The daily rotation of the Earth causes predictable 24-h environmental cycles that have resulted in the evolution of circadian clocks, endogenously maintained timing mechanisms in almost all organisms, including bacteria, fungi, plants, and metazoan (Paranjpe and Sharma, 2005; Rosbash, 2009; Eelderink-Chen et al., 2021). These internal clocks are synchronized (entrained) by environmental stimuli, such as daylight or temperature, enabling organisms to adapt to environmental changes in phase with the 24-h day. Moreover, they regulate molecular, cellular, physiological, and behavioral rhythms, which show a periodicity of ̴ 24h in the absence of any external cue (free-running condition). In insects, circadian clocks control the rhythmicity of several behaviors and physiological features, including egg-hatching, pupation, adult eclosion, locomotion, mating, feeding, and metabolism (Saunders, 2002; Tomioka and Matsumoto, 2010; Franco et al., 2018). The first circadian clock gene (period) was identified in Drosophila melanogaster (Konopka and Benzer, 1971); since then, the species has been the dominant model to elucidate the molecular genetic mechanisms underlying circadian rhythms in insects. However, studies have also been conducted in other insect species, many including Lepidoptera.

The order of Lepidoptera comprises butterflies and moths and is among the most species-rich and diverse insect groups. It includes approximately 160,000 identified species, subdivided into ̴ 43–45 superfamilies (Mitter et al., 2017; Kawahara et al., 2019; Figure 1). Lepidoptera are holometabolous insects, showing juvenile stages (larvae or caterpillars) morphologically distinct from the mature adult form (moths and butterflies). The majority of Lepidoptera are nocturnal (about 75–85% of the identified species), while the remaining 15–25% of diurnal species also includes butterflies (Papilionoidea, ̴19,000 described species; Kawahara et al., 2018). The overwhelming majority of Lepidoptera (>99%) are herbivores with over 90% having a host plant range of three or fewer species (Pierce, 1995); they are found in almost all terrestrial ecosystems and have fundamental ecosystem roles as pollinators and prey. They also have a significant impact on the human economy, as many species represent serious food crop pests, while others such as the domesticated silkworm Bombyx mori (Goldsmith et al., 2005) and Antheraea pernyi (Li et al., 2017) produce silk that is utilized by the textile industry. Moreover, silk is employed in innovative applications of both the biomedical and cosmetics sectors.

A recent phylogenomic analysis dated the most recent common ancestor of Lepidoptera in the late Carboniferous, about 300million years ago. Evolutionary studies support the hypothesis that Lepidoptera developed initially as internal feeders of non-vascular plants, then differentiated and coevolved in parallel with angiosperms (Figure 1). In this process, the larval stages became external plant feeders, while adults evolved a tube-like proboscis, used for the efficient acquisition of nutritious nectar from flowering plants (Mitter et al., 2017; Kawahara et al., 2019; Figure 1). These adaptations along with an increased ability to fly and colonize new plants are believed to be associated with the large body size observed in many moths and butterflies (Powell et al., 1998; Kawahara et al., 2019).

Several of the larger lepidopteran species have been model organisms in the study of different biological phenomena, including development, physiology, ecology, and evolution (Roe et al., 2009). Pioneering Lepidoptera-based investigations have been fundamental in the study of circadian biology, showing for the first time that the brain is the site of a light-entrainable circadian clock controlling behavioral rhythms (Williams, 1963; Truman and Riddiford, 1970).

This review presents an updated overview of the circadian clock in Lepidoptera. After a general description of the different behavioral circadian rhythms currently identified, we report the molecular and anatomical organization of circadian clocks in the adult stage and during development. We then describe the ecological importance of circadian clocks detailing the complex interplay between lepidopteran feeding behavior and plants. Finally, we discuss how the characterization of these features could be useful in both pest control and rearing improvements of beneficial Lepidoptera.

