Table S4: General linear mixed model to test the effect of EMF exposure (treatment: control, 1 mT, 7 mT) on weight loss with time (time, days 1-7) in mealworm pupae.The model included a random factor: experimental replicate, within which animals were measured on consecutive days.The dependent variable in the model was the relative weight of insects on consecutive days, expressed as the percentage of the initial weight (on day 0); Table S5: General or generalized linear models to test the effect of EMF exposure (treatment: control, 1 mT, 7 mT) on metamorphosis success measured as the day of the first emergence of a given subsequent stage (pupa from larva or adult from a pupa) or as a percentage of successfully emerging individuals (relative to the initial number of larvae); Table S6: General linear mixed models to test the effect of EMF exposure (treatment: control, 7 mT) on adult and larval mealworm metabolic rate measured as CO 2 production.The model included a random factor: individual, measured 4 consecutive times during the exposure.Time was included as a within-subject factor and individual weight as a covariate to control for its effect on metabolism.The weight and the dependent variable (CO 2 production per individual per unit time) were log-transformed to linearize potentially allometric relationships between weight and metabolism; Table S7: General linear mixed model to test the effect of EMF exposure (treatment: control, 7 mT) on sugar content in adult haemolymph.The model included a random factor: replicate (3 replicates with 6 individuals in each).
Appl.Sci.2023, 13, x FOR PEER REVIEW 3 of 16of the control group decreased to 76.15% of the initial value on the last day of the experiment.The weight of the exposed group decreased to 76.8% for 1 mT and 63.51% for 7 mT on the last day of the experiment.

Figure 1 .
Figure 1.Decrease in body weight of imago exposed to 1 and 7 mT compared to the control group.(values expressed as a percentage of initial weight, number of insects per sample: n = 10, number of independent replications: N = 10).The lines are predicted by the general linear mixed model with 95% confidence intervals (dashed lines).Lines labelled with the same letters indicate no significant differences in a decrease in body weight.Data points represent individual measurements.

Figure 2 .
Figure 2. The day of appearance of the first dead individual, the day when the mortality reached 50% and 100% (mean ± SE).The same letters indicate no significant differences between groups.

Figure 1 .
Figure 1.Decrease in body weight of imago exposed to 1 and 7 mT compared to the control group.(values expressed as a percentage of initial weight, number of insects per sample: n = 10, number of independent replications: N = 10).The lines are predicted by the general linear mixed model with 95% confidence intervals (dashed lines).Lines labelled with the same letters indicate no significant differences in a decrease in body weight.Data points represent individual measurements.

Figure 1 .
Figure 1.Decrease in body weight of imago exposed to 1 and 7 mT comp (values expressed as a percentage of initial weight, number of insects per independent replications: N = 10).The lines are predicted by the genera 95% confidence intervals (dashed lines).Lines labelled with the same lett differences in a decrease in body weight.Data points represent individua

Figure 2 .
Figure 2. The day of appearance of the first dead individual, the day w 50% and 100% (mean ± SE).The same letters indicate no significant differ

Figure 2 .
Figure 2. The day of appearance of the first dead individual, the day when the mortality reached 50% and 100% (mean ± SE).The same letters indicate no significant differences between groups.

Figure 3 .
Figure 3. Decrease in body weight of larvae (a) and pupae (b) (from exposed larvae) exposed to 1 mT and 7 mT EMF compared to the control group (values expressed as a percentage of initial weight, number of insects per sample n = 10, number of independent replications N = 6).The lines are predicted by the general linear mixed model with 95% confidence intervals (dashed lines).Lines labelled with the same letters indicate no significant differences in a decrease in body weight.Data points represent individual measurements.

Figure 3 .
Figure 3. Decrease in body weight of larvae (a) and pupae (b) (from exposed larvae) exposed to 1 mT and 7 mT EMF compared to the control group (values expressed as a percentage of initial weight, number of insects per sample n = 10, number of independent replications N = 6).The lines are predicted by the general linear mixed model with 95% confidence intervals (dashed lines).Lines labelled with the same letters indicate no significant differences in a decrease in body weight.Data points represent individual measurements.

Figure 4 .
Figure 4. Effect of electromagnetic exposure on the metabolic rate of larvae and adults (n Results are expressed as mean with a 95% confidence interval.The same letters indicate cant differences in CO2 production between groups.

Figure 5 .
Figure 5.Effect of electromagnetic exposure on haemolymph sugar content (number of sample n = 6, number of independent replications N = 3).Results are expressed as mean confidence interval.The same letters indicate no significant differences in sugar content b control and EMF-exposed groups.

Figure 4 .
Figure 4. Effect of electromagnetic exposure on the metabolic rate of larvae and adults (n = 4, N = 6).Results are expressed as mean with a 95% confidence interval.The same letters indicate no significant differences in CO 2 production between groups.

Figure 4 .
Figure 4. Effect of electromagnetic exposure on the metabolic rate of larvae and adults (n = Results are expressed as mean with a 95% confidence interval.The same letters indicate n cant differences in CO2 production between groups.

Figure 5 .
Figure 5.Effect of electromagnetic exposure on haemolymph sugar content (number of i sample n = 6, number of independent replications N = 3).Results are expressed as mean w confidence interval.The same letters indicate no significant differences in sugar content be control and EMF-exposed groups.

Figure 5 .
Figure 5.Effect of electromagnetic exposure on haemolymph sugar content (number of insects per sample n = 6, number of independent replications N = 3).Results are expressed as mean with a 95% confidence interval.The same letters indicate no significant differences in sugar content between the control and EMF-exposed groups.

Figure 6 .
Figure 6.The electromagnetic field (EMF) exposure system: (a) photograph of the coil−magnetotherapy applicator and Variac used to generate EMF; magnetic flux density distribution inside the coil for (b) 1 mT, and (c) 7 mT (schemes represent half of the cross−section of the coil, point (x = 0, y = 0) is a geometrical center of the coil, numerical simulation).

Table 1 .
The day of appearance of the first individuals of subsequent developmental phases.Results are expressed as mean ± SE.

Table 2 .
The percentage of the number of larvae that have transitioned to the next stage (relative to the original number of larvae).Results are expressed as mean ± SE.Number/Group CONTROL (%) EMF 1 mT (%) EMF 7 mT (%) PUPAE 62.7 ± 5.5 68 ± 6.7 58 ± 5.4 IMAGO 42.5 ± 3.7 39.9 ± 3.8 31.3 ± 3.6 It is worth noting that during pupal metamorphosis into the imago under exposure

Table 1 .
The day of appearance of the first individuals of subsequent developmental phases.Results are expressed as mean ± SE.

Table 2 .
The percentage of the number of larvae that have transitioned to the next stage (relative to the original number of larvae).Results are expressed as mean ± SE.