Cardiac activity and the influence of isoflurane on this parameter in Anteos menippe butterflies

Cardiac activity and the influence of isoflurane on this parameter in Anteos menippe butterflies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cardiac activity and the influence of isoflurane on this parameter in Anteos menippe butterflies Antônio Basílio Guerreiro Júnior, Deise de Lima Cardoso, Sabrina Reika Seko Kondo, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8022097/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Understanding the physiological mechanisms regulating cardiac activity in insects remains a fundamental challenge in comparative physiology. This study investigated the effects of isoflurane anesthesia on the electrocardiographic (ECG) activity of Anteos menippe butterflies during induction and recovery. Six newly emerged adults were exposed to 1 mL of isoflurane vapor, and the latency to loss and recovery of postural reflex was recorded. Nickel–chromium electrodes were implanted in the abdomen to record ECG signals for 30 min in a Faraday-shielded setup. Isoflurane produced smooth and reversible anesthesia with induction and recovery times of 48.5 ± 8.9 s and 117.7 ± 16.2 s, respectively, without excitatory responses. During anesthesia, spike frequency decreased to 35 ± 3.3 min⁻¹, returning to 64.3 ± 3.9 min⁻¹ during recovery, accompanied by progressive amplitude and power increases. Spectral analysis revealed dynamic energy redistribution associated with chronotropic modulation during anesthetic elimination. These findings provide the first direct ECG characterization in Lepidoptera, demonstrating that isoflurane is a safe and effective anesthetic and that the butterfly cardiac system follows conserved electrophysiological mechanisms among insects. Anteos menippe isoflurane electrocardiography cardiac physiology autonomic modulation invertebrate anesthesia Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The knowledge of biological parameters of urban insect species is of great importance for assessing environmental quality and for establishing procedures aimed at the preservation of ecosystems (Born & Lima, 2005 ). The order Lepidoptera , which includes butterflies and moths, presents a high diversity in Brazil (Prado & Zukovski, 2012 ) and has significant ecological importance, as these insects are highly sensitive to alterations in their habitat (Dessuy & Morais, 2007 ). Within this context, an important group of this order is the Pieridae butterflies, which exhibit migratory behavior and are geographically distributed in temperate climates, particularly in tropical regions of South America (Monteiro et al., 2009 ). Many species within this family act as pollinators. Anteos menippe belongs to this family, within the subfamily Coliadinae, and is characterized by predominantly yellow and orange coloration (Monteiro et al., 2009 ). Due to the high environmental fragility of these insects, several practices must be adopted to maximize their welfare during handling and transportation, both in captive breeding programs and in scientific experimentation. In this regard, anesthesia in invertebrates is an important process that facilitates manipulation for examinations, sampling, and transport, reducing stress and ensuring animal welfare (Cooper, 2001 ). Traditionally, insect immobilization has been performed through exposure to cold or carbon dioxide (CO₂) (Nilson; Sinclair; Roberts, 2006 ). However, related studies indicate that exposure to these agents may cause long-lasting adverse effects on insect physiology (Gooley & Gooley, 2021 ). It is therefore essential that the anesthetic process does not alter the physiological parameters under study. Thus, the use of volatile anesthetic agents, such as isoflurane, emerges as an effective alternative for managing these organisms. Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane) primarily acts as a positive modulator of GABA-A receptors, increasing chloride ion permeability and inducing neuronal hyperpolarization. These mechanisms promote general anesthesia with analgesic, hypnotic, and muscle-relaxant effects. It was employed in this study due to its ability to provide rapid anesthetic induction, predictable recovery, and a lower potential to cause prolonged stress. Its use in insects has been explored as an efficient approach to facilitate experimental management of these animals (Macmillan et al., 2017 ). From this perspective, electrophysiology can be used to assess the anesthetic state of animals under restraint stress, providing insights into the bioelectrical processes regulating cardiac and neuromuscular function both during physical restraint and under the anesthetic effects of isoflurane. In this way, it is possible to analyze how electrical impulses are generated and conducted in the cardiac system (Berul et al., 1996 ). These mechanisms are evaluated using electrocardiography (ECG), which records the electrical signals produced by cardiomyocytes. When electrodes are positioned adjacent to the heart, the ECG detects these impulses and converts them into graphical tracings, allowing the assessment of rhythm, frequency, and other features of cardiac function (Berkaya et al., 2018 ). Despite significant advances in insect neurophysiology, the understanding of cardiac electrophysiology in invertebrates remains limited, particularly regarding the direct recording and interpretation of ECG activity. Most available data rely on indirect measurements of hemolymph flow or optical methods, which provide restricted insight into the dynamics of electrical conduction and autonomic modulation of the insect heart (Sláma, 2012 ; Davis & Orakzai, 2016 ). Although the insect cardiac system is known to exhibit rhythmic contractions regulated by both myogenic and neurogenic mechanisms, the electrophysiological basis of these events has been poorly characterized compared to vertebrate models (Berul et al., 1996 ; Wang & Chahl, 2025 ). Moreover, studies integrating anesthesia and electrophysiological monitoring are scarce, and there is a notable lack of standardized protocols for ECG acquisition in Lepidoptera species. Filling this knowledge gap is essential to establish a physiological framework for assessing how anesthetics and stress conditions modulate cardiac function in insects, enabling comparative analyses of autonomic regulation and evolutionary conservation of neural control pathways across taxa (MacMillan et al., 2017 ; Li et al., 2023 ). This study aimed to evaluate the use of isoflurane for anesthesia in Anteos menippe butterflies, describing the behavioral responses during anesthetic induction and recovery and, subsequently, evaluating the electrophysiological tracings related to cardiac function during anesthesia and anesthetic recovery. 2. Materials and Methods 2.1 Animals This study used six Anteos menippe butterflies (males and females), recently emerged adults, obtained from a breeding facility located in the Environmental Park Mangal das Garças, Belém, Pará, Brazil. The butterflies were transported to the Laboratory of Pharmacology and Toxicology of Natural Products, Institute of Biological Sciences, Federal University of Pará (ICB-UFPA), in a temperature-controlled environment (25 ± 0.3°C). 2.2 Preparation of animals for experimentation The butterflies were initially anesthetized with isoflurane. A volume of 1 mL was impregnated into cotton and placed inside an anesthetic chamber with a capacity of 1500 cm³ at 25 ± 0.3°C. The latency to loss of postural reflex and immobility was recorded. Subsequently, each butterfly was removed from the isoflurane chamber and transferred to an anesthetic-free environment, where the recovery time was recorded, defined by the return of the postural reflex (Fig. 1 A and 1 B). Figure 1 (A and B) here 2.3 Electrode fabrication, implantation, and ECG recording For electrocardiographic (ECG) recording, electrodes were constructed from JST SM 2-pin jack cables, 13 cm in length. Paired electrodes were spaced 1 mm apart and built from nickel-chromium wire (Morelli Orthodontics), with 0.2 mm diameter and 2 mm length (Fig. 2 C). The electrodes were insulated with liquid insulator. The butterflies were restrained using paper strips and pins (Figs. 2 A and 2 B). After fixation, the electrodes were positioned according to the following coordinates: along the median sagittal line of the dorsal surface, adjacent to the first abdominal spiracle after the thoracic region, with a lateral displacement of ± 1 mm and a dorsoventral depth of 2 mm (Figs. 2 B and 2 C). The recording electrode was positioned on the right side and the reference electrode on the left. After electrode implantation, electrocardiograms were recorded (Figs. 2 D and 2 E). The method of Contrera et al. ( 2023 ) was adapted with modifications, according to the equipment and materials available in this study. Figure 2 (A, B, C, D and E) here All procedures were performed inside a Faraday cage with metallic shielding. The electrodes were connected to a high-impedance amplifier (Grass Technologies P511, West Warwick, USA) with a signal amplification of 50,000×, monitored by an oscilloscope (Protek, 6510). Each butterfly was recorded for a duration of 30 minutes. The animals remained in contact with isoflurane for 5 minutes for anesthetic induction, and recovery from anesthesia was subsequently observed and monitored by ECG during the remaining 25 minutes. Data were analyzed every 5 minutes of recording, as follows: 0-300s (A), 300-600s (B), 600-900s (C), 900-1200s (D), 1200-1500s (E), and 1500–1800 (F). The recordings allowed evaluating the following parameters: peak frequency per minute, peak amplitude, inter-peak interval, peak duration, and signal power. 2.4 Statistical analysis Data are presented as mean ± standard deviation (SD). A significance level of p < 0.05, p < 0.01, and p < 0.001 was considered for all analyses. Comparisons between periods were performed using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Statistical analyses were carried out using GraphPad Prism, version 8 (GraphPad Software Inc., San Diego, CA, USA). Normality and homogeneity of variances were tested using the Kolmogorov–Smirnov and Levene tests, respectively. 