Long-term exposure to extreme illumination regimes alters behavioral responses to light in the cockroach, Periplaneta americana L

Long-term exposure to extreme illumination regimes alters behavioral responses to light in the cockroach, Periplaneta americana L | 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 Long-term exposure to extreme illumination regimes alters behavioral responses to light in the cockroach, Periplaneta americana L Ekaterina S. Novikova, Marianna I. Zhukovskaya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6519676/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 The natural lighting conditions vary depending on latitude, niche and time of day; the animals are evolutionarily adapted to them. Artificial lighting along with global warming drive population ranges toward high latitudes, which creates fast-changing environments for the biota. The American cockroach is a synanthropic species with nocturnal lifestyle, rarely exposed to light. Three-month long exposure to constant light or constant darkness, in comparison with normal 12:12 day and night cycle, causes behavioral changes that is explained by two main factors: adaptation of visual system, and circadian rhythm disturbance. Freezing behavior, an indicator of circadian disturbances, appeared in groups kept under constant light regimes ant tested in the dark, as well as those subjected to experimental lighting with low intensity green light. Exposure to such light caused multidirectional behavioral changes in groups kept in different light regimes, reflecting their internal levels of arousal, stress, and light adaptation of their photoreceptor organs. Thus, altered lighting conditions impose significant challenges to different aspects of insect physiology and behavior. insect Periplaneta americana behavior light responses circadian rhythm Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Long-living animals have more chances to experience severe environmental alteration during their lifetime in comparison with short-living ones. Although some species developed genetically coded adaptations, such as diapause and aestivation, others demonstrate greater phenotypic and behavioral plasticity (Ganguly and Candolin 2023). Insects can successfully survive not only cold or dry and hot period in dormant states, but also occasional drowning (Rondeau and Raine 2024 and references therein) and other situations with oxygen deficits (Hoback and Stanely 2001). Active stages are supposed to be more susceptible to adverse conditions. One of the main factors of insect life is illumination, which regulates circadian and seasonal cycles, but also has direct action on their organisms. For example, for Drosophila eggs and pupae blue light was highly toxic: the majority of eggs died after 48-h irradiation at 5.0 * 3 10 18 photons/m 2 /s, whereas most eggs hatched under the dark; larvae were susceptible as well (Hori et al. 2014). In most of the cases the light acts through light-sensitive sensory organs, such are compound eyes, ocelli, stemmata and extraretinal eyelets (Hofbauer and Buchner 1989; Oakley et al. 2007; Friedrich et al. 2006; Land and Chittka 2013; Severina et al. 2024). This multicomponent visual system provides an insect with highly valuable information for use in spatial orientation, mate and food finding and recognition, predator avoidance, as well as circadian entrainment. Photoreceptor organs are plastic in their responses to light. Relatively short-term exposure (from seconds to minutes) to light of particular intensity and wavelength causes temporal changes in sensitivity of photoreceptor cells due to adaptation (Laughlin and Hardie 1978; Matić and Laughlin 1981; Burton 2002). Mechanisms of light adaptations are well known: temporal structural changes (Meyer-Rochow 1974; Gribakin 1979; Belušič et al. 2017) and adjustments in phototransduction cascade, such as Ca 2+ feedback, activity modulation of a number of enzymes and second messengers (Sandler and Kirschfeld 1991; Abbas and Vinber, 2021). Further neural processing adjusts the signals to get the most important information about visual surroundings (Hempel de Ibarra et al. 2014; Ketkar at al. 2023) depending on the context of internal state and self-motion (Longden et al. 2014; Kohn et al. 2018; Hindmarsh Sten et al. 2021). Longer exposures affect the biological clock and cause, in addition to above-mentioned, some other phenomena, such as masking, the reaction to unexpected fast change in ambient light conditions, which elicited behaviors characteristic for the opposite phase of circadian cycle (Aschoff 1960; Mrozovsky 1999). Fly larvae raised under different photoregimes from 0 to 24 h of light demonstrated changes in the total dendritic length of LN neurons, postsynaptic to photoreceptor of Bolwig's organ, the larval eye (Yuan et al. 2011). Honey bees are evolutionarily preadapted to the dramatic change in light exposure on transition between nursing inside the hive and foraging outside. Light exposure of nurse bees causes decrease in mushroom body microglomerulus density and increase in juvenile hormone level, showing changes similar to untreated foragers of older age (Scholl et al. 2014). Animals inhabiting high latitudes often show behavioral arrhythmicity. Larvae and adults of Antarctic midges, Belgica antarctica Jacobs , demonstrate around the clock activity at the constant temperature, regardless of the photoregime. Their clock genes, such as period , timeless , Clock , and vrille expressed no cycling in any of the experimental photoregimes (Kobelkova et al. 2015). The high-latitude fly, Drosophila lummei , shows rhythmicity under the constant light conditions, while its cosmopolitan and holotropical relatives, such as D. melanogaster , D. mercatorum, D. hydei, Z. indianus and Z. camerounensis become arrhythmic (Beauchamp et al. 2018). Chymomyza costata (Diptera: Drosophilidae) from Finland lacks circadian rhythmicity in constant darkness, whereas most known species, including the closely related Drosophilidae from temperate and tropical latitudes, demonstrate free-running along with structural changes of clock neurons network. (Bertolini et al. 2019; Meier et al. 2024). Cave dwellers, on the contrary, living in almost complete darkness, are characterized by partial or total reduction of their visual systems that mostly correlates with the loss of rhythmicity (Friedrich 2013; Heads 2010; Rampini et al. 2008; Sendi et al. 2020). Nevertheless, cave-dwelling glowworms Arachnocampa tasmaniensis (Merritt and Clarke 2011) retain visual system and light-entrainable circadian rhythms with a period of about 24h, but populations from different caves are shifted by several hours. Thus, long periods of daylight, as well as an almost complete darkness are not totally artificial conditions for insects on Earth, although most tropical insects, such as cockroaches, do not experience such conditions in their life. However, evolutionary history of cockroaches and related groups is traced from Cretaceous period, which was characterized by warm climate around the globe, including polar areas (Skelton et al. 2003) and ancient cockroaches inhabited areas with polar day and night (Grandcolas 1999). The American cockroach, Periplaneta americana L. usually avoids lit area and shows escape reactions by incident illumination (Bell and Adiyodi 1982; Okada and Toh 1998). Its visual system has two pairs of photosensory organs – compound eyes and ocelli. The compound eyes consist of ommatidia with photoreceptor cells of two spectral classes – greenlight sensitive (broadband with max 560 nm) and UV-sensitive (365 nm); while ocelli – presumably of only one class of green-sensitive photoreceptors (Goldsmith and Ruck 1958; Mote and Goldsmith 1970). Outstanding adaptability and plasticity has allowed the cockroaches to inhabit human buildings and yards (Johnson and Munshi-South 2017; Gondhalekar et al. 2021), where lighting conditions vary from bright light to almost complete darkness, and the modes of illumination can change unpredictably. The present paper is aimed to study changes in behavioral responses to light stimuli of the cockroaches kept under different illumination regimes. Materials and methods Insects. Male nymphs of P. americana L. were separated from the lab colony and kept under 3 regimes for 3 moths: 12:12 LD; 24 h dark; 24 h light, temperature 22-27 °C and humidity 60-90%. Animals were fed ad libitum (bread, oatmeal, cabbage, dandelion leafs, dry cat food). Freshly molted adults were transferred to the experimental setup designed as Plexiglas cage (350 × 250 × 105 mm) connected to the always dark shelter (150 × 150 × 100 mm) under the same light conditions as described before (Novikova et al. 2017). All groups were subjected to thermoregime 26±2 °C during the day and 22±2 °C during the night. For the 12:12 LD group testing was started at the beginning of a dark phase, for other two groups – at the start of subjective night. Behavioral responses to low intensity green light (530 nm, 10 µW/cm 2 ) were monitored as described earlier (Zhukovskaya et al. 2017). Namely, an experiment would start by entering a cockroach to the test chamber (150 × 150 × 100 mm) from the home cage, after that the chamber with the insect was separated by the sliding Plexiglas door. After 10 min adaptation period, the