Transcriptional Response of Circadian Clock Genes to an ‘Artificial Light at Night’ Pulse in the Cricket Gryllus bimaculatus

For most animal species, light detection is vital for their temporal adaptation to the earth’s diel cycles. Monitoring the annual change in the daily light duration (photoperiod) is also used by many organisms in adapting to the seasonal cycle and conditions [1,2,3]. Many behaviors depend on such cycles: daily periods of activity and rest, sleep, foraging, courtship and mating, ecdysis in insects, migration, as well as diapause, to name just a few [1,4,5,6,7]. Consequently, many living organisms have evolved sensitive mechanism for light detection, and utilize light for other functions in addition to vision, such as entrainment of the circadian clock, timing of gene expression, sexual maturation, and hormonal regulation [4,5,8,9,10].

Artificial light at night (ALAN) is a fast-growing worldwide phenomenon [11,12]. ALAN refers to both an increase in light intensity and changes in the naturally occurring light spectrum. ALAN causes changes in the behavior and temporal activity of many animals, such as birds [13,14], rodents [15], anurans [16], and insects [17,18]. ALAN impairs sleeping behavior [19], camouflage, and population synchronization [17,20,21]. Moreover, it affects predation [22,23], orientation [24], and pollination [25,26], causes high insect mortality [27,28,29], changes in community structures, and changes in biodiversity [30,31,32]. Although the specific impacts of ALAN on insects have recently received considerable attention [33], the underlying molecular mechanisms are still poorly understood. Here, we specifically focus on the effect of dim ALAN on the circadian system, which has been well studied in vertebrates [34,35,36], but to a lesser extent in insects.

The circadian clock and its light input pathways in insects have been studied extensively in the fruit fly, Drosophila melanogaster [37,38], as well as in cockroaches, crickets, and bees (reviewed in [39]). The cricket Gryllus bimaculatus has been widely used as a model for behavior and circadian activity [9,40]. In contrast to the fruit fly, whose clock neurons are located in the brain, the circadian pacemaker of the cricket is located in the optic lobes [39,41,42,43,44,45,46,47,48]. Light entrainment of the circadian system in the cricket is mediated by green-sensitive opsins in the insect’s compound eyes [43]. The pacemaker consists of two major feedback loops, one of which is based on the period (per) and timeless (tim) genes, repressing their own transcription by inhibiting the transcription of Clock (Clk) and cycle (cyc). The other feedback loop is based on the two cry genes, cry1 (a Drosophila-type cry), which is a light sensitive photopigment, and cry2, a light insensitive mammalian-type cry [39,49]. This second feedback loop involves the upregulation of PAR domain protein 1 (Pdp1) and c-fosB [49,50,51].

Recently, we have demonstrated that the lifelong exposure of male crickets to dim ALAN as low as 2 lx leads to a loss of rhythmicity and desynchronization of stridulation and locomotion behavior [17]. Stridulation serves to attract females and is therefore crucial for the species’ fitness. We have reasoned that these behavioral changes are mediated by changes in gene expression. Indeed, recent studies on birds [52,53], amphibians [54], and glow-worms [55] have revealed altered gene expression following exposure to dim ALAN, an effect that was present in both the visual system and various other tissues. Here, we sought to investigate the effect of exposure to dim ALAN on gene expression in four different tissues of the cricket G. bimaculatus. Due the effect of ALAN on male behavior (locomotion, stridulation), we have focused on transcriptional response in males. Understanding the effect of dim ALAN on internal processes and pathways is crucial for assessing the threats of ALAN to pollinators and ground-dwelling insects, as well as for predicting the long-term effects on ecosystems.

The relative levels of gene expression in the different cricket tissues and hemolymph samples following a 30 min light pulse of 2, 5, or 40 lx, and a no-pulse control, are shown in Figure 1. In the brain, the expression of most genes was affected by the different light intensities (Figure 1A). The expression levels of both per and cry2 significantly increased with increasing light pulse intensity (F_3,21 = 3.70, p = 0.028, and F_3,24 = 3.04, p = 0.048, respectively, Figure 1A). A dependence on light pulse intensity was also observed in cry1, although the effect was only marginally significant (F_3,21 = 2.96, p = 0.056). The expression of opLW differed significantly only when light treatment experiments were pooled and compared with the control (F_1,26 = 4.90, p = 0.036). The c-fosB transcription in the brain did not differ among the different treatments (F_3,24 = 1.15, p = 0.35).