3. Results During anesthetic induction with isoflurane at 25 ± 0.3°C, a gradual deepening of anesthesia was observed, characterized by the loss of postural reflex and immobility, with a latency of 48.50 ± 8.98 s (Fig. 1 A). Anesthetic recovery occurred upon removing the butterflies from the isoflurane chamber, with the return of the postural reflex observed after 117.7 ± 16.21 s. No excitability-related behaviors were detected during the recovery period, and anesthetic lightening occurred gradually and smoothly (Fig. 1 B). Figure 2 shows the physical restraint method applied to Anteos menippe butterflies for anesthetic induction with isoflurane (A), the electrode insertion site for signal acquisition (B), and the characteristics of the electrodes manufactured for the experiment (C). Representative ECG recording showing the firing of impulses that maintain cardiac rhythm (D), characterized by peaks with ascending and descending phases, as observed under amplified recording (E). The electrocardiogram illustrating the cardiac function of Anteos menippe butterflies during the first 5 seconds of recording under restraint stress without anesthesia showed a mean peak frequency of 61 ± 4.14 per minute, with an amplitude of 2.46 ± 0.466 mV (50,000× signal amplification). The mean inter-peak interval was 966 ± 86.79 ms. The contraction cycle responsible for driving the circulatory components, represented by the peak duration, had an average value of 31.33 ± 3.26 ms (Fig. 2 E). ECG recordings of Anteos menippe butterflies during anesthetic induction and recovery with isoflurane performed for 30 minutes, showing variations in the amplitude of the graphoelement signal. From each recording, 5-minute segments were extracted for analysis to compare the effects of anesthesia on hemolymph pumping. During the first 5 minutes (0-300 s), low recording amplitude was observed, followed by a progressive increase in amplitude in the period 300–600 s, while the amplitude remained stable during the periods 600–900 s, 900–1200 s, 1200–1500 s, and 1500–1800 s (Fig. 3 A). Spectrogram analysis showed an increase in energy levels during the 300–1800 s periods, which represent anesthetic recovery, demonstrating the effect of the anesthetic on butterfly hemodynamics during anesthesia (Fig. 3 B). During the 30-minute cardiac recording, differences in the mean linear power of the signals were observed. In the 0-300 s period, the mean linear power was 0.114 ± 0.015 mV²/Hz × 10⁻³, similar to the 300–600 s period (p = 0.993), but lower than in the other groups. The control group was 0.210 ± 0.029 mV²/Hz × 10⁻³ was similar to periods C (p = 0.998) and D (p = 0.166), but was lower than group E and F. Periods C (0.201 ± 0.027 mV²/Hz × 10⁻³) was similar to period D (p = 0.0571), but was lower than period E and F. Periods D (0.252 ± 0.031 mV²/Hz × 10⁻³) E and F were similar (p = 0.119)(3C), demonstrating that in the anesthetic recovery period there was a progressive increase in signal power throughout the recording. Figure 3 (A, B and C) here Each change in the ECG tracings during anesthetic induction and recovery in periods A, B, C, D, E, and F was analyzed as a pattern of cardiac activity (Fig. 4 A–F). For period A, the mean peak frequency was 35 ± 3.28 per minute, with well-defined peaks showing positive deflections, and the histogram indicated the highest concentration of energy between 3 and 4 Hz with good distribution up to 25 Hz (Fig. 4 A). In interval B, a change in peak direction was observed, with negative deflections predominating; the peak frequency increased to 41.0 ± 4.85 per minute, and the histogram showed maximum power at 4 Hz (Fig. 4 B). In period C (600–900 s), maintenance in signal amplitude was observed, with a peak frequency of 45.0 ± 3.2 per minute, exhibiting positive and negative deflections, and the histogram showed peak power at 3 Hz (Fig. 4 C). During period D (900 to 1200 s), the peak frequency was 59.67 ± 4.63 per minute, with positive and negative peaks and a predominance of 3 Hz in the histogram (Fig. 4 D). For period E (1200-1500s) the frequency was 60.33 ± 4.8 per minute, with a predominance in the frequencies of 3 to 6 Hz (Fig. 4 E) and Period F (1500-1800s) presented an average peak frequency of 64.33 ± 3.88 per minute with a predominance in the frequency range between 2–4 Hz (Figure F). The frequency of peaks varied throughout the anesthetic induction and recovery periods. The control group presented a mean frequency of 61.0 ± 4.14 per minute, which was similar to periods D (p = 0.997), E (p = 0.999), and F (p = 0.806), but was higher than periods A, B, and C. Period A (35 ± 3.2 per minute) was similar to period B (p = 0.194). Period B (41 ± 4.8 per minute) was similar to period C (p = 0.646), but was lower than the subsequent periods. Periods D, E, and F were similar (p = 0.471) (Fig. 4 G). The signal amplitude increased throughout recovery. For the control group, the mean amplitude was 2.46 ± 0.46 mV, similar to periods A (p = 0.762), B (p = 0.996), C (p = 0.482), D (p = 0.969) and E (p = 0.563). However, it was lower than period F. Period A (1.89 ± 0.35 mV) was similar to periods B (p = 0.400) and D (p = 0.240), however, it was lower than the other periods (Fig. 4 H). For the inter-peak intervals, the control presented a mean of 963 ± 83.79 ms, which was lower than periods A, B and C, but was similar to periods D (p = 0.992), E (p = 0.999) and F (p = 0.998). Period A (1718 ± 163.8 ms) was higher than all other intervals. Period B (1479 ± 158.8 ms) was similar to period C (p = 0.411). Period D (1010 ± 77.62 ms) was similar to periods E (p = 0.999) and F (p = 0.898) (Fig. 4 I). The peak duration increased during the anesthesia period. The control had a mean peak duration of 31.33 ± 3.26 ms, which was lower than periods A and B, but was similar to groups C (p = 0.0512), D (p = 0.231), E (p = 0.953), and F (p = 0.968). Periods A (66 ± 11.22 ms) were similar to period B (p = 0.086), but were higher than the other periods. Period B (53.33 ± 10.39 ms) was similar to periods C (p = 0.4938) and D (p = 0.1503). Periods C, D, E, and F were similar (p = 0.319) (Fig. 4 J). Figure 4 (A, B, C, D, E, F, G, H and I) here 4. Discussion Our study represents the first attempt to investigate the effects of isoflurane, along with electrocardiographic assessment, during anesthetic induction and recovery in Anteos menippe butterflies. The anesthetic produced the expected results, inducing sedation with gradual deepening, as evidenced by the loss and subsequent recovery of postural reflex. The butterflies did not exhibit behaviors indicative of excitability caused by the anesthetic, demonstrating a positive outcome with gradual and smooth induction. Although specific studies on butterfly species are lacking, isoflurane has a well-established use in invertebrate anesthesia and is considered superior to traditional methods such as cold exposure or CO₂ (MacMillan et al., 2017 ). Its sedative efficacy has been reported in other species, including button-wing mantises ( Parasphendale agrionina ), emperor scorpions ( Pandinus imperator ), and yellow uruçu bees ( Melipona flavolineata ), showing mild induction and positive prognosis without observable adverse effects, consistent with our findings (D’Ovidio, Monticelli, & Adami, 2021 ; Gaudette et al., 2022 ; Contrera et al., 2023 ). Therefore, isoflurane represents a highly effective and valuable anesthetic for invertebrates, with applications in scientific, commercial, and veterinary contexts. The GABAergic system is highly conserved among insects (Li et al., 2023 ). Sedation is mediated by activation of the GABA system, leading to general inhibition of the central nervous system, resulting in reduced motor activity and loss of sensitivity to stimuli via mechanisms like those observed in vertebrates under anesthetic action. According to McClure and Heberlein ( 2013 ), these effects suggest that GABAergic modulation is an evolutionarily conserved pathway that regulates behaviors associated with torpor or deep rest, particularly during physiological processes such as diapause. Studies in Drosophila have shown that GABAergic activity regulates the release of neuropeptides, such as corazonin (Crz), which influence reproductive cycle behaviors and rest states, including the control of diapause phases. These findings suggest that GABAergic modulation can impact physiological and behavioral processes associated with sedation, providing a conceptual basis for hypotheses regarding the physiological responses of Anteos menippe to sedatives. Additionally, the spatial distribution of GABA receptor subunits in the brain may explain differential sensitivity of various neural centers to sedative drugs (Yu et al., 2010). This study represents the first direct electrocardiographic evaluation of butterflies, focusing on the effects of isoflurane during anesthetic induction and recovery in Anteos menippe. It is important to note that the peaks observed in insects are not functional equivalents of the human QRS complex, although they can be used for physiological analogies. Our findings are consistent with those reported by Davis & Orakzai ( 2016 ), who documented mean resting heart rates of 63 bpm in butterflies, supporting the physiological congruence between lepidopterans from different biomes. Furthermore, the characterization of electrophysiological phases in butterflies aligns with observations in other arthropods, as reported by Sláma et al. (2012), revealing homologous patterns to the cardiac complexes of arthropods when compared to vertebrates and reinforcing the evolutionary conservation of autonomic nervous system mechanisms in cardiac contraction. Methodologically, our study complements the approach proposed by Wang & Chahl ( 2025 ), highlighting the value of high-resolution electrophysiological monitoring to ensure animal welfare and detailed spectral analysis. Regarding the spectrographic analysis, the increase in spike frequency over time, associated with postural recovery after anesthesia, suggests a physiological behavior similar to that observed in bees by Contrera et al. ( 2023 ), indicating a shortening of the interspike interval during the recovery period. Furthermore, oscillations in amplitude and energy over time may reflect changes in cardiac activity after anesthetic removal, indicating alterations in cardiac electrical conduction and hemodynamic effects. Overall, these results demonstrate that the cardiac activity of A. menippe follows universal patterns of chronotropic variability described in insects, while also extending the physiological framework to a broader range of species. Conclusion The results of this study demonstrate, for the first time, that isoflurane is an effective and safe anesthetic agent for managing Anteos menippe butterflies, providing gradual induction, predictable recovery, and the absence of undesirable excitatory effects. Electrocardiographic characterization performed during anesthetic induction and recovery revealed physiological patterns consistent with autonomic modulation previously described in other insects, demonstrating that the cardiac system of this species follows conserved mechanisms of