From embryo to adulthood, both nocturnal and diurnal Lepidoptera exhibit multiple daily behavioral rhythms restricted to species-specific daytimes. Furthermore, for some species, these rhythmic activities have been demonstrated to be maintained under free-running conditions, indicating they are controlled by an endogenous circadian clock (Merlin and Reppert, 2009; Groot, 2014).

One of the first described circadian events occurring during lepidopteran development is egg-hatching, i.e., the emergence of the larva from the protective envelopes of the egg. In several moth species, egg-hatching occurs in the first hours of the day with a peak anticipating morning light-on, when maintained in light/dark (LD) entraining conditions (Minis and Pittendrigh, 1968; Sakamoto and Shimizu, 1994; Sauman and Reppert, 1998). This egg-hatching rhythm was demonstrated to be truly circadian as it was entrainable by environmental stimuli (such as light and/or temperature) from mid embryogenesis and was maintained in constant darkness (DD) regimes in the pink bollworm Pectinophora gossypiella (superfamily Gelechioidea; Minis and Pittendrigh, 1968) and the Chinese oak silk moth Antheraea pernyi (superfamily Bombycoidea; Sauman et al., 1996; Sauman and Reppert, 1998). Circadian egg-hatching rhythms have been described in other lepidopteran species, including the domesticated silkworm B. mori (superfamily Bombycoidea; Sakamoto and Shimizu, 1994; Table 1).

During post-embryonic development, lepidopteran larvae are characterized by intense feeding and recent studies demonstrated that for some Lepidoptera, feeding behavior in the last larval instar occurs with a daily rhythm in entraining conditions (Goodspeed et al., 2012; Suszczynska et al., 2017; Niepoth et al., 2018; Zhang et al., 2021). Studies on different pest species belonging to the Noctuoidea superfamily, including the cabbage looper Trichoplusia ni, as well as the cotton leafworms Spodoptera littoralis and Spodoptera litura, analyzed feeding behavior in both LD and DD conditions. They indicated that in these species larval feeding is under circadian control (Goodspeed et al., 2012; Suszczynska et al., 2017; Zhang et al., 2021). Moreover, S. litura larvae showed rhythmic circadian locomotor and defecation activities, respectively peaking during the night and the day, in both LD and DD conditions (Zhang et al., 2021; Table 1). Additionally, gut purging, which occurs at the end of larval development to empty the gut before entering the pupal stage, was shown to be under circadian control in the silkmoth Samia cynthia var. ricini (superfamily Bombycoidea; Mizoguchi and Ishizaki, 1982; Table 1).

Adult eclosion, i.e., adult emergence from the pupal exuvia, is one of the most vulnerable events during an insect’s life cycle, and in many species is controlled by the circadian clock. Eclosion occurs at species-specific times of the day in different lepidopteran species when maintained in LD conditions and likely restricts the timing of this behavior to the most advantageous moments of the day (Merlin and Reppert, 2009). In constant conditions, adult eclosion maintains a 24-h rhythmic profile, as demonstrated for species belonging to the superfamilies of Bombycoidea (the giant silkmoth Hyalophora cecropia; Truman and Riddiford, 1970; B. mori; Truman, 1972; Shimizu and Matsui, 1983; A. pernyi; Truman and Riddiford, 1970), Noctuoidea (the fall webworm Hyphantria cunea; Morris and Takeda, 1994), Pyraloidea (the southwestern corn borer Diatraea grandiosella; Takeda, 1983), and Papilionoidea (the monarch butterfly Danaus plexippus; Froy et al., 2003; Table 1).

Adult flight is related to vital activities for both moths and butterflies, including nectar feeding and mating. To date, the circadian control of flight behavior has been evaluated in relatively few Lepidoptera. Nevertheless, a flight circadian rhythmicity has been demonstrated in species belonging to the superfamily Bombycoidea, initially in Hyalophora cecropia, S. cynthia var. ricini, A. pernyi (Truman, 1974), and more recently in the hawkmoths Manduca sexta and Hyles lineata (Broadhead et al., 2017). In LD conditions, the nocturnal moths H. cecropia, S. cynthia var. ricini, A. pernyi, and M. sexta display rhythmic flight restricted to the dark period of the day, while the crepuscular H. lineata shows a bimodal activity, with a first peak after light-on and a second after light-off. In DD, all these species maintain 24h rhythmic flight, although with some modifications in their profiles (Truman, 1974; Broadhead et al., 2017; Table 1).

