Exposure to a nocturnal light pulse simultaneously and differentially affects stridulation and locomotion behaviors in crickets

The entrainment of a living organism’s circadian clock is mostly accomplished by means of synchronization to light (Aschoff, 1960; Saunders et al., 2002; Merrow et al., 2005; Tomioka and Matsumoto, 2019; Helfrich-Förster, 2020), which is the most reliable environmental signal of the Earth’s cycle. Most animals depend on perceiving the light’s rhythmicity for the anticipation and regulation of their activities: for choosing their foraging and sleep times, and for predator avoidance, as well as for the regulation of internal, hormonal, and molecular processes (Saunders et al., 2002; Kronfeld-Schor et al., 2013; Saunders, 2013; Kuhlman et al., 2018). In accordance with the species’ diurnal, nocturnal, or crepuscular nature, the same input may trigger different internal, and behavioral reactions (Mrosovsky, 1999; Helm et al., 2017).

Any obstruction of the light-dark cycle (or its perception) may affect daily activity rhythms, manifested in either entrainment to the new conditions, free-running behavior (an endogenous internal rhythm, that is not synchronized to the environment), or arrhythmic behavior. Yet another effect may be that of masking (see Aschoff, 1960; Mrosovsky, 1999; Kronfeld-Schor et al., 2017): i.e., an instantaneous reaction to an external signal that does not entrain the rhythm but overrides the circadian clock circuits. Such triggered interference may lead to an increase in activity (i.e., positive masking) or to a decrease in activity (i.e., negative masking), depending on the threshold and susceptibility of the relevant species and specific behavior to the stimuli (Mrosovsky, 1999). For example, light may trigger activity in diurnal species while eliciting an urge to rest in nocturnal species (Helm et al., 2017), a phenomenon that has been described in various species (Aschoff and von Goetz, 1988; Germ and Tomioka, 1998; Cohen et al., 2010; Rotics et al., 2011; Erkert et al., 2012; Spoelstra et al., 2018).

An important and constantly increasing source of obstruction of the light-dark cycle is that of artificial light at night (Hölker et al., 2010; Falchi et al., 2016; Falchi et al., 2019). ALAN reduces (even eliminates) the nocturnal darkness and thus disturbs the circadian entrainment to the daily periodicity. It modifies the time, duration, and intensity of the surrounding lighting conditions, as well as the light spectrum. Each of these parameters may distinctly affect a susceptible animal, depending on the species’ photoreceptive properties and sensitivity. The reported negative impacts of ALAN on the natural behavior of various animals include sleep disruption (Bedrosian et al., 2013; Raap et al., 2015), changes in foraging activity and predation (Sanders et al., 2018; Sanders et al., 2021), loss of spatial orientation (Eisenbeis, 2006; Foster et al., 2021), and disturbance to temporal partitioning and synchronization of activity (Amichai and Kronfeld-Schor, 2019; Levy et al., 2021).

Crickets (Gryllidae) have been used as insect models for behavioral research, neuroethology and, specifically, studies of circadian activity (Huber et al., 1989; Tomioka and Chiba, 1989; Horch et al., 2017). The nocturnal field cricket Gryllus bimaculatus expresses temporal shifts in its locomotor activity, stridulatory activity, or circadian gene expression following exposure to changes in illumination patterns (Loher, 1972; Loher, 1989; Abe et al., 1997; Uryu, 2014; Kutaragi et al., 2018; Tomioka and Matsumoto, 2019). Moreover, in constant darkness the compound eyes of G. bimaculatus display a clear circadian rhythm in their response to stimuli, with a diurnal minimum and a nocturnal maximum (Tomioka and Chiba, 1982), as well as a circadian sensitivity of visual interneurons and serotonin levels (Tomioka et al., 1993). We recently demonstrated the negative impact of ALAN on male field crickets (Levy et al., 2021). Lifelong exposure to even dim ALAN intensity had a desynchronizing effect on the crickets’ stridulation and locomotion behaviors. Recently, we also found tissue-, genes- and light-intensity-dependent changes in circadian gene expression in various body parts of the cricket, including the brain and optic lobes (Levy et al., 2022).

Here we studied the effects of exposing male G. bimaculatus to a pulse of ALAN of different intensities on stridulation and locomotion behavior. We further investigated the effects of a transition from light-dark conditions to constant light on the two behaviors in the same individual. We describe a light-intensity-dependent behavioral reaction, presenting simultaneous changes in distinct activities’ levels, or simultaneous negative and positive masking effects on stridulation and locomotion behavior, respectively. We also demonstrate light-intensity-dependent changes in the period of daily activity rhythms following transition from light:dark (LD) to light:ALAN (LA) or constant light (LL) treatments. The findings from this work emphasize the importance of utilizing a multi-behavioral approach in order to obtain a more complete understanding of the impact of ALAN, and its related mechanisms.