chronotropic variability. The progression of electrical responses, reflected in changes in the frequency, amplitude, and energy distribution of the recorded peaks, reinforces the applicability of the ECG as a monitoring tool in invertebrates, expanding the possibilities for investigating physiological parameters in experimental and conservation contexts. Thus, this work not only contributes to the refinement of anesthetic practices in lepidopterans, but also establishes a relevant methodological basis for future studies aimed at understanding cardiac physiology in insects, strengthening the scientific framework necessary for the preservation and well-being of these organisms in laboratory and management environments. Declarations CRediT authorship statement Conceptualization and Methodology: Antônio Basílio Guerreiro Júnior, Deise de Lima Cardoso, Nilton Akio Muto and Moisés Hamoy. Performed the experiments: Antônio Basílio Guerreiro Júnior, Deise de Lima Cardoso and Moisés Hamoy. Writing-original draft and editing: Axell Lins, Sabrina Reika Seko Kondo, Eduardo Machado da Silva Zamian, Alexa Camila Lopes de Assis, Diogo Macola Felix, Eva Vitória Ferreira Viana, Daniella Bastos de Araújo, Clarissa Araújo da Paz and Diva Anelie de Araújo Guimarães. Financial support and administrative support: Moisés Hamoy. All authors have read and agreed to the published version of the manuscript. Clinical trial number The study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable. Consent to participate No human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable. Consent publish All authors have read and approved the final version of the manuscript and consent to its publication. Consent to Participate This study did not involve human participants. As the experimental model consisted exclusively of invertebrates ( Anteos menippe butterflies), no institutional ethics approval or individual consent was required. Therefore, this item is not applicable. DAS statement request All data generated or analyzed during this study are available in the public repository at the following link: https://drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing . The datasets support the findings of this study and include the original electrophysiological and behavioral data. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author Contribution Conceptualization and Methodology: Antônio Basílio Guerreiro Júnior, Deise de Lima Cardoso, Nilton Akio Muto and Moisés Hamoy. Performed the experiments: Antônio Basílio Guerreiro Júnior, Deise de Lima Cardoso and Moisés Hamoy. Writing-original draft and editing: Axell Lins, Sabrina Reika Seko Kondo, Eduardo Machado da Silva Zamian, Alexa Camila Lopes de Assis, Diogo Macola Felix, Eva Vitória Ferreira Viana, Daniella Bastos de Araújo, Clarissa Araújo da Paz and Diva Anelie de Araújo Guimarães. Financial support and administrative support: Moisés Hamoy. All authors have read and agreed to the published version of the manuscript. Data Availability All data generated or analyzed during this study are available in the public repository at the following link: [https://drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing](https:/drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing) . The datasets support the findings of this study and include the original electrophysiological and behavioral data. References Berkaya SK, Uysal AK, Gunal ES, Ergin S, Gunal S, Gulmezoglu MB. A survey on ECG analysis. Biomed Signal Process Control. 2018;43:216–35. https://doi.org/10.1016/j.bspc.2018.03.003 . Berul CI, Aronovitz MJ, Wang PJ, Mendelsohn ME. In vivo cardiac electrophysiology studies in the mouse. Circulation. 1996;94:2641–8. https://doi.org/10.1161/01.cir.94.10.2641 . Born FS, Lima IMM. Development stadia of Anteos menippe (Hübner) (Lepidoptera, Pieridae) on Cassia ferruginea Shrad. (Caesalpinaceae), in laboratory. Rev Bras Entomol. 2005;49:522–6. https://doi.org/10.1590/S0085-56262005000400011 . Contrera FAL, Lopes BSC, Paz CA, Hamoy MKO, Santos MF, Barbosa GB, et al. First records of heartbeats via ECG in a stingless bee, Melipona flavolineata (Apidae, Meliponini), during contention stress using isoflurane as an anesthetic. Insects. 2023;14:696. https://doi.org/10.3390/insects14080696 . Cooper JE. Invertebrate anesthesia. Vet Clin North Am Exot Anim Pract. 2001;4:57–67. https://doi.org/10.1016/S1094-9194(17)30051-8 . Davis AK, Orakzai S. A simple method for observing and measuring heart rates in live adult monarchs ( Danaus plexippus ) and other large butterflies. J Lepid Soc. 2016;70:96–8. https://doi.org/10.18473/lepi.70i2.a3 . Dessuy MB, Morais ABB. Diversidade de borboletas (Lepidoptera, Papilionoidea e Hesperioidea) em fragmentos de Floresta Estacional Decidual em Santa Maria, Rio Grande do Sul, Brasil. Rev Bras Zool. 2007;24:108–20. https://doi.org/10.1590/S0101-81752007000100014 . D’Ovidio D, Monticelli P, Adami C. Isoflurane anesthesia in budwing praying mantises ( Parasphendale agrionina ). J Zoo Wildl Med. 2021;52:1013–7. https://doi.org/10.1638/2020-0195 . Gaudette C, Johnson B, Bakal R, Dombrowski DS. Hemolymph collection and isoflurane anesthesia of the emperor scorpion ( Pandinus imperator ). J Zoo Wildl Med. 2022;53:573–7. https://doi.org/10.1638/2020-0095 . Gooley ZC, Gooley AC. Metabolic effects of anesthetics (cold, CO₂, and isoflurane) and captivity conditions in isolated honey bee ( Apis mellifera ) foragers under different ambient temperatures. J Apic Res. 2021;62:1–9. https://doi.org/10.1080/00218839.2021.1950994 . Guh YJ, Tamai TK, Yoshimura T. The underlying mechanisms of vertebrate seasonal reproduction. Proc Jpn Acad Ser B Phys Biol Sci. 2019;95:343–57. https://doi.org/10.2183/pjab.95.024 . Li H, et al. A tomato nitrogen-dependent GABA pathway confers resistance to a globally invasive fruit fly. Front Plant Sci. 2023;14:1252455. https://doi.org/10.3389/fpls.2023.1252455 . MacMillan HA, Nørgård M, MacLean HJ, Overgaard J, Williams CJA. A critical test of Drosophila anaesthetics: Isoflurane and sevoflurane are benign alternatives to cold and CO₂. J Insect Physiol. 2017;101:97–106. https://doi.org/10.1016/j.jinsphys.2017.07.005 . McClure KD, Heberlein U. A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila . J Neurosci. 2013;33:4044–54. https://doi.org/10.1523/JNEUROSCI.3140-12.2013 . Monteiro RF, Freitas AVL, Costa Filho MAF, Nascimento MS, Alves TG, Brown KS Jr, et al. Borboletas da Mata Atlântica do Estado do Rio de Janeiro: Pieridae (Lepidoptera). Arq Mus Nac Rio J. 2009;67:283–9. Nilson TL, Sinclair BJ, Roberts SP. The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster . J Insect Physiol. 2006;52:1027–33. https://doi.org/10.1016/j.jinsphys.2006.07.001 . Prado LEPA, Zukovski L. A importância da conservação de lepidópteras para os processos ecológicos. Rev Terra Cult Cad Ens Pesqui. 2012;28:69–78. Sláma K. A new look at the comparative physiology of insect and human hearts. J Insect Physiol. 2012;58:1072–81. https://doi.org/10.1016/j.jinsphys.2012.04.014 . Waldvogel HJ, Baer K, Faull RLM. Distribuição de subunidades receptoras GABA no cérebro humano. In: Monti J, Pandi-Perumal S, Möhler H, editors. GABA e Sono. Basel: Springer; 2010. pp. 45–67. https://doi.org/10.1007/978-3-0346-0226-6_3 . Watanabe M, Fukuda A, Nabekura J. The role of GABA in the regulation of GnRH neurons. Front Neurosci. 2014;8:387. https://doi.org/10.3389/fnins.2014.00387 . Wang D, Chahl J. Extracting cardiac activity for arthropods using digital cameras: Insights from a pilot study. Arch Insect Biochem Physiol. 2025;119:e70076. https://doi.org/10.1002/arch.70076 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8022097","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":545856543,"identity":"b4f4d3fb-1a43-4bf4-8d0f-58002089c357","order_by":0,"name":"Antônio Basílio Guerreiro Júnior","email":"","orcid":"","institution":"Federal University of Pará (UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Antônio","middleName":"Basílio Guerreiro","lastName":"Júnior","suffix":""},{"id":545856544,"identity":"5c4b4371-b2c3-44e8-b14d-67cc0fc8fec8","order_by":1,"name":"Deise de Lima Cardoso","email":"","orcid":"","institution":"Federal University of Pará (UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Deise","middleName":"de Lima","lastName":"Cardoso","suffix":""},{"id":545856545,"identity":"5d11eb58-4c0c-42c8-9f40-89c4f5213896","order_by":2,"name":"Sabrina Reika Seko Kondo","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"Reika Seko","lastName":"Kondo","suffix":""},{"id":545856546,"identity":"30699824-6ab7-4b68-a26e-85fd81474fba","order_by":3,"name":"Axell Lins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYHACxgMPGBh4+JmZDwA5EjJE6TmQwGDAI9nelgDSwkO0FgaDM2cMQBzCWvhnJB8Aavkjw3Aj5/OrGzUWPAzsh49uwKdF4syxBLDDGGfkbrPOOQZ0GE9a2g281hzvMQBrYZbI3WacwwbUIsFjhleL/GH+D2AtbBI5z4xz/hGhxeB4DyTEeHjOMD/ObSNCi+GZY0CHGRjzSLC3mTHn9knwsBHyi9yN5IcPPlTI2dsfZn78OedbnRw/++Fj+L0PcR6YZJMAk4SVIwDzB1JUj4JRMApGwcgBAOHwRcMGOts+AAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":true,"prefix":"","firstName":"Axell","middleName":"","lastName":"Lins","suffix":""},{"id":545856547,"identity":"06457d0e-d35a-4bf9-b030-c30d0facb68f","order_by":4,"name":"Naoto da Cunha Hayasaki","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Naoto","middleName":"da Cunha","lastName":"Hayasaki","suffix":""},{"id":545856548,"identity":"fefcb857-07f3-49cd-be28-a4716e402140","order_by":5,"name":"Eduardo Machado Silva Zamian","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"Machado Silva","lastName":"Zamian","suffix":""},{"id":545856549,"identity":"1125a912-5cf0-4007-b72d-30661415541f","order_by":6,"name":"Alexa Camila Lopes de Assis","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Alexa","middleName":"Camila Lopes","lastName":"de Assis","suffix":""},{"id":545856550,"identity":"82b867a1-c177-47e7-b120-645ddc4818ae","order_by":7,"name":"Diogo Macola Felix","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Diogo","middleName":"Macola","lastName":"Felix","suffix":""},{"id":545856551,"identity":"30adddc2-4f0b-4c47-b055-86d019f05dce","order_by":8,"name":"Eva Vitória Ferreira Viana","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"Vitória Ferreira","lastName":"Viana","suffix":""},{"id":545856552,"identity":"a8fded6b-e1cb-4194-a238-adba63b32261","order_by":9,"name":"Clarissa Paz","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Clarissa","middleName":"","lastName":"Paz","suffix":""},{"id":545856553,"identity":"f518208e-daae-4044-a708-340c9cee437e","order_by":10,"name":"Daniella Bastos","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Daniella","middleName":"","lastName":"Bastos","suffix":""},{"id":545856554,"identity":"b1a276ae-8e10-4afd-8451-870fa295e771","order_by":11,"name":"Diva Anelie Araújo Guimarães","email":"","orcid":"","institution":"Federal University of Pará (UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Diva","middleName":"Anelie Araújo","lastName":"Guimarães","suffix":""},{"id":545856555,"identity":"52561905-d0ba-4e85-8f32-931b2636eeec","order_by":12,"name":"Nilton Akio Muto","email":"","orcid":"","institution":"Federal University of Pará (UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Nilton","middleName":"Akio","lastName":"Muto","suffix":""},{"id":545856556,"identity":"8e951d6d-6145-4baf-be5d-2cdbec93cf18","order_by":13,"name":"Moisés Hamoy","email":"","orcid":"","institution":"Federal University of Pará (ICB-UFPA)","correspondingAuthor":false,"prefix":"","firstName":"Moisés","middleName":"","lastName":"Hamoy","suffix":""}],"badges":[],"createdAt":"2025-11-03 19:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8022097/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8022097/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96085836,"identity":"9d1202ad-4065-4537-b554-47ad94eabb58","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"tiff","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":418860,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2ABCDE.