In moths, several behavioral activities related to reproduction have been shown to have a rhythmic daily species-specific pattern (reviewed in Groot, 2014). In these species, reproduction is characterized by stereotypical behaviors anticipating actual mating, associated with the production and/or release of sex pheromones by females (reviewed in Groot, 2014; Stepien et al., 2020). During sex pheromone emission, females adopt typical postures (named calling), characterized by a modification of the abdomen position and the extrusion of the pheromone gland to better disperse sex pheromones in the environment (Stepien et al., 2020). Males perceive sex pheromones through the antennae and fly toward the female for mating (Stepien et al., 2020). In several moths, female calling, male response to female sex pheromones, and mating have been shown to have a rhythmic daily species-specific pattern in LD conditions (Groot, 2014). Some studies examined these behaviors under constant conditions, demonstrating a circadian control of these daily activities. For example, female calling behavior was shown to maintain its rhythmicity in DD in species of the superfamily Noctuoidea (the true armyworm moth Pseudaletia unipuncta, the oriental tobacco budworm moth Helicoverpa assulta, and S. littoralis; Turgeon and McNeil, 1982; Delisle and McNeil, 1987; Kamimura and Tatsuki, 1993; Silvegren et al., 2005; Sadek et al., 2012), in some Tortricoidea (the oriental fruit moth Grapholita molesta and the codling moth Cydia pomonella; Baker and Cardé, 1979; Castrovillo and Cardé, 1979), and in the Mediterranean flour moth Ephestia kuehniella (superfamily Pyraloidea; Závodská et al., 2012; Table 1). A 24h rhythmic male response to female sex pheromones was demonstrated in constant conditions in some Noctuoidea moths (the turnip moth Agrotis segetum, T. ni, Heliothis virescens, Spodoptera exigua, and S. littoralis; Shorey and Gaston, 1965; Castrovillo and Cardé, 1979; Linn et al., 1996; Rosén et al., 2003; Silvegren et al., 2005) and in the Tortricoidea C. pomonella (Castrovillo and Cardé, 1979; Table 1). A 24h rhythm was detected in mating frequency of S. littoralis in both 12:12 LD and DD conditions (Silvegren et al., 2005). Additionally, in S. littoralis, optimized mating efficiency requires synchronized circadian clocks in phase between the two sexes, as a decreased frequency in mating was detected when males and females possessed out of phase circadian clocks (Silvegren et al., 2005).

Various lepidopteran species show a migratory behavior due to seasonally changing environments (Williams, 1957; Walker, 1980). Migration has been extensively studied in the eastern North American populations of the monarch butterfly D. plexippus, which are characterized by oriented flight and each Autumn can travel up to 4,500km to reach wintering sites in central Mexico. Migrants traveling southward are characterized by a reproductive diapause and enhanced longevity. In Spring, adults exit from reproductive diapause, mate, and remigrate to the north, with females laying fertilized eggs in the southern United States. The progeny of these butterflies continues migrating North, reaching the original sites in North America, in a mostly multigenerational migratory phenomenon (Perez et al., 1997; recently reviewed in Reppert et al., 2016; Reppert and de Roode, 2018; Merlin et al., 2020). To generate accurate orientation during migration, monarchs possess well-characterized sun and magnetic compasses, which incorporate daylight cues and the Earth’s magnetic field information, respectively (Mouritsen and Frost, 2002; Froy et al., 2003; Guerra et al., 2014). The sun compass requires the circadian clock to compensate for the apparent movement of the sun in the sky during the day (Mouritsen and Frost, 2002; Froy et al., 2003; Merlin et al., 2009). Additionally, a specific molecular clock element with a clock-independent role (DpCRYPTOCHROME1, DpCRY1) is involved in the proper functioning of the magnetic compass (Gegear et al., 2008, 2010; Wan et al., 2021). As the role of DpCRY1 in D. plexippus magnetoreception is not related to its function in the circadian clock, this aspect is not further discussed in this review (for interested readers: Reppert et al., 2016; Reppert and de Roode, 2018; Merlin et al., 2020; Wan et al., 2021).