first session (S1) of video recording was performed for 30 min under dim red light (690 nm, 0.13 µW/cm 2 ). The second session (S2) of recording started in 10 min after the end of the first one under green light illumination. Locomotor activity and grooming were assessed, as well as stops (no movements, antennae are waving) freezings – periods of complete immobility (Zhukovskaya 2014; Zhukovskaya et al. 2017). Following series of experiments were performed with special conditions in S2: 1. “Control LD”. Cockroaches maintained in 12:12 LD regime, no addition lighting (dark) was presented in the S2; 2. “Green LD”. Cockroaches maintained in 12:12 LD regime, green light was applied at the S2; 3. “Green, DD.” Cockroaches were maintained in the constant darkness, green light was applied at the S2; 4. “Green, LL”. Cockroaches were maintained in the constant light; green light was applied at the S2; 5. Additional observations were performed on the cockroaches maintained in 12:12 LD regime. The behavior of a group of males was monitored inside the darkened shelter 30 min after light “On” under infrared (850 nm) light with irradiance of 7.4 µW/cm 2 (“Daytime LD” series). Raw data of the series “Control LD” and “Green LD” were earlier used in the paper of Zhukovskaya et al. (2017). Locomotor activity was evaluated for the series 1-4 as follows: the image of the test chamber on the computer screen was divided into quadrants and the number of them crossed by cockroach for the session was counted. Grooming behavior data were processed as described earlier (Zhukovskaya 2014, Zhukovskaya et al. 2017), briefly, cleaning of body parts was timed, and grooming bout was defined as cleaning of several body parts without interruption by any other behavior. Frequencies and timing of stops (cockroach stands still, only antennae are moving) and freezings (no detectable movements) were calculated. Also, the share time for different activities was accessed. The data obtained were tested for normality with Kolmogorov-Smirnov test using online calculator (https://contchart.com/goodness-of-fit.aspx). Parametric statistics was used for normally distributed data (Student’s T-test, ANOVA, Tukey post-hoc pairwise comparisons http://vassarstats.net/); non-parametric tests were applied to data, whose distribution was significantly different from normal (Wilcoxon signed rank test, Mann-Whitney U-test (http://vassarstats.net/), Kruskal-Wallis Test, followed by Dunn’s post hoc pairwise comparisons (https://www.statskingdom.com/kruskal-wallis-calculator.html). Results First, the locomotor activity of the cockroach groups maintained in 12:12 LD was tested. Control group (dark red light in both sessions) and insects illuminated with green light at the S2 of the experiment did not differ: they both declined their walking levels between S1 and S2 (2-way ANOVA with repeated values, F 1/28 =22.39, p <0.0001, the data were mostly distributed normally). Three months of darkness did not alter cockroach locomotion in the S1, but 3 months long exposure to white light significantly decreased the activity (Mann-Whitney U-test with Bonferroni correction, p =0.04, Fig.1 a). Changes in locomotion under green light exposure in the S2 the experiments were unidirectional (decrease) for all series excluding that, in which cockroaches were kept under 24 h of light. Statistical evaluation of the differences calculated for sessions of each series (S1-S2) gives a number of significant values (one-way ANOVA, F 3/52 =7.3, p =0.000352, post hoc Tukey HSD Test returns significant values for pairs: control LD versus green LD, p <0.05; control LD versus LL, P<0.01; DD versus LL, p <0.05) (Fig. 1 b). Analysis of freezing frequencies was performed using non-parametric Kruskal-Wallis test, since all the data did not pass normality test. For the S1 cockroaches demonstrated significant amount of freezing only if they were kept in the constant light (green LL, Kruskal-Wallis Test, H 3 =10.439364, p =0.015178 followed by Dunn’s post hoc test) (Fig. 2). Rare freezings shown by cockroaches kept in the dark did not differ significantly from both of LD series. In the S2, freezing occurred most frequently for cockroaches kept in the dark (DD), significant differences were found with both series kept under LD regime (Kruskal-Wallis Test, H3=8.32; p =0.04, Dunn’s post hoc test, p <0.05). Frequency of freezing for light-kept insects was in between, differences with other groups were non-significant. Time spent frozen followed the same pattern (Kruskal-Wallis Test, H 3 =10.55; p =0.014, Dunn’s post hoc test, p <0.05); and the duration of the single freezing episode was similar for all treatments and was on average 73.8±16.5 s. Cockroaches kept in the dark stopped in response to green light in the S2 more frequently and spent more time resting, than those kept in the LD conditions (Fig. 3). Neither frequency of stops nor total duration of stops in the first sessions differed across all series (Kruskal-Wallis Test, p >0.05, Fig. 3). LD kept cockroaches demonstrated strong difference in time spent stopped between control LD and green LD series at the second sessions of the experiments (Kruskal-Wallis Test, H 3 =17.484712, p =0.000562, followed by Dunn’s post hoc comparisons, p =0.000145). As reported earlier, low intensity green light causes restlessness in the cockroaches (Zhukovskaya et al. 2017). Cockroaches exposed to constant light or dark rested about the same amount of time (Fig. 3; Dunn’s post hoc comparisons, p =0.153). Although total grooming time did not differ significantly between treatments and between two consecutive sessions (Two-way ANOVA, p >0.05 for both factors), but thorough evaluations of parameters of this behavior revealed some significant effects. Grooming patterns, reflecting the stress level (Kalueff et al, 2007; Root-Bernstein 2010; Zhukovskaya et al. 2013; Novikova and Zhukovskaya 2015), showed prolonged grooming bouts only for the cockroaches of LL series at the first session of experiments (Kruskal-Wallis Test, H 3 = 8.6929. p = 0.03366, Post hoc Dunn’s test returns p <0.05 for all groups in compare with LL, Fig. 4a). Second sessions did not differ significantly. Grooming bouts were found to be longer in the second session as compared to the first session for controls and DD kept insects (Wilcoxon test, p < 0.0349 and p < 0.0067, respectively). Grooming of various appendages changed differently between treatments, antennae were groomed longer in the S2 of control experiment ( p <0.01, Student’s t-test) and in DD series ( p <0.001, Student’s t-test), insects kept under LL showed equally high level of grooming in both sessions and those kept under LD maintained intermediate duration of antennal grooming (Fig 4c). Two-way ANOVA proved differences between series (F 3/99 =6.55, p =0.0004) and between sessions (F 1/99 =5.32, p =0.0232), interaction between factors was non-significant. Post hoc Tukey test revealed, that LL series differed from control (p p <0.01), as well as LD ( p <0.01), both of which testing cockroaches kept under normal 12:12 photoregime. Only DD animals showed a big difference between first and second sessions of experiment (Fig. 4c). Cockroaches exposed to LL cleaned their foreleg more slowly than under all other treatments (F 3/ 9=5.4, p =0.0018, Tukey post hoc with LD groups p <0.01, with DD p <0.05, Fig. 4d). All other parameters did not show any statistical significance. Midleg and hindleg grooming took longer at the second sessions (Two-way ANOVA, F 1/92 = 9.29, p = 0.003, Fig. 4e), mostly because of prominent differences in Green LD and DD series (post hoc Tukey, p <0.05 for green LD and p <0.01 for DD series). Since illumination during the night may cause masking effect – manifestation of behaviors characteristic of the light phase of the 24h cycle (Mrosovsky 1999), the additional experiments with the group of male cockroaches were performed to recognize the behavior inherent for the light part of the day (daytime LD). The most prominent behavior was freezing. Insects demonstrated more freezings than at any other series, the frequency was 8.4±0.6 per session (in compare with 3.89±1.1 in DD series, Fig. 4), average length of a freezing episode was 83.4±13.9 s, the total time of immobility – 398±47 s for 30 min observation period in compare with 283± 96 s in DD series. Since the experimental protocol was quite different, performing the direct statistical comparison is believed to be inadequate. Anyway, we can see, that frequent and long freezings are the hallmark of daytime behavior. Cleaning sweeps of particular body parts, such as antennae and legs, slows down (Fig 4 c-e), and the S1 of LL series was the closest in values to “daytime LD”. The number of cleaned body parts per one grooming sequence was equal to 6.8±1.6, which is closer to 5.2±0.6 in cockroaches of LL series than to 4.2±0.4 for the first session of combined 12:12 LD series and 4.35±0.56 for DD. The number of cleaned body parts was about the same for all series other than daytime LD. The longest grooming bouts were observed for the cockroaches at the beginning of the light phase in the shelter (daytime LD), but they always resided in well-known living quarters under the lowest stress level. Two-way ANOVA comparing four series (control LD, green LD. green LL and green DD) revealed significant