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/1c074ddad957157a6e8c62be.tiff"},{"id":96247597,"identity":"337d7ced-cd01-4d20-a54f-b8048f362266","added_by":"auto","created_at":"2025-11-19 07:27:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":796795,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/e74c495735324b2b29a1e8e0.docx"},{"id":96248186,"identity":"af9f74d5-ccca-4f30-84a9-cddd2b21a364","added_by":"auto","created_at":"2025-11-19 07:28:08","extension":"tiff","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":396732,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3ABC.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/0220c46ae5e0f4d85ed8f111.tiff"},{"id":96247635,"identity":"6ba90c61-a292-4439-8d6c-ab2c7573300c","added_by":"auto","created_at":"2025-11-19 07:27:38","extension":"tiff","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":215514,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4ABCDEF.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/305d59b3c919abaf6ac228e0.tiff"},{"id":96248298,"identity":"723c9d78-2107-4db7-9de4-546cbdc8bff0","added_by":"auto","created_at":"2025-11-19 07:28:16","extension":"tiff","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34918,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4GH.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/006a10053a13dcdd2fd0edbb.tiff"},{"id":96249674,"identity":"f1d9765a-93cc-4631-a60f-5e23a05d9598","added_by":"auto","created_at":"2025-11-19 07:35:57","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32904,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4IJ.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/d3c13a4e66e9fd949d598333.tiff"},{"id":96085848,"identity":"560ab325-880d-4a75-9693-1f2192ab62c2","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"tiff","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":361532,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1AB.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/bb4ed17d2200af21a79c71a3.tiff"},{"id":96085847,"identity":"8a9e3a3e-c5c6-442f-93c1-ea3c21f6b9a5","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"json","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13769,"visible":true,"origin":"","legend":"","description":"","filename":"b8c331ee72c94b288285d5f324d0e279.json","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/6633ac4e5f3557b7e70df956.json"},{"id":96085844,"identity":"425b3d9f-c810-46b5-b2bd-2734ec81e912","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":80758,"visible":true,"origin":"","legend":"","description":"","filename":"b8c331ee72c94b288285d5f324d0e2791enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/996bef6c7f1b4cba5ca2eb31.xml"},{"id":96249789,"identity":"9aad97f4-5321-4341-bb79-85348122367f","added_by":"auto","created_at":"2025-11-19 07:36:14","extension":"tiff","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":418860,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2ABCDE.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/8ff7d237082fcd32b3391197.tiff"},{"id":96249416,"identity":"04b5d5ec-3f39-4a82-a583-045fc01c5c6b","added_by":"auto","created_at":"2025-11-19 07:33:29","extension":"tiff","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":396732,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3ABC.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/3cfa8916b65e758822f0737e.tiff"},{"id":96085859,"identity":"c986c2c3-d745-4316-b43f-4c106aa466d9","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"tiff","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":215514,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4ABCDEF.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/1369de3e8f7d6ed2fea8178e.tiff"},{"id":96247876,"identity":"5c6ab702-f192-4bac-8caa-50d7172c60fa","added_by":"auto","created_at":"2025-11-19 07:27:48","extension":"tiff","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":34918,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4GH.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/12884c52553009d3bda40514.tiff"},{"id":96249410,"identity":"81a3ec84-851b-484f-b5c5-595c094e162e","added_by":"auto","created_at":"2025-11-19 07:33:27","extension":"tiff","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":32904,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4IJ.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/bc8bf2c487861f756e251067.tiff"},{"id":96246843,"identity":"10313e03-f579-44ce-a696-c6c2f47a1996","added_by":"auto","created_at":"2025-11-19 07:26:46","extension":"tiff","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":361532,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1AB.tiff","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/333b9b2d075c841a13455ff7.tiff"},{"id":96249780,"identity":"282f74d2-f7c0-4ddf-a4de-fae3bb2f15ce","added_by":"auto","created_at":"2025-11-19 07:36:13","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128050,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/bdbedc36541e7d356699684f.jpeg"},{"id":96085841,"identity":"a307ff87-e108-4525-8447-1e1d30f7b173","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpeg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":170901,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/674ddc284186769f61aa70fd.jpeg"},{"id":96085850,"identity":"a00d19ee-3283-43a7-af16-198b4f2bb516","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169771,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/156ccf019c8561b32a448c6e.jpeg"},{"id":96085857,"identity":"407ea342-b977-49d7-8664-2140c691b655","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpeg","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140365,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/f737d6d21f94a2e40e9f2e6e.jpeg"},{"id":96085856,"identity":"03ad1a3c-ebf5-4ef8-8f5d-e592d267d85a","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpeg","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":76053,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/042eb4fdc7df2170866e6a62.jpeg"},{"id":96085867,"identity":"a7b00aec-af09-4d37-9632-9ea886bc7dd8","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68118,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/bb0f7e47c4796504f6ffb206.jpeg"},{"id":96085851,"identity":"d37e2f52-95fa-4e68-8b26-599faeb770f3","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83721,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2ABCDE.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/8c7a646837da37262d4149f5.png"},{"id":96249827,"identity":"acfc890b-907a-441f-8dc6-748491efa9fd","added_by":"auto","created_at":"2025-11-19 07:36:21","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59868,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3ABC.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/7df3ca0ff14c6fd9533c116a.png"},{"id":96246808,"identity":"4075bcc6-c8ed-4f9c-bd81-3df31608b028","added_by":"auto","created_at":"2025-11-19 07:26:43","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":53862,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4ABCDEF.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/3489aa3c0633a50e1c800877.png"},{"id":96085870,"identity":"6915be1d-470f-4281-9840-7382a3c3d6b8","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14098,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4GH.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/27eacbac5bd065c6e90de562.png"},{"id":96249425,"identity":"72a79db6-a2fc-4cf9-9091-270d0cd21c3b","added_by":"auto","created_at":"2025-11-19 07:33:31","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12378,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4IJ.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/4913d1cfcfbbf23caa2ebb28.png"},{"id":96085860,"identity":"4fd9f721-a924-4706-92f1-90b45418c82b","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68704,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1AB.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/548d3003b926feb5d6523ea1.png"},{"id":96085866,"identity":"3dc6b86c-c6f3-4496-b2b9-80fdff873900","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77779,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/378de9129496caf6bb84d326.png"},{"id":96085865,"identity":"09b868a9-4fc3-4a33-ae06-df52505e4127","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94538,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/79da0d4d44bd49f0aacbbf30.png"},{"id":96085861,"identity":"6a55c6d9-ec9b-410f-894a-a04e717408ce","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67361,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/fbe3c8fe961f9c98c4b82a21.png"},{"id":96249983,"identity":"fad12e58-6453-4f47-847b-e3659cec57b2","added_by":"auto","created_at":"2025-11-19 07:36:58","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":51443,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/c781b8fab5bdd030544616f9.png"},{"id":96249707,"identity":"19e8fed4-8e6f-495e-a1f3-ad74546ca4fc","added_by":"auto","created_at":"2025-11-19 07:36:04","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16241,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/4441af984dc3be9d5ab8fe44.png"},{"id":96085863,"identity":"70229779-c8cd-4780-8660-c17b2f060cbd","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15074,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/0d4f476a1a5b2462b0f64603.png"},{"id":96247378,"identity":"f1dd0083-f975-455e-8078-d002f77a6e07","added_by":"auto","created_at":"2025-11-19 07:27:25","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79433,"visible":true,"origin":"","legend":"","description":"","filename":"b8c331ee72c94b288285d5f324d0e2791structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/7de0ec919aad0f4e7705559e.xml"},{"id":96085868,"identity":"8f07bbc2-932b-4764-907e-85113bf771b8","added_by":"auto","created_at":"2025-11-17 12:24:36","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92168,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/fb097d0aa2ae4c9ac120df13.html"},{"id":96248357,"identity":"24bcde75-6753-45e7-a60e-a7c2ae00bb91","added_by":"auto","created_at":"2025-11-19 07:28:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43995,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral characteristics of \u003cem\u003eAnteos menippe\u003c/em\u003ebutterflies during anesthetic induction and recovery with isoflurane: butterfly with normal postural reflex (A); butterfly with loss of postural reflex and immobility (B).