In this study, in order to provide novel insights into the adverse effects of ALAN and into the mechanisms of these effects on animal behavior, we investigated the impact of a pulse of light at night on stridulation and locomotion behavior in the field cricket G. bimaculatus.

Under exposure to 40 lux we identified simultaneous changes in specific activity levels in the same individual crickets; simultaneous negative and positive masking effects on the cricket’s stridulation and locomotion behavior, respectively. Such findings, to the best of our knowledge, have never been described in an insect. The masking phenomenon reflects a flexible, immediate reaction to a stimulus without affecting the animal’s endogenous rhythm (Kuhlman et al., 2018). Aschoff (1960) suggested that light increases diurnal activity in light-active animals and decreases it in dark-active animals. In accord with the Aschoff’s rule, the light pulse evoked a negative masking effect in the crickets’ nocturnal behavior, reflected in a decrease and even cessation of stridulation; while the diurnal behavior (locomotion) increased (positive masking) under the light pulse. Similarly, in rodents, exposure to ALAN was reported to result in negative masking in a nocturnal rodent, while no effect was observed in a diurnal rodent (Rotics et al., 2011). In addition, the presented increase in crickets’ diurnal locomotion activity during the light pulse is in agreement with the previously described increase in burst and intensity of locomotion during a pulse of light (Germ and Tomioka, 1998; though this was demonstrated under constant darkness). The inter-individual variations and light-intensity dependent percentage of crickets presenting a masking response in the current work (as seen in Figure 3), may indicate both an individual and a population-related illumination-sensitivity threshold.

It should be noted, that we had a preference in the current study to present the ALAN-pulse 1–2 h after lights off, as the early and late subjective night were reported to be more affected by a pulse of light (Kutaragi et al., 2016). However, the pulse was not submitted at a specific Zeitgeber time, but rather at the individual’s timing of previous consistent stridulation. These differences may have affected the responsiveness of the experimental individuals, leading to an increased variance.

The reactions of animals to different light stimuli are affected by many factors, such as the species’ way of life (i.e., nocturnal, diurnal; Helm et al., 2017; Kronfeld-Schor and Dayan, 2003; Mrosovsky, 1999), the specific properties of the animal’s visual system (Warrant and Nilsson, 2006; Land and Nilsson, 2012), visual sensory processing (Blum and Labhart, 2000; Okamoto et al., 2001; Mappes and Homberg, 2004), and of course the nature of the specific stimuli. G. bimaculatus is a nocturnal species, which possesses three types of visual receptors: UV (peak: 332 nm), blue (peak: 445 nm) and green (peak: 515 nm) (Zufall et al., 1989), and whose visual system is adapted for signal processing in dim light (Zufall et al., 1989; Sakura et al., 2003; Frolov et al., 2014). While the blue receptor has been reported to be responsible for polarization vision (Labhart et al., 1984; Herzmann and Labhart, 1989), the green-sensitive opsin-long wavelength (OpLW) was described to be the major circadian photoreceptor molecule, responsible for photic entrainment (Komada et al., 2015). Both, the visual and the circadian pathways start at the compound eye. Information from the eye is then transmitted to the optic lobe, where the circadian clock is assumed to reside, and processed in parts of the lamina, medulla and lobula of the optic lobe (Tomioka and Chiba, 1982; Tomioka and Chiba, 1985; Tomioka and Chiba, 1989; Tomioka and Chiba, 1992).

Insects, such as crickets, locusts, and cockroaches, may have several parallel neuronal pathways reaching from the compound eye to the brain, serving for similar or different functions (Okamoto et al., 2001; Homberg et al., 2003; Homberg et al., 2011; Homberg, 2004; Helfrich-Förster, 2020). Whether masking and entrainment use the same neuronal pathway or different ones, is still far from fully resolved. However, the current findings together with available other data suggest a common pathway for both: here we induced masking by a single light pulse, shown previously to affect circadian clock related transcriptional responses in the cricket one hour following the pulse (Levy et al., 2022). Our observations also revealed some residual effects of the pulse, expressed in a change in the percentage of normalized behavioral level one hour after pulse termination. This persisted even under light intensities for which the pulse itself seemed to have no effect. Such a lingering effect has not been described for rodents (Rotics et al., 2011). In a previous study on crickets, repetitive 15 min pulses (at 4, or 8 h intervals) under constant darkness evoked rhythm synchronization to the timing of the light pulses (Germ and Tomioka, 1998). It is therefore probable that in addition to the observed transient change (masking), a 3 h pulse of light at night triggers additional effects, which may be involved in the observed residual changes and maybe also in the entrainment to repetitive stimuli. These results are in line with Aschoff (1960) who suggested a connection between masking and entrainment, which may depend on the frequency and intensity of the stimuli.