differences between sessions (F 1/98 =7.95, p <0.01), as well as series (F 3/98 =5.41, p <0.01). Post hoc Tukey test showed that LL group was different from both LD series ( p <0.01), but not from DD. Student’s t-test for pairs revealed significant increase in bout time in the second session only for DD cockroaches ( p <0.05). Discussion Cockroaches kept in constant light conditions for 3 months before the test demonstrated behavior different from LD groups in the first sessions of the experiments, without illumination. The most distinctive behavior was demonstrated by light-kept insects; their locomotion was noticeably lower than of all other treatments, these cockroaches displayed prominent freezing behavior before the light was turned on, and their grooming was the slowest. Dark-kept insects behaved almost like 12:12 LD ones, namely they showed similar level of locomotion and grooming. (Figs 1, 4). Further analyzing cockroach behavior, the proportions of time allocated to each activity (activity budgets) were calculated (Fig.5). Only cockroaches of green LL series spent noticeable amount of time frozen in the S1. Freezings, as was shown earlier, are a sign of the masking effect, if they are observed under sudden illumination during the dark phase of the 24 h cycle (Novikova and Zhukovskaya 2017; Zhukovskaya et al. 2018; Novikova et al. 2021; Zhukovskaya et al. 2024) and are presumably a part of inactive daytime behavioral repertoire (see below). Second sessions of both DD and LL series were even more similar, which may be explained by direct effect of light. An earlier study demonstrated, that 3-months long exposure of the cockroaches to LL significantly decreased light responses of their green-sensitive photoreceptors in comparison with both DD and LD groups. Photoreceptor sensitivity to light was shown to be decreased in those cockroaches kept in the constant illumination (Frolov et al. 2018), which is in part attributed to well-known phenomenon of physiological and structural. adaptation (Wolken and Gupta 1961; Butler and Horridge 1973; Ferrell and Reitcheck 1993, Frolov et al. 2022). On the contrary, 3 months of DD regime strongly up-regulated the main green-sensitive opsin GO1 (but not the ultraviolet sensitive UVO) in the compound eyes of P. americana cockroaches (Frolov et al. 2018). Anticipated oppositely directed changes in eye sensitivity due to prolonged exposure to light or darkness should have resulted in oppositely directed changes in behavior, namely light-kept insects were supposed to rest a lot, similar with controls (Control LD), due to decrease in photoreceptor sensitivity (Frolov et al. 2018), and dark-kept ones should have shown responses like 12:12 LD reared insects exposed to brighter light (Zhukovskaya et al. 2017). Indeed, dark-kept animals demonstrated significant amount of stops (resting) and freezing at the expense of locomotion under low-intensity light in the S2 of the experiment, similar with responses of 12:12 LD cockroaches to high intensity light (Zhukovskaya et al. 2017, Fig. 2 b). Also difference in total freezing time clearly corresponded to subjective brightness of light stimulus (Fig. 6, Kruskal-Wallis Test, p =0.12, post-hoc Dunn’s test revealed differences between green DD and both control and LL, between bright green LD and both control and LL. Raw data of bright green LD series were taken from Zhukovskaya et al. (2017)). Insects of LL group demonstrated bidirectional changes in freezing time between sessions, giving average and median close to zero. Thus, the results of the present study suggest that the discrepancy between the expected and observed effects of different illumination regimes is a result of other factor(s) influencing cockroach behavior as opposed to changes in photoreceptor sensitivity. To solve this disagreement, we compared the behavior of all experimental groups in the first sessions of experiments, under dim red light. The first sessions of all the experiments with individual cockroaches reveal that freezings – periods of absolute immobility, appear only at the series, in which cockroaches were kept under constant light conditions. Together with the data on cockroach behavior in the shelter during the light phase of 12:12h LD regime (Daytime LD) becomes clear, that freezings are the attribute of inactive phase of circadian cycle (sleep). The biological clock can run with different speed, this species-specific and individual feature is determined by the internal circadian period τ (tau) and vary in the range of ~24±4 hours, which is largely predetermined by polymorphism of core clock and some other related genes (Dibner et al. 2010; Doi et al. 2011). In this study, cockroaches kept in the constant dark show only minor signs of circadian rhythm disturbance, and only one individual was on subjective day phase in our experiment, showing 6 episodes of freezing with total time of 269 s, likely because of the differences in τ between individuals (Lipton and Sutherland 1970; Bertossa et al. 2013; Shinkawa et al. 1994; Pivarciova et al. 2016). So, to check if the circadian state is solely responsible for the locomotor activity level, the cockroaches of LL series were regrouped according to the presence or absence of freezing behavior in the S1 of experiment, which was always conducted under dim red light. Interestingly, if a cockroach demonstrated freezing behavior at the S1, it froze at the S2 as well, and vice versa. The individual of DD series that froze, was excluded from the analysis (Fig. 7). As a result of such recalculations, LL cockroaches that did not freeze demonstrated practically identical locomotor activity with DD individuals, as under dark red light in the S1, as under green illumination at the S2. Those, who showed freezings crossed significantly less quadrants (two-way ANOVA, F 1/33 =5.79, p =0.029). Moreover, the walking speed, (quadrants/(time of locomotion, s)) showed the same pattern, namely, the walking speed was slowest for the cockroaches of LL series, that showed freezings (Kruskal-Wallis Test, p =0.0084, Dunns post hoc test revealed that LL subgroup that froze was significantly different from all other series, P<0.05, which did not differ from each other). Thus, locomotor activity level and walking speed mostly reflects the circadian phase of an insect, rather than the state of its photoreceptors. Interestingly, the cockroaches of the LL subgroup that showed freezings, demonstrated some but non-significant raise in the speed of locomotion at S2 (Wilcoxon Signed-Rank Test, p =0.0549, Fig. 7). Thermoregime in which the insects were kept (see “Material and Methods”) probably entrained DD cockroaches, but failed to do the same for LL group. It is clear when the data for the S1 was analyzed, namely parameters of behavior for DD cockroaches were similar with those for LD series for all but one cockroach, which demonstrated freezing in the first session. Temperature cycles are known to be a second robust Zeitgeber for behavioral rhythms of model and non-model insect, such as fruit fly D. melanogaster (Wheeler et al., 1993; Busza et al, 2007; Wolfgang et al, 2013), linden bug Pyrrhocoris apterus ( Kaniewska et al. 2020), cockroaches (Page. 1985) and crickets (Rence and Loher 1975). The direct effect of light adaptation or decreased photoreceptor sensitivity due to structural changes was not clearly recognized in our experiments with LL cockroaches likely due to arrythmicity caused by constant light exposure (Konopka et al. 1989; Hong and Saunders 1994; Saunders and Cymborowski 2008). In any case, cockroaches from LL subgroup, that froze stopped more frequently (Mann-Whitney U-test, p <0.05) and longer (Mann-Whitney U-test, p <0.05) then those that did not freeze under green light stimulation at the S2 (Fig. 8. Total time of stops). Resting behavior of non-freezing cockroaches of LL series was similar with control LD insects not exposed to light. The level of stress, evaluated by the shortening of grooming sequences (Zhukovskaya 2014) depended on rearing conditions, namely, cockroaches kept in the dark (DD) were comparable with control (LD) animals, not exposed to light (Fig. 4 b), but light kept (LL) cockroaches demonstrated sequence length similar with LD ones under experimental light of the same intensity. Bright light was shown to stress cockroaches (Zhukovskaya et al. 2017), leading to decrease of sequence time. Similar data were earlier obtained for the bank voles Myodes glareolus exposed to extremely long day at the laboratory or natural environment at high latitude in summer period, where short and not really dark nights caused increased predation risk and lower foraging level (Bleicher et al. 2019; van Manen and Smaal 2021). Nevertheless, grooming sequences took the longest time during the daytime (daytime LD series), not due to the cleaning of more body parts, but because of general slowdown of all movements. The proportion of time spent on different activities, as was earlier shown (Zhukovskaya et al. 2017) depends on the intensity of light. Comparing that data with one obtained from cockroaches of LL and DD series (Fig. 5), it is easy to notice that the S1 of DD series was similar with S1 of LD series, while LL insects demonstrated freezings, indicative of the subjective day. Detailed studies on behavior of insect exposed to the long periods