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/3909fe0627764c1880a56f27.jpg"},{"id":96085834,"identity":"b7fa1e40-2198-41c3-8c46-6352f2d09966","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":47136,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation for ECG recording in \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies. Butterfly restraint for electrode positioning (A); magnified view showing the first abdominal spiracle and the positions of the recording and reference electrodes (B); model of the electrode used and its characteristics for implantation (C); representative cardiac recording of \u003cem\u003eAnteos menippe\u003c/em\u003e (D); 5-second segment of the recording and a 0.12-second amplified trace of the ECG showing the ascending and descending phases of the spike, spike frequency per minute, spike amplitude (mV), interspike interval (ms), and spike duration (ms) (indicated by red arrows) (E). Signal amplification: 50,000×; characteristic signal observed on the monitor.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/dd4945ef09e1bce52825bfe7.jpg"},{"id":96085837,"identity":"89a3c954-efb8-45c1-b525-75fd4bc6e7b9","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46043,"visible":true,"origin":"","legend":"\u003cp\u003eElectrocardiographic trace of \u003cem\u003eAnteos menippe\u003c/em\u003ebutterflies recorded over 30 minutes, showing the segments analyzed every 10 minutes (red dashed lines) (A); spectrogram of energy distribution illustrating regions with different energy intensities during restraint stress (B); Linear power graph of cardiac activity recorded during anesthetic induction and recovery (C) (after ANOVA applied by Tukey's test; ***p \u0026lt; 0.001, n = 6).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/54adb2f0fc3c05d12eef0041.jpg"},{"id":96085838,"identity":"6c5ea8b6-51c9-4993-af92-bf917850172e","added_by":"auto","created_at":"2025-11-17 12:24:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":237905,"visible":true,"origin":"","legend":"\u003cp\u003eECG tracing patterns recorded over 30 min, analyzed in 5-min segments, with corresponding power distribution histograms up to 40 Hz: 0–300 s (A); 300–600 s (B); 600–900 s (C); 900–1200 s (D); 1200–1500 s (E); and 1500–1800 s (F). Graphs show mean spike frequency per minute (G), spike amplitude (mV) (H), interspike interval (ms) (I), and spike duration (ms) (J). Data were analyzed using ANOVA followed by Tukey's test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (n = 6).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/9fa10d2a990fb10bc558d595.jpg"},{"id":99321442,"identity":"fc08cf23-d7b5-4b02-a4e5-1fbfc0f8ddfd","added_by":"auto","created_at":"2025-12-31 16:39:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1025935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8022097/v1/1493eb8f-d957-4daf-91dc-a8d6378a1415.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cardiac activity and the influence of isoflurane on this parameter in Anteos menippe butterflies","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe knowledge of biological parameters of urban insect species is of great importance for assessing environmental quality and for establishing procedures aimed at the preservation of ecosystems (Born \u0026amp; Lima, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The order \u003cem\u003eLepidoptera\u003c/em\u003e, which includes butterflies and moths, presents a high diversity in Brazil (Prado \u0026amp; Zukovski, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and has significant ecological importance, as these insects are highly sensitive to alterations in their habitat (Dessuy \u0026amp; Morais, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Within this context, an important group of this order is the Pieridae butterflies, which exhibit migratory behavior and are geographically distributed in temperate climates, particularly in tropical regions of South America (Monteiro et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMany species within this family act as pollinators. \u003cem\u003eAnteos menippe\u003c/em\u003e belongs to this family, within the subfamily Coliadinae, and is characterized by predominantly yellow and orange coloration (Monteiro et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Due to the high environmental fragility of these insects, several practices must be adopted to maximize their welfare during handling and transportation, both in captive breeding programs and in scientific experimentation.\u003c/p\u003e\u003cp\u003eIn this regard, anesthesia in invertebrates is an important process that facilitates manipulation for examinations, sampling, and transport, reducing stress and ensuring animal welfare (Cooper, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Traditionally, insect immobilization has been performed through exposure to cold or carbon dioxide (CO₂) (Nilson; Sinclair; Roberts, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, related studies indicate that exposure to these agents may cause long-lasting adverse effects on insect physiology (Gooley \u0026amp; Gooley, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is therefore essential that the anesthetic process does not alter the physiological parameters under study. Thus, the use of volatile anesthetic agents, such as isoflurane, emerges as an effective alternative for managing these organisms.\u003c/p\u003e\u003cp\u003eIsoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane) primarily acts as a positive modulator of GABA-A receptors, increasing chloride ion permeability and inducing neuronal hyperpolarization. These mechanisms promote general anesthesia with analgesic, hypnotic, and muscle-relaxant effects. It was employed in this study due to its ability to provide rapid anesthetic induction, predictable recovery, and a lower potential to cause prolonged stress. Its use in insects has been explored as an efficient approach to facilitate experimental management of these animals (Macmillan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom this perspective, electrophysiology can be used to assess the anesthetic state of animals under restraint stress, providing insights into the bioelectrical processes regulating cardiac and neuromuscular function both during physical restraint and under the anesthetic effects of isoflurane. In this way, it is possible to analyze how electrical impulses are generated and conducted in the cardiac system (Berul et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). These mechanisms are evaluated using electrocardiography (ECG), which records the electrical signals produced by cardiomyocytes. When electrodes are positioned adjacent to the heart, the ECG detects these impulses and converts them into graphical tracings, allowing the assessment of rhythm, frequency, and other features of cardiac function (Berkaya et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite significant advances in insect neurophysiology, the understanding of cardiac electrophysiology in invertebrates remains limited, particularly regarding the direct recording and interpretation of ECG activity. Most available data rely on indirect measurements of hemolymph flow or optical methods, which provide restricted insight into the dynamics of electrical conduction and autonomic modulation of the insect heart (Sl\u0026aacute;ma, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Davis \u0026amp; Orakzai, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Although the insect cardiac system is known to exhibit rhythmic contractions regulated by both myogenic and neurogenic mechanisms, the electrophysiological basis of these events has been poorly characterized compared to vertebrate models (Berul et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Wang \u0026amp; Chahl, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Moreover, studies integrating anesthesia and electrophysiological monitoring are scarce, and there is a notable lack of standardized protocols for ECG acquisition in Lepidoptera species. Filling this knowledge gap is essential to establish a physiological framework for assessing how anesthetics and stress conditions modulate cardiac function in insects, enabling comparative analyses of autonomic regulation and evolutionary conservation of neural control pathways across taxa (MacMillan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aimed to evaluate the use of isoflurane for anesthesia in \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies, describing the behavioral responses during anesthetic induction and recovery and, subsequently, evaluating the electrophysiological tracings related to cardiac function during anesthesia and anesthetic recovery.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eThis study used six \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies (males and females), recently emerged adults, obtained from a breeding facility located in the Environmental Park Mangal das Gar\u0026ccedil;as, Bel\u0026eacute;m, Par\u0026aacute;, Brazil. The butterflies were transported to the Laboratory of Pharmacology and Toxicology of Natural Products, Institute of Biological Sciences, Federal University of Par\u0026aacute; (ICB-UFPA), in a temperature-controlled environment (25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of animals for experimentation\u003c/h2\u003e\u003cp\u003eThe butterflies were initially anesthetized with isoflurane. A volume of 1 mL was impregnated into cotton and placed inside an anesthetic chamber with a capacity of 1500 cm\u0026sup3; at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C. The latency to loss of postural reflex and immobility was recorded. Subsequently, each butterfly was removed from the isoflurane chamber and transferred to an anesthetic-free environment, where the recovery time was recorded, defined by the return of the postural reflex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003e(A and B) here\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Electrode fabrication, implantation, and ECG recording\u003c/h2\u003e\u003cp\u003eFor electrocardiographic (ECG) recording, electrodes were constructed from JST SM 2-pin jack cables, 13 cm in length. Paired electrodes were spaced 1 mm apart and built from nickel-chromium wire (Morelli Orthodontics), with 0.2 mm diameter and 2 mm length (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The electrodes were insulated with liquid insulator.