In the optic lobe, the expression of opLW was 100-fold higher compared to that seen in the brain but did not change significantly following exposure to light (Figure 1B). In contrast to the brain (Figure 1A), in which most genes exhibited light-induced upregulation, in the optic lobe the light stimuli elicited a decrease in the expression of most genes. Downregulation was observed in cry2 although only marginally significant (F_3,16 = 2.86, p = 0.07) and a similar trend was observed in cry1 (Figure 1B). No change in per and c-fosB expression was observed (F_3,17 = 1.45, p = 0.26, F_3,18 = 0.98, p = 0.42, respectively).

In the Malpighian tubules most gene expression did not change with increased light pulse intensity (Figure 1C). A marginally significant effect was observed for opLW (F_3,42 = 2.58, p = 0.066) and no differences were found for cry2 (F_3,42 = 0.80, p = 0.501), per (F_3,40 = 1.14, p = 0.34), or c-fosB (F_3,42 = 0.18, p = 0.91). However, opLW expression was higher in the control, compared to the light treatments pooled together (F_1,44 = 6.84, p = 0.012), and the expression of cry1 significantly diminished following exposure to any of the light pulses (F_3,41 = 7.21, p = 0.0005, Figure 1C).

Interestingly, we found traces of expression of all genes in the hemolymph (Figure 1D). Gene expression did not differ significantly among samples. However, when light treatments were pooled together, upregulation in per was observed (F_1,18 = 5.83, p = 0.027, Figure 1D).

In order to enable a better comparison of gene expression among tissues, we repeated the analysis using within-tissue normalization of the expression values (relative to the control mean expression values in each tissue, Figure 2). Gene expression patterns varied substantially among tissues. The expression of opLW was strongly upregulated in the brain and in the optic lobe throughout all three light treatments, while being downregulated in the Malpighian tubules (Figure 2A). The light stimuli elicited upregulation of cry2 in the brain and downregulation in the optic lobe (Figure 2B). The transcriptional response of per showed a similar pattern, being upregulated in the brain and downregulated in the Malpighian tubules (Figure 2C). The expression of c-fosB showed no significant changes in the brain, while being downregulated in the optic lobe. c-fosB was the only gene that was upregulated in the Malpighian tubules (Figure 2D). Expression of cry1 was downregulated in all tissues except the brain, where it was upregulated under 40 lx only (F_3,23 = 7.77, p < 0.001. Figure 2E). The PCA of the relative gene expression clearly separated the optic lobe and the hemolymph from the brain and the Malpighian tubules, while the latter two mostly overlapped (Figure 2F).

To simultaneously analyze the transcriptional differences between the treatments, we used Linear Discriminant Analysis (LDA). The first two LD functions explained 67.12% of the variation in the brain, 73.29% in the optic lobes, 66.87% in the Malpighian tubules and, 73.76% in the hemolymph (Figure 3). In the brain, the control group was clearly separated from the 5 and 40 lx treatments, but not from the 2 lx one (Figure 3A). In contrast, the clustering of the treatments in the optic lobe revealed a substantial difference between the control and all ALAN treatments (Figure 3B). In the Malpighian tubules and in the hemolymph, the discriminant map did not reveal any significant difference between the control and the ALAN treatments (Figure 3C,D).

Insects have served for almost a century as successful models in laboratory studies on the role of light in regulating circadian behavior [7], and in raising questions about the underlying molecular mechanisms. Here, we investigated the effect of a short, dim ALAN pulse on the gene expression of five genes associated with the circadian system of the cricket Gryllus bimaculatus.

This work substantiates the findings of previous studies [56] that uncovered the heterogeneity of circadian pacemakers across various tissues in G. bimaculatus (see PCA analysis, Figure 2F), and demonstrates the tissue-specific effects of ALAN on transcription. In the optic lobe, where the cricket’s central pacemaker is located [46,48,57,58], the response to ALAN exposures was clearly distinct from that of the control, presumably due to the strong effect of dim ALAN on opLW expression. In the brain, transcriptional responses of the control and the 2 lx treatment were largely overlapping, and to a lesser extent also with those of the 5 lx and 40 lx treatments. The Malpighian tubules and hemolymph seemed less affected by the light exposures. Indeed, a previous study [56] suggested that G. bimaculatus tubules do not harbor a circadian pacemaker, as per transcript levels lacked any diurnal rhythm (in contrast to in Drosophila [59]).