of darkness or light of different intensity performed with insects are rare. Moreover, it is difficult to compare the results of different studies because of high variation in light intensity, spectral characteristics, time of exposure and variety of species. Fire ants Solenopsis invicta demonstrate disturbance in foraging behavior being exposed to 24:0 and 0:24 L:D photoperiods, accompanied by shift in foraging sifor gene expression (Lei et al. 2019). Not only periodicity, but also subjective intensity of light, is shown to cause altered circadian rhythm, for example, in laboratory mice mutant with decreased retinal sensitivity behavioral pattern shifts to diurnality (Mrosovsky and Hattar 2005). Circadian system was shown to be affected by photoperiod, namely cockroaches Rhyparobia maderae reared for a long period in 16:8 LD had significantly different pattern of PDF immunoreactive neuron number and arborization from both 12:12 and 8:16 LD regimes (Wey and Stengl 2011). Our data show greater effects of extreme long day (24:0 LD) than complete darkness in comparison with more natural for a tropical insect 12:12 LD. Light around the clock is likely to uncouple some or all physiological function from the circadian oscillator present in the optic lobe of cockroaches (Page 1982; Althaus et al. 2022), similar with other invertebrates and vertebrates (Oster et al. 2002; Helfrich-Förster 2004; Häfker et al. 2024; Meier et al. 2024). In crickets, electrical discharges in the optic lobes are greatest at night both in the nymphs, which are nocturnal and in the adults which are diurnal (Tomioka and Chiba 1992). Also, the phase difference, that we observed in LL kept cockroaches, is possibly not caused by their circadian clocks but rather by how the clock couples to output mechanisms (Mrosovsky and Hattar 2005). The mechanisms of masking described previously for cockroaches under illumination during the dark phase of the 24h cycle, and manifested as some diurnal behaviors, appear to play some role under constant illumination. Thus, the data presented here demonstrate, that light conditions alters the cockroach behavior trough three main mechanisms – light adaptation of their photoreceptor organs, biological clock, and masking effect. Declarations Ethical note For the experiments on cockroach behavior, the insects were reared and tested according to Guidelines for the ethical treatment of nonhuman animals in behavioral research and teaching. The experimental animals used for the study were bred in captivity, and no pain was induced during testing. Competing Interests: Authors declare no competing interests Significance Statement Visual systems of animals adapt to lighting conditions to provide the most valuable information of the surrounding. Whereas short-term exposure adjusts photoreceptor organ sensitivity, long-term exposure also interacts with biological clocks. In this work, freezings – periods of total immobility – appeared under invisible for the cockroaches dim red light only if they were kept in the constant light conditions, indicating the inactive state (sleep) of the nocturnal insect corresponding to their subjective day. On the other hand, the increase in total freezing time under the green light illumination of the same intensity clearly corresponded to photoreceptor adaptation level. Thus, the results of experiments presented here demonstrate that some parameters of the cockroach behavior correspond to the eye adaptation state, while others are caused by disturbed biological clocks. Funding The study was supported by the IEPhB RAS Research Program № 075-00263-25-00. Author Contribution M. Z. planned the experiments and wrote the draft, E. N. conducted the experiments. Both authors analyzed the data, and contributed critically to the drafts and gave final approval for publication. Acknowledgement Authors are thankful to Dr. Roman V. Frolov for the initial idea of the experiments, and to Dr. Boris F. Gribakin (Laboratoire Charles Coulomb, UMR 5221 CNRS/Université de Montpellier, France) for the English editing. 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S1 – dark red light; S2 – dim green light. (a) – freezings count for a session (30 min); (b) – total time frozen. Statistical significance: different letters denote statistically significant differences between sample. More information is supplied in the text.\u003c/p\u003e","description":"","filename":"Onlinefloatimage29.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/752d071dcf9704f2d2b2f0c7.png"},{"id":81572510,"identity":"0fe800df-8564-4418-ab08-15b0d5f7aa35","added_by":"auto","created_at":"2025-04-28 16:39:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14213,"visible":true,"origin":"","legend":"\u003cp\u003eParameters of resting behavior of the cockroaches, kept in different light regimes. S1 – dark red light; S2 – dim green light. (a) – the stops count for a session (30 min); (b) – total time of stops. Statistical significance: different letters denote statistically significant differences between sample. More information is supplied in the text.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/ce5e80b5bf2f8d892fbd936f.png"},{"id":81572518,"identity":"c40a9bcb-aa55-4a67-a49f-812530415da2","added_by":"auto","created_at":"2025-04-28 16:39:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17521,"visible":true,"origin":"","legend":"\u003cp\u003eGrooming. S1 – dark red light; S2 – experimental illumination. (a) – Bout time; (b) – Increase in bout time between sessions ((S2/S1)*100). Raw data for LD series, other than daytime, were used earlier (Zhukovskaya et al. 2018). Experimental light intensity in S2: control – dim red light; DD, LL, LD - 4.1·10\u003csup\u003e12\u003c/sup\u003e LD medium -9.7·10\u003csup\u003e13\u003c/sup\u003e; LD bright -1.1·10\u003csup\u003e15 \u003c/sup\u003ephotons s\u003csup\u003e-1\u003c/sup\u003e cm\u003csup\u003e-2\u003c/sup\u003e). (c), (d), (e) grooming of body parts, the average time of sweeps. Data are shown as mean ± SE.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/5d4d99bac77639f88dc88c15.png"},{"id":81572514,"identity":"850c7337-3bda-4634-a222-503b453810e2","added_by":"auto","created_at":"2025-04-28 16:39:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21086,"visible":true,"origin":"","legend":"\u003cp\u003eTime, allocated for different behaviors, % to session time.\u003c/p\u003e","description":"","filename":"Onlinefloatimage54.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/bd142047eb02e0113f6487eb.png"},{"id":81572511,"identity":"ae0093c0-634e-4fe6-ad5d-7272eba09a7e","added_by":"auto","created_at":"2025-04-28 16:39:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12485,"visible":true,"origin":"","legend":"\u003cp\u003eChange in freezing time between sessions, s. Raw data for green LD and bright green LD series were used earlier (Zhukovskaya et al. 2018).\u003c/p\u003e","description":"","filename":"Onlinefloatimage63.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/986f6e615ecde87c19a5f036.png"},{"id":81572515,"identity":"b9fa486a-0cec-4f1d-a7c9-6552440f3983","added_by":"auto","created_at":"2025-04-28 16:39:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6976,"visible":true,"origin":"","legend":"\u003cp\u003eSpeed of locomotion (quadrant/s).\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/bc155a669d70b97fb1c7c9c9.png"},{"id":81572892,"identity":"70bff215-2033-4d16-86be-ea9ffbdf918c","added_by":"auto","created_at":"2025-04-28 16:47:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5981,"visible":true,"origin":"","legend":"\u003cp\u003eTotal time of stops (s) for the subgroups of LL series.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/a65d0c739120e6b365ec4478.png"},{"id":83343053,"identity":"da3f8e13-df46-4bd2-af13-3426c8bd7acb","added_by":"auto","created_at":"2025-05-23 11:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":704939,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/4c64036e-1456-4855-aafd-903b8d13380e.pdf"},{"id":81572891,"identity":"321cfe84-f61b-4ff5-b3f9-65f2a88add95","added_by":"auto","created_at":"2025-04-28 16:47:51","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25232,"visible":true,"origin":"","legend":"","description":"","filename":"data.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6519676/v1/c70f288515a71c1fd5dd1bf3.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Long-term exposure to extreme illumination regimes alters behavioral responses to light in the cockroach, Periplaneta americana L","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLong-living animals have more chances to experience severe environmental alteration during their lifetime in comparison with short-living ones. Although some species developed genetically coded adaptations, such as diapause and aestivation, others demonstrate greater phenotypic and behavioral plasticity (Ganguly and Candolin 2023). Insects can successfully survive not only cold or dry and hot period in dormant states, but also occasional drowning (Rondeau and Raine 2024 and references therein) and other situations with oxygen deficits (Hoback and Stanely 2001). Active stages are supposed to be more susceptible to adverse conditions. One of the main factors of insect life is illumination, which regulates circadian and seasonal cycles, but also has direct action on their organisms. For example, for \u003cem\u003eDrosophila\u003c/em\u003e eggs and pupae blue light was highly toxic: the majority of eggs died after 48-h irradiation at 5.0 * 3 10\u003csup\u003e18\u003c/sup\u003e photons/m\u003csup\u003e2\u003c/sup\u003e/s, whereas most eggs hatched under the dark; larvae were susceptible as well (Hori et al. 2014). In most of the cases the light acts through light-sensitive sensory organs, such are compound eyes, ocelli, stemmata and extraretinal eyelets (Hofbauer and Buchner 1989;\u0026nbsp;Oakley\u0026nbsp;et al. 2007; Friedrich et al. 2006; Land and Chittka 2013; Severina et al. 2024). This multicomponent visual system provides an insect with highly valuable information for use in spatial orientation, mate and food finding and recognition, predator avoidance, as well as circadian entrainment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhotoreceptor organs are plastic in their responses to light.