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe butterflies were restrained using paper strips and pins (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). After fixation, the electrodes were positioned according to the following coordinates: along the median sagittal line of the dorsal surface, adjacent to the first abdominal spiracle after the thoracic region, with a lateral displacement of \u0026plusmn;\u0026thinsp;1 mm and a dorsoventral depth of 2 mm (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The recording electrode was positioned on the right side and the reference electrode on the left. After electrode implantation, electrocardiograms were recorded (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eThe method of Contrera et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) was adapted with modifications, according to the equipment and materials available in this study.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003e(A, B, C, D and E) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll procedures were performed inside a Faraday cage with metallic shielding. The electrodes were connected to a high-impedance amplifier (Grass Technologies P511, West Warwick, USA) with a signal amplification of 50,000\u0026times;, monitored by an oscilloscope (Protek, 6510). Each butterfly was recorded for a duration of 30 minutes. The animals remained in contact with isoflurane for 5 minutes for anesthetic induction, and recovery from anesthesia was subsequently observed and monitored by ECG during the remaining 25 minutes. Data were analyzed every 5 minutes of recording, as follows: 0-300s (A), 300-600s (B), 600-900s (C), 900-1200s (D), 1200-1500s (E), and 1500\u0026ndash;1800 (F). The recordings allowed evaluating the following parameters: peak frequency per minute, peak amplitude, inter-peak interval, peak duration, and signal power.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). A significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was considered for all analyses. Comparisons between periods were performed using two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. Statistical analyses were carried out using GraphPad Prism, version 8 (GraphPad Software Inc., San Diego, CA, USA). Normality and homogeneity of variances were tested using the Kolmogorov\u0026ndash;Smirnov and Levene tests, respectively.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eDuring anesthetic induction with isoflurane at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026deg;C, a gradual deepening of anesthesia was observed, characterized by the loss of postural reflex and immobility, with a latency of 48.50\u0026thinsp;\u0026plusmn;\u0026thinsp;8.98 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Anesthetic recovery occurred upon removing the butterflies from the isoflurane chamber, with the return of the postural reflex observed after 117.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.21 s. No excitability-related behaviors were detected during the recovery period, and anesthetic lightening occurred gradually and smoothly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the physical restraint method applied to \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies for anesthetic induction with isoflurane (A), the electrode insertion site for signal acquisition (B), and the characteristics of the electrodes manufactured for the experiment (C). Representative ECG recording showing the firing of impulses that maintain cardiac rhythm (D), characterized by peaks with ascending and descending phases, as observed under amplified recording (E).\u003c/p\u003e\u003cp\u003eThe electrocardiogram illustrating the cardiac function of \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies during the first 5 seconds of recording under restraint stress without anesthesia showed a mean peak frequency of 61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.14 per minute, with an amplitude of 2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.466 mV (50,000\u0026times; signal amplification). The mean inter-peak interval was 966\u0026thinsp;\u0026plusmn;\u0026thinsp;86.79 ms. The contraction cycle responsible for driving the circulatory components, represented by the peak duration, had an average value of 31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26 ms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eECG recordings of \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies during anesthetic induction and recovery with isoflurane performed for 30 minutes, showing variations in the amplitude of the graphoelement signal. From each recording, 5-minute segments were extracted for analysis to compare the effects of anesthesia on hemolymph pumping. During the first 5 minutes (0-300 s), low recording amplitude was observed, followed by a progressive increase in amplitude in the period 300\u0026ndash;600 s, while the amplitude remained stable during the periods 600\u0026ndash;900 s, 900\u0026ndash;1200 s, 1200\u0026ndash;1500 s, and 1500\u0026ndash;1800 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Spectrogram analysis showed an increase in energy levels during the 300\u0026ndash;1800 s periods, which represent anesthetic recovery, demonstrating the effect of the anesthetic on butterfly hemodynamics during anesthesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDuring the 30-minute cardiac recording, differences in the mean linear power of the signals were observed. In the 0-300 s period, the mean linear power was 0.114\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;, similar to the 300\u0026ndash;600 s period (p\u0026thinsp;=\u0026thinsp;0.993), but lower than in the other groups. The control group was 0.210\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3; was similar to periods C (p\u0026thinsp;=\u0026thinsp;0.998) and D (p\u0026thinsp;=\u0026thinsp;0.166), but was lower than group E and F. Periods C (0.201\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) was similar to period D (p\u0026thinsp;=\u0026thinsp;0.0571), but was lower than period E and F. Periods D (0.252\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031 mV\u0026sup2;/Hz \u0026times; 10⁻\u0026sup3;) E and F were similar (p\u0026thinsp;=\u0026thinsp;0.119)(3C), demonstrating that in the anesthetic recovery period there was a progressive increase in signal power throughout the recording.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003e(A, B and C) here\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEach change in the ECG tracings during anesthetic induction and recovery in periods A, B, C, D, E, and F was analyzed as a pattern of cardiac activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;F). For period A, the mean peak frequency was 35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28 per minute, with well-defined peaks showing positive deflections, and the histogram indicated the highest concentration of energy between 3 and 4 Hz with good distribution up to 25 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In interval B, a change in peak direction was observed, with negative deflections predominating; the peak frequency increased to 41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.85 per minute, and the histogram showed maximum power at 4 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In period C (600\u0026ndash;900 s), maintenance in signal amplitude was observed, with a peak frequency of 45.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 per minute, exhibiting positive and negative deflections, and the histogram showed peak power at 3 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). During period D (900 to 1200 s), the peak frequency was 59.67\u0026thinsp;\u0026plusmn;\u0026thinsp;4.63 per minute, with positive and negative peaks and a predominance of 3 Hz in the histogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). For period E (1200-1500s) the frequency was 60.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 per minute, with a predominance in the frequencies of 3 to 6 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and Period F (1500-1800s) presented an average peak frequency of 64.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.88 per minute with a predominance in the frequency range between 2\u0026ndash;4 Hz (Figure F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe frequency of peaks varied throughout the anesthetic induction and recovery periods. The control group presented a mean frequency of 61.