The expression of circadian clock genes in G. bimaculatus in tissues other than the optic lobes has been previously reported [56]. Particularly interesting is the role of these genes in the brain. Expression of per oscillates under constant darkness (DD), and this oscillation persists after removal of the optic lobes. However, in the absence of the optic lobes, the phase of the brain per mRNA rhythm is aberrant. These results allude to a brain circadian pacemaker that is subordinate to the central clock in the optic lobes [56].

In the hemolymph, we found minute levels of circadian clock transcripts, which to the best of our knowledge is the first report of extracellular RNA (exRNA) in circadian clock transcripts. exRNA is emerging as a newly discovered form of intracellular signaling [60]. It is secreted to the biofluids, encapsulated by protecting extracellular vesicles. The exRNA is largely composed of small non-coding RNA, and to a lesser extent messenger RNA [60]. exRNA have been shown to be an effective biomarker for diagnostic purposes, but their functional role awaits further study.

The rather rapid transcriptional response to light of clock genes such as per or cry, is intriguing, and the underlying molecular mechanism warrants further investigation. In Drosophila, a similar rapid change of per mRNA following a light pulse at zeitgeber time (ZT) 15 has been previously reported [61,62], although, in contrast to the cricket, light evoked downregulation of per. It was suggested that the response to light is mediated by an histone acetylation mechanism. In accord with studies in other insects (e.g., ants [63] and lepidoptera [64]), we observed very high levels of opLW in the crickets’ optic lobe. All ALAN treatments induced some upregulation in opLW expression in the optic lobe, although not significant (we sampled 4.5 h post lights-off, while 6 h after onset of darkness a substantial downregulation has been reported [43], Figure 1 therein). Given that the circadian photosensitivity of G. bimaculatus varies during the day, as was shown by the phase response curve of this species [65,66], one can predict that the effect of transient ALAN would be time dependent.

The disruption caused by ALAN to the circadian transcriptional cycle (Figure 1) is likely to disrupt the diurnal pattern of behavior (locomotor, stridulation), but the specific behavioral output awaits further study.

The circadian disruption caused by dim (2–5 lx) ALAN may have ecological implications, which should be taken into consideration when designing regulations concerning suitable light intensity for park and city lights [67]. Not only flying insects, which are attracted to light [28], are threatened by ALAN, but also ground-dwelling insects [21,24,68]. The deep-level effect of ALAN may therefore threaten many more species of insects than currently assumed, and add to the ongoing insect decline described in the last century [69,70]. Our own work in crickets [17] demonstrated that ALAN can lead to arrhythmicity in locomotory behavior and male stridulation and therefore may affect the survival and reproduction of exposed individuals.

It should also be noted that outdoors, especially in proximity to cities and streetlights, insects are not exposed to a single light pulse, but rather to night-long, and even lifelong ALAN [17]. Populations of the small ermine moth for example, were found to reduce their flight-to-light behavior following long-term exposure to ALAN, which may result in lower predation risk and mortality in this population [71]. It remains unknown whether crickets, and indeed other insects, have developed similar adaptations to chronic ALAN exposure.

We thank Odelia Pisanti, Yael Diesendruck, Daniel Knebel, David Burg, Alex Slavenko, Lilach Raijman, Omer Lavy, Michaela Kolker-Ghatan, Izhak David, and Talma and Rami Zelingher for their assistance. This work is dedicated to our mentor Meir Paul Pener (1930–2021).

The supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms231911358/s1↗.

Conceptualization, A.B., A.A., E.T. and K.L.; methodology, A.A., E.T. and B.F.; validation, K.L., B.F. and E.T.; formal analysis, K.L. and E.T.; investigation, K.L.; resources, A.B. and E.T.; writing—original draft preparation, K.L.; writing—review and editing, E.T., A.A. and A.B.; funding acquisition, A.B. All authors have read and agreed to the published version of the manuscript.

Not applicable.

Not applicable.

The data presented in this study are openly available in FigShare at doi: 10.6084/m9.figshare.20721265 [75].

The authors declare no conflict of interest.

This research was funded by The Open University of Israel Research Fund and by the Constantiner Institute for Molecular Genetics of Tel Aviv University.