\u0026nbsp;Relatively short-term exposure (from seconds to minutes) to light of particular intensity and wavelength causes temporal changes in sensitivity of photoreceptor cells due to adaptation (Laughlin and Hardie 1978;\u0026nbsp;Matić and Laughlin 1981;\u0026nbsp;Burton 2002). Mechanisms of light adaptations are well known: temporal structural changes (Meyer-Rochow\u0026nbsp;1974; Gribakin 1979; Belu\u0026scaron;ič et al. 2017) and adjustments in phototransduction cascade, such as Ca\u003csup\u003e2+\u003c/sup\u003e feedback, activity modulation of a number of enzymes and second messengers (Sandler and Kirschfeld 1991; Abbas and Vinber, 2021). Further neural processing adjusts the signals to get the most important information about visual surroundings (Hempel de Ibarra et al. 2014; Ketkar at al. 2023) depending on the context of internal state and self-motion (Longden et al. 2014; Kohn et al. 2018; Hindmarsh Sten et al. 2021). Longer exposures affect the biological clock and cause, in addition to above-mentioned, some other phenomena, such as masking, the reaction to unexpected fast change in ambient light conditions, which elicited behaviors characteristic for the opposite phase of circadian cycle (Aschoff 1960; Mrozovsky 1999). Fly larvae raised under different photoregimes from 0 to 24 h of light demonstrated changes in the total dendritic length of LN neurons, postsynaptic to photoreceptor of Bolwig\u0026apos;s organ, the larval eye (Yuan et al. 2011). Honey bees are evolutionarily preadapted to the dramatic change in light exposure on transition between nursing inside the hive and foraging outside. Light exposure of nurse bees causes decrease in mushroom body microglomerulus density and increase in juvenile hormone level, showing changes similar to untreated foragers of older age (Scholl et al. 2014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals inhabiting high latitudes often show behavioral arrhythmicity. Larvae and adults of Antarctic midges, \u003cem\u003eBelgica antarctica\u0026nbsp;\u003c/em\u003eJacobs\u003cem\u003e,\u0026nbsp;\u003c/em\u003edemonstrate around the clock activity at the constant temperature, regardless of the photoregime. Their clock genes, such as \u003cem\u003eperiod\u003c/em\u003e, \u003cem\u003etimeless\u003c/em\u003e, \u003cem\u003eClock\u003c/em\u003e, and \u003cem\u003evrille\u003c/em\u003e expressed no cycling in any of the experimental photoregimes (Kobelkova et al. 2015). The high-latitude fly, \u003cem\u003eDrosophila lummei\u003c/em\u003e, shows rhythmicity under the constant light conditions, while its cosmopolitan and holotropical relatives, such as \u003cem\u003eD. melanogaster\u003c/em\u003e, \u003cem\u003eD. mercatorum, D. hydei, Z. indianus and Z. camerounensis\u003c/em\u003e become arrhythmic (Beauchamp et al. 2018). \u003cem\u003eChymomyza costata\u003c/em\u003e (Diptera: Drosophilidae) from Finland lacks circadian rhythmicity in constant darkness, whereas most known species, including the closely related Drosophilidae from temperate and tropical latitudes, demonstrate free-running along with structural changes of clock neurons network. (Bertolini et al. 2019; Meier et al. 2024).\u003c/p\u003e\n\u003cp\u003eCave dwellers, on the contrary, living in almost complete darkness, are characterized by partial or total reduction of their visual systems that mostly correlates with the loss of rhythmicity (Friedrich 2013; Heads 2010; Rampini et al. 2008; Sendi et al. 2020). Nevertheless, cave-dwelling glowworms \u003cem\u003eArachnocampa tasmaniensis\u003c/em\u003e (Merritt and Clarke 2011) retain visual system and light-entrainable circadian rhythms with a period of about 24h, but populations from different caves are shifted by several hours.\u003c/p\u003e\n\u003cp\u003eThus, long periods of daylight, as well as an almost complete darkness are not totally artificial conditions for insects on Earth, although most tropical insects, such as cockroaches, do not experience such conditions in their life. However, evolutionary history of cockroaches and related groups is traced from Cretaceous period, which was characterized by warm climate around the globe, including polar areas (Skelton et al. 2003) and ancient cockroaches inhabited areas with polar day and night (Grandcolas 1999). The American cockroach, \u003cem\u003ePeriplaneta americana\u003c/em\u003e L. usually avoids lit area and shows escape reactions by incident illumination (Bell and Adiyodi 1982; Okada and Toh 1998). Its visual system has two pairs of photosensory organs \u0026ndash; compound eyes and ocelli. The compound eyes consist of ommatidia with photoreceptor cells of two spectral classes \u0026ndash; greenlight sensitive (broadband with max 560 nm) and UV-sensitive (365 nm); while ocelli \u0026ndash; presumably of only one class of green-sensitive photoreceptors (Goldsmith and Ruck 1958; Mote and Goldsmith 1970). Outstanding adaptability and plasticity has allowed the cockroaches to inhabit human buildings and yards (Johnson and Munshi-South 2017; Gondhalekar et al. 2021), where lighting conditions vary from bright light to almost complete darkness, and the modes of illumination can change unpredictably. The present paper is aimed to study changes in behavioral responses to light stimuli of the cockroaches kept under different illumination regimes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eInsects. Male nymphs of \u003cem\u003eP. americana\u003c/em\u003e L. were separated from the lab colony and kept under 3 regimes for 3 moths: 12:12 LD; 24 h dark; 24 h light, temperature 22-27 \u0026deg;C and humidity 60-90%. Animals were fed ad libitum (bread, oatmeal, cabbage, dandelion leafs, dry cat food).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFreshly molted adults were transferred to the experimental setup designed as Plexiglas cage (350 \u0026times; 250 \u0026times; 105 mm) connected to the always dark shelter (150 \u0026times; 150 \u0026times; 100 mm) under the same light conditions as described before (Novikova et al. 2017). All groups were subjected to thermoregime 26\u0026plusmn;2 \u0026deg;C during the day and 22\u0026plusmn;2 \u0026deg;C during the night. For the 12:12 LD group testing was started at the beginning of a dark phase, for other two groups \u0026ndash; at the start of subjective night.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBehavioral responses to low intensity green light (530 nm, 10 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e) were monitored as described earlier (Zhukovskaya et al. 2017). Namely, an experiment would start by entering a cockroach to the test chamber (150 \u0026times; 150 \u0026times; 100 mm) from the home cage, after that the chamber with the insect was separated by the sliding Plexiglas door. After 10 min adaptation period, the first session (S1) of video recording was performed for 30 min under dim red light (690 nm, 0.13 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e). The second session (S2) of recording started in 10 min after the end of the first one under green light illumination. Locomotor activity and grooming were assessed, as well as stops (no movements, antennae are waving) freezings \u0026ndash; periods of complete immobility (Zhukovskaya 2014; Zhukovskaya et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing series of experiments were performed with special conditions in S2:\u003c/p\u003e\n\u003cp\u003e1. \u0026ldquo;Control LD\u0026rdquo;. Cockroaches maintained in 12:12 LD regime, no addition lighting (dark) was presented in the S2;\u003c/p\u003e\n\u003cp\u003e2. \u0026ldquo;Green LD\u0026rdquo;. Cockroaches maintained in 12:12 LD regime, green light was applied at the S2;\u003c/p\u003e\n\u003cp\u003e3. \u0026ldquo;Green, DD.\u0026rdquo; Cockroaches were maintained in the constant darkness, green light was applied at the S2;\u003c/p\u003e\n\u003cp\u003e4. \u0026ldquo;Green, LL\u0026rdquo;. Cockroaches were maintained in the constant light; green light was applied at the S2;\u003c/p\u003e\n\u003cp\u003e5. Additional observations were performed on the cockroaches maintained in 12:12 LD regime. The behavior of a group of males was monitored inside the darkened shelter 30 min after light \u0026ldquo;On\u0026rdquo; under infrared (850 nm) light with irradiance of 7.4 \u0026micro;W/cm\u003csup\u003e2\u003c/sup\u003e (\u0026ldquo;Daytime LD\u0026rdquo; series).