The transition to LA or LL evoked a reaction in at least one of the two tested behaviors in all the experimental crickets, including in individuals that seemed unaffected by the earlier pulse. Our findings suggest, therefore, a light-intensity as well as a light-duration-dependent effect of ALAN on cricket behavior. This is in agreement with Germ and Tomioka’s (1998) findings, showing that in crickets frequent light pulses modulated the free-running period, depending on the interval of the pulses. The herein described differences in the median of the period of stridulation and locomotion activity patterns are in agreement with our previous experimental findings regarding lifelong exposure to ALAN illumination (Levy et al., 2021). These differences and the lack of correlation among the two behaviors could indicate a different susceptibility of stridulation and locomotion behaviors to the same light signals. It may also be that the differences reflect a more pronounced effect on the nocturnal than the diurnal behavior, simply since the light signal was presented at night. Our findings indicate no correlation between the stridulation and locomotion activity periods. No correlation was also found between the change during the pulse treatment and the LA/LL-induced change in activity periods for both behaviors. These findings are in accord with those of Levy et al. (2021), suggesting that the desynchronization of stridulation and locomotion activity rhythms reflect control by several peripheral clocks. We suggest that these different results for stridulation and locomotion indicate that the crickets’ behavioral reactions to light may rely on several mechanisms, rather than just one, but remain unclear to date and require further study.

Interestingly, in our previous study (Levy et al., 2021), lifelong illumination patterns evoked a high percentage of arrhythmic stridulation and locomotion behavior (29%, and 42.8%, respectively. In contrast, here, only one individual (14%) subjected to 40 lux (LL) presented arrhythmic behavior. This may be due to one or more of several reasons: First, the complexity of the experiments described in the current report have resulted in a relative small sample size, and it may be possible that a larger sample size would have included some additional arrhythmic individuals. Second, the experiments were conducted only with crickets which demonstrated consistent stridulation behavior during three consecutive nights. This pre-requisite may have filtered out individuals with a stronger tendency for arrhythmicity. Third, the crickets in (Levy et al., 2021) were submitted to lifelong ALAN, whereas the crickets in this study were raised under LD and transferred to ALAN as adults. It may be that the exposure to lifelong ALAN resulted in more arrhythmic crickets.

In summary, to date the response to a light pulse has been mostly studied in relation to phase shifts or entrainment under constant darkness. Here, we present instantaneous masking effects in two key insect behaviors. We describe for the first time a simultaneous pulse-induced negative and positive reaction, as well as a residual effect. We also demonstrate a change in behavior generated by transition to LA or LL conditions. This study and its findings add to the alarming effects of ALAN on living organisms, both individuals and populations. Our findings indicate the importance of darkness for timekeeping in nocturnal insects, and present the harsh, and fast effects of a pulse of light or chronic dim-ALAN on activity rhythms and courtship behavior of the crickets. The findings indicate that not only can life-long or night-long exposure to ALAN alter animal behavior (Rich and Longcore, 2006; Levy et al., 2021; Borges, 2022; Dominoni et al., 2022), but so too can a transient light exposure, such as a single pulse of ALAN (Rotics et al., 2011; Spoelstra et al., 2018; Levy et al., 2022). This aspect should be added to the other possible effects of ALAN, as it may become a stressor when frequently experienced, or even entrain individual behaviors, as shown for the cricket (Germ and Tomioka, 1998). The findings from this study provide further new insights into the importance of a multi-modal approach in order to more fully uncover the effects of ALAN on animal behavior and populations.

We thank Yoav Gothilf, Noga Kronfeld-Schor, and Michal Segoli for their seminal idea and contribution to the conceptualization and analysis of the study. We are grateful to Yoav Wegrzyn for his assistance with the analysis and Ronny Efronny for developing the code for the actograms.

This research was funded by The Open University of Israel Research Fund.

The original contributions presented in the study are included in the article/, further inquiries can be directed to the corresponding author. Supplementary Materials

AA, AB, and KL conceived the study and wrote the manuscript; KL carried out experiments and conducted the data analysis. AB and AA secured the funding.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys.2023.1151570/full#supplementary-material↗