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.14 per minute, which was similar to periods D (p\u0026thinsp;=\u0026thinsp;0.997), E (p\u0026thinsp;=\u0026thinsp;0.999), and F (p\u0026thinsp;=\u0026thinsp;0.806), but was higher than periods A, B, and C. Period A (35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 per minute) was similar to period B (p\u0026thinsp;=\u0026thinsp;0.194). Period B (41\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8 per minute) was similar to period C (p\u0026thinsp;=\u0026thinsp;0.646), but was lower than the subsequent periods. Periods D, E, and F were similar (p\u0026thinsp;=\u0026thinsp;0.471) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eThe signal amplitude increased throughout recovery. For the control group, the mean amplitude was 2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 mV, similar to periods A (p\u0026thinsp;=\u0026thinsp;0.762), B (p\u0026thinsp;=\u0026thinsp;0.996), C (p\u0026thinsp;=\u0026thinsp;0.482), D (p\u0026thinsp;=\u0026thinsp;0.969) and E (p\u0026thinsp;=\u0026thinsp;0.563). However, it was lower than period F. Period A (1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 mV) was similar to periods B (p\u0026thinsp;=\u0026thinsp;0.400) and D (p\u0026thinsp;=\u0026thinsp;0.240), however, it was lower than the other periods (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eFor the inter-peak intervals, the control presented a mean of 963\u0026thinsp;\u0026plusmn;\u0026thinsp;83.79 ms, which was lower than periods A, B and C, but was similar to periods D (p\u0026thinsp;=\u0026thinsp;0.992), E (p\u0026thinsp;=\u0026thinsp;0.999) and F (p\u0026thinsp;=\u0026thinsp;0.998). Period A (1718\u0026thinsp;\u0026plusmn;\u0026thinsp;163.8 ms) was higher than all other intervals. Period B (1479\u0026thinsp;\u0026plusmn;\u0026thinsp;158.8 ms) was similar to period C (p\u0026thinsp;=\u0026thinsp;0.411). Period D (1010\u0026thinsp;\u0026plusmn;\u0026thinsp;77.62 ms) was similar to periods E (p\u0026thinsp;=\u0026thinsp;0.999) and F (p\u0026thinsp;=\u0026thinsp;0.898) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eThe peak duration increased during the anesthesia period. The control had a mean peak duration of 31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26 ms, which was lower than periods A and B, but was similar to groups C (p\u0026thinsp;=\u0026thinsp;0.0512), D (p\u0026thinsp;=\u0026thinsp;0.231), E (p\u0026thinsp;=\u0026thinsp;0.953), and F (p\u0026thinsp;=\u0026thinsp;0.968). Periods A (66\u0026thinsp;\u0026plusmn;\u0026thinsp;11.22 ms) were similar to period B (p\u0026thinsp;=\u0026thinsp;0.086), but were higher than the other periods. Period B (53.33\u0026thinsp;\u0026plusmn;\u0026thinsp;10.39 ms) was similar to periods C (p\u0026thinsp;=\u0026thinsp;0.4938) and D (p\u0026thinsp;=\u0026thinsp;0.1503). Periods C, D, E, and F were similar (p\u0026thinsp;=\u0026thinsp;0.319) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003e(A, B, C, D, E, F, G, H and I) here\u003c/b\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study represents the first attempt to investigate the effects of isoflurane, along with electrocardiographic assessment, during anesthetic induction and recovery in \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies. The anesthetic produced the expected results, inducing sedation with gradual deepening, as evidenced by the loss and subsequent recovery of postural reflex. The butterflies did not exhibit behaviors indicative of excitability caused by the anesthetic, demonstrating a positive outcome with gradual and smooth induction.\u003c/p\u003e\u003cp\u003eAlthough specific studies on butterfly species are lacking, isoflurane has a well-established use in invertebrate anesthesia and is considered superior to traditional methods such as cold exposure or CO₂ (MacMillan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Its sedative efficacy has been reported in other species, including button-wing mantises (\u003cem\u003eParasphendale agrionina\u003c/em\u003e), emperor scorpions (\u003cem\u003ePandinus imperator\u003c/em\u003e), and yellow uru\u0026ccedil;u bees (\u003cem\u003eMelipona flavolineata\u003c/em\u003e), showing mild induction and positive prognosis without observable adverse effects, consistent with our findings (D\u0026rsquo;Ovidio, Monticelli, \u0026amp; Adami, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gaudette et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Contrera et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, isoflurane represents a highly effective and valuable anesthetic for invertebrates, with applications in scientific, commercial, and veterinary contexts.\u003c/p\u003e\u003cp\u003eThe GABAergic system is highly conserved among insects (Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Sedation is mediated by activation of the GABA system, leading to general inhibition of the central nervous system, resulting in reduced motor activity and loss of sensitivity to stimuli via mechanisms like those observed in vertebrates under anesthetic action. According to McClure and Heberlein (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), these effects suggest that GABAergic modulation is an evolutionarily conserved pathway that regulates behaviors associated with torpor or deep rest, particularly during physiological processes such as diapause.\u003c/p\u003e\u003cp\u003eStudies in \u003cem\u003eDrosophila\u003c/em\u003e have shown that GABAergic activity regulates the release of neuropeptides, such as corazonin (Crz), which influence reproductive cycle behaviors and rest states, including the control of diapause phases. These findings suggest that GABAergic modulation can impact physiological and behavioral processes associated with sedation, providing a conceptual basis for hypotheses regarding the physiological responses of \u003cem\u003eAnteos menippe\u003c/em\u003e to sedatives. Additionally, the spatial distribution of GABA receptor subunits in the brain may explain differential sensitivity of various neural centers to sedative drugs (Yu et al., 2010).\u003c/p\u003e\u003cp\u003eThis study represents the first direct electrocardiographic evaluation of butterflies, focusing on the effects of isoflurane during anesthetic induction and recovery in Anteos menippe. It is important to note that the peaks observed in insects are not functional equivalents of the human QRS complex, although they can be used for physiological analogies. Our findings are consistent with those reported by Davis \u0026amp; Orakzai (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who documented mean resting heart rates of 63 bpm in butterflies, supporting the physiological congruence between lepidopterans from different biomes. Furthermore, the characterization of electrophysiological phases in butterflies aligns with observations in other arthropods, as reported by Sl\u0026aacute;ma et al. (2012), revealing homologous patterns to the cardiac complexes of arthropods when compared to vertebrates and reinforcing the evolutionary conservation of autonomic nervous system mechanisms in cardiac contraction. Methodologically, our study complements the approach proposed by Wang \u0026amp; Chahl (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), highlighting the value of high-resolution electrophysiological monitoring to ensure animal welfare and detailed spectral analysis.\u003c/p\u003e\u003cp\u003eRegarding the spectrographic analysis, the increase in spike frequency over time, associated with postural recovery after anesthesia, suggests a physiological behavior similar to that observed in bees by Contrera et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), indicating a shortening of the interspike interval during the recovery period. Furthermore, oscillations in amplitude and energy over time may reflect changes in cardiac activity after anesthetic removal, indicating alterations in cardiac electrical conduction and hemodynamic effects. Overall, these results demonstrate that the cardiac activity of A. menippe follows universal patterns of chronotropic variability described in insects, while also extending the physiological framework to a broader range of species.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this study demonstrate, for the first time, that isoflurane is an effective and safe anesthetic agent for managing Anteos menippe butterflies, providing gradual induction, predictable recovery, and the absence of undesirable excitatory effects. Electrocardiographic characterization performed during anesthetic induction and recovery revealed physiological patterns consistent with autonomic modulation previously described in other insects, demonstrating that the cardiac system of this species follows conserved mechanisms of chronotropic variability. The progression of electrical responses, reflected in changes in the frequency, amplitude, and energy distribution of the recorded peaks, reinforces the applicability of the ECG as a monitoring tool in invertebrates, expanding the possibilities for investigating physiological parameters in experimental and conservation contexts. Thus, this work not only contributes to the refinement of anesthetic practices in lepidopterans, but also establishes a relevant methodological basis for future studies aimed at understanding cardiac physiology in insects, strengthening the scientific framework necessary for the preservation and well-being of these organisms in laboratory and management environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eCRediT authorship statement\u003c/h3\u003e\n\u003cp\u003eConceptualization and Methodology: Ant\u0026ocirc;nio Bas\u0026iacute;lio Guerreiro J\u0026uacute;nior, Deise de Lima Cardoso, Nilton Akio Muto and Mois\u0026eacute;s Hamoy. Performed the experiments: Ant\u0026ocirc;nio Bas\u0026iacute;lio Guerreiro J\u0026uacute;nior, Deise de Lima Cardoso and Mois\u0026eacute;s Hamoy. Writing-original draft and editing: Axell Lins, Sabrina Reika Seko Kondo, Eduardo Machado da Silva Zamian, Alexa Camila Lopes de Assis, Diogo Macola Felix, Eva Vit\u0026oacute;ria Ferreira Viana, Daniella Bastos de Ara\u0026uacute;jo, Clarissa Ara\u0026uacute;jo da Paz and Diva Anelie de Ara\u0026uacute;jo Guimar\u0026atilde;es. Financial support and administrative support: Mois\u0026eacute;s Hamoy. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical trial number\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study was experimental with fish and does not involve clinical trials on humans or companion animals, therefore is not applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cp\u003eNo human participants were involved; the study was conducted under institutional ethics approval (CEUA/UFPA), which already covers the consent requirement, therefore it is not applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent publish\u003c/h2\u003e\u003cp\u003e All authors have read and approved the final version of the manuscript and consent to its publication.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent to Participate\u003c/h2\u003e\u003cp\u003eThis study did not involve human participants. As the experimental model consisted exclusively of invertebrates (\u003cem\u003eAnteos menippe\u003c/em\u003e butterflies), no institutional ethics approval or individual consent was required. Therefore, this item is not applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eDAS statement request\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are available in the public repository at the following link: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing\u003c/span\u003e\u003cspan address=\"https://drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The datasets support the findings of this study and include the original electrophysiological and behavioral data.