\u003c/p\u003e\n\u003cp\u003eRaw data of the series \u0026ldquo;Control LD\u0026rdquo; and \u0026ldquo;Green LD\u0026rdquo; were earlier used in the paper of Zhukovskaya et al. (2017).\u003c/p\u003e\n\u003cp\u003eLocomotor activity was evaluated for the series 1-4 as follows: the image of the test chamber on the computer screen was divided into quadrants and the number of them crossed by cockroach for the session was counted. Grooming behavior data were processed as described earlier (Zhukovskaya 2014, Zhukovskaya et al. 2017), briefly, cleaning of body parts was timed, and grooming bout was defined as cleaning of several body parts without interruption by any other behavior. Frequencies and timing of stops (cockroach stands still, only antennae are moving) and freezings (no detectable movements) were calculated. Also, the share time for different activities was accessed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data obtained were tested for normality with Kolmogorov-Smirnov test using online calculator (https://contchart.com/goodness-of-fit.aspx). Parametric statistics was used for normally distributed data (Student\u0026rsquo;s T-test, ANOVA, Tukey post-hoc pairwise comparisons http://vassarstats.net/); non-parametric tests were applied to data, whose distribution was significantly different from normal (Wilcoxon signed rank test, Mann-Whitney U-test (http://vassarstats.net/), Kruskal-Wallis Test, followed by Dunn\u0026rsquo;s post hoc pairwise comparisons (https://www.statskingdom.com/kruskal-wallis-calculator.html).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFirst, the locomotor activity of the cockroach groups maintained in 12:12 LD was tested. Control group (dark red light in both sessions) and insects illuminated with green light at the S2 of the experiment did not differ: they both declined their walking levels between S1 and S2 (2-way ANOVA with repeated values, F\u003csub\u003e1/28\u003c/sub\u003e=22.39, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, the data were mostly distributed normally). Three months of darkness did not alter cockroach locomotion in the S1, but 3 months long exposure to white light significantly decreased the activity (Mann-Whitney U-test with Bonferroni correction, \u003cem\u003ep\u003c/em\u003e =0.04, Fig.1 a). Changes in locomotion under green light exposure in the S2 the experiments were unidirectional (decrease) for all series excluding that, in which cockroaches were kept under 24 h of light. Statistical evaluation of the differences calculated for sessions of each series (S1-S2) gives a number of significant values (one-way ANOVA, F\u003csub\u003e3/52\u003c/sub\u003e=7.3, \u003cem\u003ep\u003c/em\u003e =0.000352, post hoc Tukey HSD Test returns significant values for pairs: control LD versus green LD, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; control LD versus LL, P\u0026lt;0.01; DD versus LL, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05) (Fig. 1 b).\u003c/p\u003e\n\u003cp\u003eAnalysis of freezing frequencies was performed using non-parametric Kruskal-Wallis test, since all the data did not pass normality test. For the S1 cockroaches demonstrated significant amount of freezing only if they were kept in the constant light (green LL, Kruskal-Wallis Test, H\u003csub\u003e3\u003c/sub\u003e=10.439364, \u003cem\u003ep\u003c/em\u003e =0.015178 followed by Dunn\u0026rsquo;s post hoc test) (Fig. 2). Rare freezings shown by cockroaches kept in the dark did not differ significantly from both of LD series. In the S2, freezing occurred most frequently for cockroaches kept in the dark (DD), significant differences were found with both series kept under LD regime (Kruskal-Wallis Test, H3=8.32; \u003cem\u003ep\u003c/em\u003e =0.04, Dunn\u0026rsquo;s post hoc test, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05). Frequency of freezing for light-kept insects was in between, differences with other groups were non-significant. Time spent frozen followed the same pattern (Kruskal-Wallis Test, H\u003csub\u003e3\u003c/sub\u003e=10.55; \u003cem\u003ep\u003c/em\u003e =0.014, Dunn\u0026rsquo;s post hoc test, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05); and the duration of the single freezing episode was similar for all treatments and was on average 73.8\u0026plusmn;16.5 s.\u003c/p\u003e\n\u003cp\u003eCockroaches kept in the dark stopped in response to green light in the S2 more frequently and spent more time resting, than those kept in the LD conditions (Fig. 3). Neither frequency of stops nor total duration of stops in the first sessions differed across all series (Kruskal-Wallis Test, \u003cem\u003ep\u003c/em\u003e \u0026gt;0.05, Fig. 3). LD kept cockroaches demonstrated strong difference in time spent stopped between control LD and green LD series at the second sessions of the experiments (Kruskal-Wallis Test, H\u003csub\u003e3\u003c/sub\u003e=17.484712, \u003cem\u003ep\u003c/em\u003e =0.000562, followed by Dunn\u0026rsquo;s post hoc comparisons, \u003cem\u003ep\u003c/em\u003e =0.000145). As reported earlier, low intensity green light causes restlessness in the cockroaches (Zhukovskaya et al. 2017). Cockroaches exposed to constant light or dark rested about the same amount of time (Fig. 3; Dunn\u0026rsquo;s post hoc comparisons, \u003cem\u003ep\u003c/em\u003e =0.153).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough total grooming time did not differ significantly between treatments and between two consecutive sessions (Two-way ANOVA, \u003cem\u003ep\u003c/em\u003e \u0026gt;0.05 for both factors), but thorough evaluations of parameters of this behavior revealed some significant effects. Grooming patterns, reflecting the stress level (Kalueff et al, 2007; Root-Bernstein 2010;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZhukovskaya et al. 2013; Novikova and Zhukovskaya 2015), showed prolonged grooming bouts only for the cockroaches of LL series at the first session of experiments (Kruskal-Wallis Test, H\u003csub\u003e3\u003c/sub\u003e= 8.6929. \u003cem\u003ep\u003c/em\u003e = 0.03366, Post hoc Dunn\u0026rsquo;s test returns \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 for all groups in compare with LL, Fig. 4a). Second sessions did not differ significantly. Grooming bouts were found to be longer in the second session as compared to the first session for controls and DD kept insects (Wilcoxon test, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0349 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0067, respectively).\u003c/p\u003e\n\u003cp\u003eGrooming of various appendages changed differently between treatments, antennae were groomed longer in the S2 of control experiment (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, Student\u0026rsquo;s t-test) and in DD series (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, Student\u0026rsquo;s t-test), insects kept under LL showed equally high level of grooming in both sessions and those kept under LD maintained intermediate duration of antennal grooming (Fig 4c). Two-way ANOVA proved differences between series (F\u003csub\u003e3/99\u003c/sub\u003e=6.55, \u003cem\u003ep\u003c/em\u003e =0.0004) and between sessions (F\u003csub\u003e1/99\u003c/sub\u003e=5.32, \u003cem\u003ep\u003c/em\u003e =0.0232), interaction between factors was non-significant. Post hoc Tukey test revealed, that LL series differed from control (p\u003cem\u003e\u0026nbsp;p\u003c/em\u003e \u0026lt;0.01), as well as LD (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01), both of which testing cockroaches kept under normal 12:12 photoregime. Only DD animals showed a big difference between first and second sessions of experiment (Fig. 4c).\u003c/p\u003e\n\u003cp\u003eCockroaches exposed to LL cleaned their foreleg more slowly than under all other treatments (F\u003csub\u003e3/\u003c/sub\u003e9=5.4, \u003cem\u003ep\u003c/em\u003e =0.0018, Tukey post hoc with LD groups \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, with DD \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, Fig. 4d). All other parameters did not show any statistical significance. Midleg and hindleg grooming took longer at the second sessions (Two-way ANOVA, F\u003csub\u003e1/92\u003c/sub\u003e= 9.29, \u003cem\u003ep\u003c/em\u003e = 0.003, Fig. 4e), mostly because of prominent differences in Green LD and DD series (post hoc Tukey, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05 for green LD and \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01 for DD series).\u003c/p\u003e\n\u003cp\u003eSince illumination during the night may cause masking effect \u0026ndash; manifestation of behaviors characteristic of the light phase of the 24h cycle (Mrosovsky 1999), the additional experiments with the group of male cockroaches were performed to recognize the behavior inherent for the light part of the day (daytime LD). The most prominent behavior was freezing. Insects demonstrated more freezings than at any other series, the frequency was 8.4\u0026plusmn;0.6 per session (in compare with 3.89\u0026plusmn;1.1 in DD series, Fig. 4), average length of a freezing episode was 83.4\u0026plusmn;13.9 s, the total time of immobility \u0026ndash; 398\u0026plusmn;47 s for 30 min observation period in compare with 283\u0026plusmn; 96 s in DD series. Since the experimental protocol was quite different, performing the direct statistical comparison is believed to be inadequate. Anyway, we can see, that frequent and long freezings are the hallmark of daytime behavior. Cleaning sweeps of particular body parts, such as antennae and legs, slows down (Fig 4 c-e), and the S1 of LL series was the closest in values to \u0026ldquo;daytime LD\u0026rdquo;. The number of cleaned body parts per one grooming sequence was equal to 6.8\u0026plusmn;1.6, which is closer to 5.2\u0026plusmn;0.6 in cockroaches of LL series than to 4.2\u0026plusmn;0.4 for the first session of combined 12:12 LD series and 4.35\u0026plusmn;0.56 for DD.\u003c/p\u003e\n\u003cp\u003eThe number of cleaned body parts was about the same for all series other than daytime LD. The longest grooming bouts were observed for the cockroaches at the beginning of the light phase in the shelter (daytime LD), but they always resided in well-known living quarters under the lowest stress level. Two-way ANOVA comparing four series (control LD, green LD. green LL and green DD) revealed significant differences between sessions (F\u003csub\u003e1/98\u003c/sub\u003e=7.95, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01), as well as series (F\u003csub\u003e3/98\u003c/sub\u003e=5.41, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01). Post hoc Tukey test showed that LL group was different from both LD series (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01), but not from DD. Student\u0026rsquo;s t-test for pairs revealed significant increase in bout time in the second session only for DD cockroaches (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCockroaches kept in constant light conditions for 3 months before the test demonstrated behavior different from LD groups in the first sessions of the experiments, without illumination. The most distinctive behavior was demonstrated by light-kept insects; their locomotion was noticeably lower than of all other treatments, these cockroaches displayed prominent freezing behavior before the light was turned on, and their grooming was the slowest. Dark-kept insects behaved almost like 12:12 LD ones, namely they showed similar level of locomotion and grooming. (Figs 1, 4). Further analyzing cockroach behavior, the proportions of time allocated to each activity (activity budgets) were calculated (Fig.5). Only cockroaches of green LL series spent noticeable amount of time frozen in the S1. Freezings, as was shown earlier, are a sign of the masking effect, if they are observed under sudden illumination during the dark phase of the 24 h cycle (Novikova and Zhukovskaya 2017; Zhukovskaya et al. 2018; Novikova et al. 2021; Zhukovskaya et al. 2024) and are presumably a part of inactive daytime behavioral repertoire (see below). Second sessions of both DD and LL series were even more similar, which may be explained by direct effect of light. An earlier study demonstrated, that 3-months long exposure of the cockroaches to LL significantly decreased light responses of their green-sensitive photoreceptors in comparison with both DD and LD groups. Photoreceptor sensitivity to light was shown to be decreased in those cockroaches kept in the constant illumination (Frolov et al. 2018), which is in part attributed to well-known phenomenon of physiological and structural.\u003c/p\u003e\n\u003cp\u003eadaptation (Wolken and Gupta 1961; Butler and Horridge 1973; Ferrell and Reitcheck 1993, Frolov et al. 2022). On the contrary, 3 months of DD regime strongly up-regulated the main green-sensitive opsin GO1 (but not the ultraviolet sensitive UVO) in the compound eyes of \u003cem\u003eP. americana\u003c/em\u003e cockroaches (Frolov et al. 2018). Anticipated oppositely directed changes in eye sensitivity due to prolonged exposure to light or darkness should have resulted in oppositely directed changes in behavior, namely light-kept insects were supposed to rest a lot, similar with controls (Control LD), due to decrease in photoreceptor sensitivity (Frolov et al. 2018), and dark-kept ones should have shown responses like 12:12 LD reared insects exposed to brighter light (Zhukovskaya et al. 2017). Indeed, dark-kept animals demonstrated significant amount of stops (resting) and freezing at the expense of locomotion under low-intensity light in the S2 of the experiment, similar with responses of 12:12 LD cockroaches to high intensity light (Zhukovskaya et al. 2017, Fig. 2 b). Also difference in total freezing time clearly corresponded to subjective brightness of light stimulus (Fig. 6, Kruskal-Wallis Test, \u003cem\u003ep\u003c/em\u003e =0.12, post-hoc Dunn\u0026rsquo;s test revealed differences between green DD and both control and LL, between bright green LD and both control and LL. Raw data of bright green LD series were taken from Zhukovskaya et al. (2017)). Insects of LL group demonstrated bidirectional changes in freezing time between sessions, giving average and median close to zero.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThus, the results of the present study suggest that the discrepancy between the expected and observed effects of different illumination regimes is a result of other factor(s) influencing cockroach behavior as opposed to changes in photoreceptor sensitivity. To solve this disagreement, we compared the behavior of all experimental groups in the first sessions of experiments, under dim red light. The first sessions of all the experiments with individual cockroaches reveal that freezings \u0026ndash; periods of absolute immobility, appear only at the series, in which cockroaches were kept under constant light conditions. Together with the data on cockroach behavior in the shelter during the light phase of 12:12h LD regime (Daytime LD) becomes clear, that freezings are the attribute of inactive phase of circadian cycle (sleep). The biological clock can run with different speed, this species-specific and individual feature is determined by the internal circadian period \u003cstrong\u003e\u0026tau;\u003c/strong\u003e (tau) and vary in the range of ~24\u0026plusmn;4 hours, which is largely predetermined by polymorphism of core clock and some other related genes (Dibner et al. 2010; Doi et al. 2011). In this study, cockroaches kept in the constant dark show only minor signs of circadian rhythm disturbance, and only one individual was on subjective day phase in our experiment, showing 6 episodes of freezing with total time of 269 s, likely because of the differences in \u003cstrong\u003e\u0026tau;\u003c/strong\u003e between individuals (Lipton and Sutherland 1970; Bertossa et al. 2013; Shinkawa et al. 1994; Pivarciova et al. 2016). So, to check if the circadian state is solely responsible for the locomotor activity level, the cockroaches of LL series were regrouped according to the presence or absence of freezing behavior in the S1 of experiment, which was always conducted under dim red light. Interestingly, if a cockroach demonstrated freezing behavior at the S1, it froze at the S2 as well, and vice versa. The individual of DD series that froze, was excluded from the analysis (Fig. 7). As a result of such recalculations, LL cockroaches that did not freeze demonstrated practically identical locomotor activity with DD individuals, as under dark red light in the S1, as under green illumination at the S2. Those, who showed freezings crossed significantly less quadrants (two-way ANOVA, F\u003csub\u003e1/33\u003c/sub\u003e=5.79, \u003cem\u003ep\u003c/em\u003e =0.029). Moreover, the walking speed, (quadrants/(time of locomotion, s)) showed the same pattern, namely, the walking speed was slowest for the cockroaches of LL series, that showed freezings (Kruskal-Wallis Test, \u003cem\u003ep\u003c/em\u003e =0.0084, Dunns post hoc test revealed that LL subgroup that froze was significantly different from all other series, P\u0026lt;0.05, which did not differ from each other). Thus, locomotor activity level and walking speed mostly reflects the circadian phase of an insect, rather than the state of its photoreceptors. Interestingly, the cockroaches of the LL subgroup that showed freezings, demonstrated some but non-significant raise in the speed of locomotion at S2 (Wilcoxon Signed-Rank Test, \u003cem\u003ep\u003c/em\u003e =0.0549, Fig. 7).\u003c/p\u003e\n\u003cp\u003eThermoregime in which the insects were kept (see \u0026ldquo;Material and Methods\u0026rdquo;) probably entrained DD cockroaches, but failed to do the same for LL group. It is clear when the data for the S1 was analyzed, namely parameters of behavior for DD cockroaches were similar with those for LD series for all but one cockroach, which demonstrated freezing in the first session. Temperature cycles are known to be a second robust Zeitgeber for behavioral rhythms of model and non-model insect, such as fruit fly \u003cem\u003eD. melanogaster\u003c/em\u003e (Wheeler et al., 1993; Busza et al, 2007; Wolfgang et al, 2013), linden bug \u003cem\u003ePyrrhocoris apterus (\u003c/em\u003eKaniewska et al. 2020), cockroaches (Page. 1985) and crickets (Rence and Loher 1975).