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003ePublisher\u0026rsquo;s note\u003c/h2\u003e\u003cp\u003e All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization and Methodology: Ant\u0026ocirc;nio Bas\u0026iacute;lio Guerreiro J\u0026uacute;nior, Deise de Lima Cardoso, Nilton Akio Muto and Mois\u0026eacute;s Hamoy. Performed the experiments: Ant\u0026ocirc;nio Bas\u0026iacute;lio Guerreiro J\u0026uacute;nior, Deise de Lima Cardoso and Mois\u0026eacute;s Hamoy. Writing-original draft and editing: Axell Lins, Sabrina Reika Seko Kondo, Eduardo Machado da Silva Zamian, Alexa Camila Lopes de Assis, Diogo Macola Felix, Eva Vit\u0026oacute;ria Ferreira Viana, Daniella Bastos de Ara\u0026uacute;jo, Clarissa Ara\u0026uacute;jo da Paz and Diva Anelie de Ara\u0026uacute;jo Guimar\u0026atilde;es. Financial support and administrative support: Mois\u0026eacute;s Hamoy. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are available in the public repository at the following link: [https://drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing](https:/drive.google.com/file/d/1tXFolmPTa7sO4esOJahb4U2E6W1DuXz_/view?usp=sharing) . The datasets support the findings of this study and include the original electrophysiological and behavioral data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBerkaya SK, Uysal AK, Gunal ES, Ergin S, Gunal S, Gulmezoglu MB. A survey on ECG analysis. Biomed Signal Process Control. 2018;43:216\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bspc.2018.03.003\u003c/span\u003e\u003cspan address=\"10.1016/j.bspc.2018.03.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBerul CI, Aronovitz MJ, Wang PJ, Mendelsohn ME. In vivo cardiac electrophysiology studies in the mouse. Circulation. 1996;94:2641\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/01.cir.94.10.2641\u003c/span\u003e\u003cspan address=\"10.1161/01.cir.94.10.2641\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBorn FS, Lima IMM. Development stadia of \u003cem\u003eAnteos menippe\u003c/em\u003e (H\u0026uuml;bner) (Lepidoptera, Pieridae) on \u003cem\u003eCassia ferruginea\u003c/em\u003eShrad. (Caesalpinaceae), in laboratory. Rev Bras Entomol. 2005;49:522\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0085-56262005000400011\u003c/span\u003e\u003cspan address=\"10.1590/S0085-56262005000400011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eContrera FAL, Lopes BSC, Paz CA, Hamoy MKO, Santos MF, Barbosa GB, et al. First records of heartbeats via ECG in a stingless bee, \u003cem\u003eMelipona flavolineata\u003c/em\u003e (Apidae, Meliponini), during contention stress using isoflurane as an anesthetic. Insects. 2023;14:696. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects14080696\u003c/span\u003e\u003cspan address=\"10.3390/insects14080696\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCooper JE. Invertebrate anesthesia. Vet Clin North Am Exot Anim Pract. 2001;4:57\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1094-9194(17)30051-8\u003c/span\u003e\u003cspan address=\"10.1016/S1094-9194(17)30051-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavis AK, Orakzai S. A simple method for observing and measuring heart rates in live adult monarchs (\u003cem\u003eDanaus plexippus\u003c/em\u003e) and other large butterflies. J Lepid Soc. 2016;70:96\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18473/lepi.70i2.a3\u003c/span\u003e\u003cspan address=\"10.18473/lepi.70i2.a3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDessuy MB, Morais ABB. Diversidade de borboletas (Lepidoptera, Papilionoidea e Hesperioidea) em fragmentos de Floresta Estacional Decidual em Santa Maria, Rio Grande do Sul, Brasil. Rev Bras Zool. 2007;24:108\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0101-81752007000100014\u003c/span\u003e\u003cspan address=\"10.1590/S0101-81752007000100014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;Ovidio D, Monticelli P, Adami C. Isoflurane anesthesia in budwing praying mantises (\u003cem\u003eParasphendale agrionina\u003c/em\u003e). J Zoo Wildl Med. 2021;52:1013\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1638/2020-0195\u003c/span\u003e\u003cspan address=\"10.1638/2020-0195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGaudette C, Johnson B, Bakal R, Dombrowski DS. Hemolymph collection and isoflurane anesthesia of the emperor scorpion (\u003cem\u003ePandinus imperator\u003c/em\u003e). J Zoo Wildl Med. 2022;53:573\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1638/2020-0095\u003c/span\u003e\u003cspan address=\"10.1638/2020-0095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGooley ZC, Gooley AC. Metabolic effects of anesthetics (cold, CO₂, and isoflurane) and captivity conditions in isolated honey bee (\u003cem\u003eApis mellifera\u003c/em\u003e) foragers under different ambient temperatures. J Apic Res. 2021;62:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/00218839.2021.1950994\u003c/span\u003e\u003cspan address=\"10.1080/00218839.2021.1950994\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGuh YJ, Tamai TK, Yoshimura T. The underlying mechanisms of vertebrate seasonal reproduction. Proc Jpn Acad Ser B Phys Biol Sci. 2019;95:343\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2183/pjab.95.024\u003c/span\u003e\u003cspan address=\"10.2183/pjab.95.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi H, et al. A tomato nitrogen-dependent GABA pathway confers resistance to a globally invasive fruit fly. Front Plant Sci. 2023;14:1252455. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2023.1252455\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2023.1252455\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMacMillan HA, N\u0026oslash;rg\u0026aring;rd M, MacLean HJ, Overgaard J, Williams CJA. A critical test of \u003cem\u003eDrosophila\u003c/em\u003e anaesthetics: Isoflurane and sevoflurane are benign alternatives to cold and CO₂. J Insect Physiol. 2017;101:97\u0026ndash;106. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2017.07.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2017.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcClure KD, Heberlein U. A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in \u003cem\u003eDrosophila\u003c/em\u003e. J Neurosci. 2013;33:4044\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.3140-12.2013\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.3140-12.2013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMonteiro RF, Freitas AVL, Costa Filho MAF, Nascimento MS, Alves TG, Brown KS Jr, et al. Borboletas da Mata Atl\u0026acirc;ntica do Estado do Rio de Janeiro: Pieridae (Lepidoptera). Arq Mus Nac Rio J. 2009;67:283\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNilson TL, Sinclair BJ, Roberts SP. The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. J Insect Physiol. 2006;52:1027\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2006.07.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2006.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrado LEPA, Zukovski L. A import\u0026acirc;ncia da conserva\u0026ccedil;\u0026atilde;o de lepid\u0026oacute;pteras para os processos ecol\u0026oacute;gicos. Rev Terra Cult Cad Ens Pesqui. 2012;28:69\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSl\u0026aacute;ma K. A new look at the comparative physiology of insect and human hearts. J Insect Physiol. 2012;58:1072\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2012.04.014\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2012.04.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWaldvogel HJ, Baer K, Faull RLM. Distribui\u0026ccedil;\u0026atilde;o de subunidades receptoras GABA no c\u0026eacute;rebro humano. In: Monti J, Pandi-Perumal S, M\u0026ouml;hler H, editors. GABA e Sono. Basel: Springer; 2010. pp. 45\u0026ndash;67. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-0346-0226-6_3\u003c/span\u003e\u003cspan address=\"10.1007/978-3-0346-0226-6_3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatanabe M, Fukuda A, Nabekura J. The role of GABA in the regulation of GnRH neurons. Front Neurosci. 2014;8:387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnins.2014.00387\u003c/span\u003e\u003cspan address=\"10.3389/fnins.2014.00387\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang D, Chahl J. Extracting cardiac activity for arthropods using digital cameras: Insights from a pilot study. Arch Insect Biochem Physiol. 2025;119:e70076. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/arch.70076\u003c/span\u003e\u003cspan address=\"10.1002/arch.70076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anteos menippe, isoflurane, electrocardiography, cardiac physiology, autonomic modulation, invertebrate anesthesia","lastPublishedDoi":"10.21203/rs.3.rs-8022097/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8022097/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the physiological mechanisms regulating cardiac activity in insects remains a fundamental challenge in comparative physiology. This study investigated the effects of isoflurane anesthesia on the electrocardiographic (ECG) activity of \u003cem\u003eAnteos menippe\u003c/em\u003e butterflies during induction and recovery. Six newly emerged adults were exposed to 1 mL of isoflurane vapor, and the latency to loss and recovery of postural reflex was recorded. Nickel\u0026ndash;chromium electrodes were implanted in the abdomen to record ECG signals for 30 min in a Faraday-shielded setup. Isoflurane produced smooth and reversible anesthesia with induction and recovery times of 48.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9 s and 117.7\u0026thinsp;\u0026plusmn;\u0026thinsp;16.2 s, respectively, without excitatory responses. During anesthesia, spike frequency decreased to 35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 min⁻\u0026sup1;, returning to 64.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 min⁻\u0026sup1; during recovery, accompanied by progressive amplitude and power increases. Spectral analysis revealed dynamic energy redistribution associated with chronotropic modulation during anesthetic elimination. These findings provide the first direct ECG characterization in Lepidoptera, demonstrating that isoflurane is a safe and effective anesthetic and that the butterfly cardiac system follows conserved electrophysiological mechanisms among insects.\u003c/p\u003e","manuscriptTitle":"Cardiac activity and the influence of isoflurane on this parameter in Anteos menippe butterflies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 12:24:30","doi":"10.21203/rs.3.rs-8022097/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d13fd820-ae03-4da8-8648-9c1caf1502b3","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-31T13:54:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 12:24:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8022097","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8022097","identity":"rs-8022097","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}