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe direct effect of light adaptation or decreased photoreceptor sensitivity due to structural changes was not clearly recognized in our experiments with LL cockroaches likely due to arrythmicity caused by constant light exposure (Konopka et al. 1989; Hong and Saunders 1994; Saunders and Cymborowski 2008). In any case, cockroaches from LL subgroup, that froze stopped more frequently (Mann-Whitney U-test, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05) and longer (Mann-Whitney U-test, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05) then those that did not freeze under green light stimulation at the S2 (Fig. 8. Total time of stops). Resting behavior of non-freezing cockroaches of LL series was similar with control LD insects not exposed to light.\u003c/p\u003e\n\u003cp\u003eThe level of stress, evaluated by the shortening of grooming sequences (Zhukovskaya 2014) depended on rearing conditions, namely, cockroaches kept in the dark (DD) were comparable with control (LD) animals, not exposed to light (Fig. 4 b), but light kept (LL) cockroaches demonstrated sequence length similar with LD ones under experimental light of the same intensity. Bright light was shown to stress cockroaches (Zhukovskaya et al. 2017), leading to decrease of sequence time. Similar data were earlier obtained for the bank voles \u003cem\u003eMyodes glareolus\u0026nbsp;\u003c/em\u003eexposed to extremely long day at the laboratory or natural environment at high latitude in summer period, where short and not really dark nights caused increased predation risk and lower foraging level (Bleicher et al. 2019; van Manen and Smaal 2021). Nevertheless, grooming sequences took the longest time during the daytime (daytime LD series), not due to the cleaning of more body parts, but because of general slowdown of all movements.\u003c/p\u003e\n\u003cp\u003eThe proportion of time spent on different activities, as was earlier shown (Zhukovskaya et al. 2017) depends on the intensity of light. Comparing that data with one obtained from cockroaches of LL and DD series (Fig. 5), it is easy to notice that the S1 of DD series was similar with S1 of LD series, while LL insects demonstrated freezings, indicative of the subjective day.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDetailed studies on behavior of insect exposed to the long periods of darkness or light of different intensity performed with insects are rare. Moreover, it is difficult to compare the results of different studies because of high variation in light intensity, spectral characteristics, time of exposure and variety of species. Fire ants \u003cem\u003eSolenopsis invicta\u003c/em\u003e demonstrate disturbance in foraging behavior being exposed to 24:0 and 0:24 L:D photoperiods, accompanied by shift in foraging \u003cem\u003esifor\u0026nbsp;\u003c/em\u003egene expression (Lei et al. 2019). Not only periodicity, but also subjective intensity of light, is shown to cause altered circadian rhythm, for example, in laboratory mice mutant with decreased retinal sensitivity behavioral pattern shifts to diurnality (Mrosovsky and Hattar 2005). Circadian system was shown to be affected by photoperiod, namely cockroaches \u003cem\u003eRhyparobia maderae\u003c/em\u003e reared for a long period in 16:8 LD had significantly different pattern of PDF immunoreactive neuron number and arborization from both 12:12 and 8:16 LD regimes (Wey and Stengl 2011). Our data show greater effects of extreme long day (24:0 LD) than complete darkness in comparison with more natural for a tropical insect 12:12 LD. Light around the clock is likely to uncouple some or all physiological function from the circadian oscillator present in the optic lobe of cockroaches (Page 1982; Althaus et al. 2022), similar with other invertebrates and vertebrates (Oster et al. 2002; Helfrich-F\u0026ouml;rster 2004; H\u0026auml;fker et al. 2024; Meier et al. 2024). In crickets, electrical discharges in the optic lobes are greatest at night both in the nymphs, which are nocturnal and in the adults which are diurnal (Tomioka and Chiba 1992). Also, the phase difference, that we observed in LL kept cockroaches, is possibly not caused by their circadian clocks but rather by how the clock couples to output mechanisms (Mrosovsky and Hattar 2005). The mechanisms of masking described previously for cockroaches under illumination during the dark phase of the 24h cycle, and manifested as some diurnal behaviors, appear to play some role under constant illumination.\u003c/p\u003e\n\u003cp\u003eThus, the data presented here demonstrate, that light conditions alters the cockroach behavior trough three main mechanisms \u0026ndash; light adaptation of their photoreceptor organs, biological clock, and masking effect.\u003c/h1\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical note\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the experiments on cockroach behavior, the insects were reared and tested according to Guidelines for the ethical treatment of nonhuman animals in behavioral research and teaching. The experimental animals used for the study were bred in captivity, and no pain was induced during testing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSignificance Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVisual systems of animals adapt to lighting conditions to provide the most valuable information of the surrounding. Whereas short-term exposure adjusts photoreceptor organ sensitivity, long-term exposure also interacts with biological clocks. In this work, freezings \u0026ndash; periods of total immobility \u0026ndash; appeared under invisible for the cockroaches dim red light only if they were kept in the constant light conditions, indicating the inactive state (sleep) of the nocturnal insect corresponding to their subjective day. On the other hand, the increase in total freezing time under the green light illumination of the same intensity clearly corresponded to photoreceptor adaptation level. Thus, the results of experiments presented here demonstrate that some parameters of the cockroach behavior correspond to the eye adaptation state, while others are caused by disturbed biological clocks.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe study was supported by the IEPhB RAS Research Program № 075-00263-25-00.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. Z. planned the experiments and wrote the draft, E. N. conducted the experiments. Both authors analyzed the data, and contributed critically to the drafts and gave final approval for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors are thankful to Dr. Roman V. Frolov for the initial idea of the experiments, and to Dr. Boris F. Gribakin (Laboratoire Charles Coulomb, UMR 5221 CNRS/Universit\u0026eacute; de Montpellier, France) for the English editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbas F, Vinberg F (2021) Transduction and adaptation mechanisms in the cilium or microvilli of photoreceptors and olfactory receptors from insects to humans. Front Cell Neurosci 15:662453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fncel.2021.662453\u003c/span\u003e\u003cspan address=\"10.3389/fncel.2021.662453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAschoff J (1960) Exogenous and endogenous components in circadian rhythms. 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Science 333(6048):1458\u0026ndash;1462. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1207121\u003c/span\u003e\u003cspan address=\"10.1126/science.1207121\" 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":"insect, Periplaneta americana, behavior, light responses, circadian rhythm","lastPublishedDoi":"10.21203/rs.3.rs-6519676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6519676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe natural lighting conditions vary depending on latitude, niche and time of day; the animals are evolutionarily adapted to them. Artificial lighting along with global warming drive population ranges toward high latitudes, which creates fast-changing environments for the biota. The American cockroach is a synanthropic species with nocturnal lifestyle, rarely exposed to light. Three-month long exposure to constant light or constant darkness, in comparison with normal 12:12 day and night cycle, causes behavioral changes that is explained by two main factors: adaptation of visual system, and circadian rhythm disturbance. Freezing behavior, an indicator of circadian disturbances, appeared in groups kept under constant light regimes ant tested in the dark, as well as those subjected to experimental lighting with low intensity green light. Exposure to such light caused multidirectional behavioral changes in groups kept in different light regimes, reflecting their internal levels of arousal, stress, and light adaptation of their photoreceptor organs. Thus, altered lighting conditions impose significant challenges to different aspects of insect physiology and behavior.\u003c/p\u003e","manuscriptTitle":"Long-term exposure to extreme illumination regimes alters behavioral responses to light in the cockroach, Periplaneta americana L","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 16:39:46","doi":"10.21203/rs.3.rs-6519676/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":"3f8f0de7-0233-4462-abf8-ae2dc973c1fc","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-07T09:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-28 16:39:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6519676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6519676","identity":"rs-6519676","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}