The involvement of the circadian clock for time-compensated navigation during migration has been hypothesized in other butterflies such as Aphrissa statira and Phoebis argante (Oliveira et al., 1998; Table 1). Migration has great relevance for the functionality of an ecosystem since newly arrived migrants can have a role as pollinators, representing a food source for insectivores and in turn for the other components of a trophic system. As migration is typical of several butterflies and moths (Williams, 1957), it would be interesting to understand whether the circadian clock is involved in orientation during navigation of other lepidopteran species.

In nature, adult moths and butterflies are crucial pollinators feeding on nectar, and in some cases, on pollen. However, almost all lepidopteran larvae are voraciously phytophagous, feeding on leaves and, less frequently, on stems, flowers, fruits, seeds, or wood. Complex plant-Lepidoptera interactions have evolved, with plants having developed defense strategies to protect from caterpillar attacks (Howe and Jander, 2008), while also attracting adult lepidopteran pollinators (Raguso, 2008; Rausher, 2008). Conversely, caterpillars have developed mechanisms to avoid plant defense systems (e.g., expansion of detoxification genes; Cheng et al., 2017; Gouin et al., 2017; Pearce et al., 2017) and adult moths and butterflies have evolved structures to efficiently reach their food sources, working at a same time as pollinators (Powell et al., 1998; Mitter et al., 2017; Kawahara et al., 2019; Figure 1). In this survival game, circadian systems of both plants and Lepidoptera appear to have played a fundamental role in controlling the timing of different activities/physiological phenomena, such as the emission (plants)/perception (moths/butterflies) of attraction signals, or the production of toxic substances (plants) and detoxification enzyme (caterpillars) at the proper time of the day.

A series of studies aimed at exploring the relationship between M. sexta moth and the plant Petunia axillaris indicate that an interaction between their respective circadian-controlled phenotypes is ecologically and evolutionary important for an efficient mutualistic relationship in which a plant requiring out-crossing provides nectar as a source of food to adult Lepidoptera which act as pollinators (Hoballah et al., 2005; Broadhead et al., 2017; Fenske et al., 2018; Figure 4A). Although, the M. sexta larval form is a pest for Solanaceae crops, adult moths are generally known as crepuscular/nocturnal nectivores of different plants species, including P. axillaris (Metcalf et al., 1993; Hoballah et al., 2005; Riffell and Hildebrand, 2016). Petunia axillaris flowers show clear rhythmicity in scent emission, with a peak during the night, in a mechanism under circadian control (Hoballah et al., 2005; Fenske and Imaizumi, 2016). Manduca sexta shows circadian rhythmicity in flight behavior, with higher activity during the night in both LD and DD (Broadhead et al., 2017; Fenske et al., 2018). Manipulating the circadian system of both moths and plants, Fenske et al. (2018) demonstrated that the moth and petunia circadian clocks must be in phase to ensure an efficient level of flower visits by the moths (Figure 4A). In fact, when the moth clocks were out of phase compared to that of the plant and vice versa, the number of flower visits was reduced compared to a condition in which the plant and moth circadian systems oscillated in phase. The importance of the circadian clock was emphasized by the fact that moth flight activity increased significantly when exposed to flower scent during the subjective night compared to when the same odor signal was administered during the subjective day. It has been demonstrated that the ability to perceive odors resides in the antenna, which also contains a peripheral clock in M. sexta moths (Schuckel et al., 2007). Additionally, in response to synthetic flower scent, M. sexta antennae show circadian rhythmicity in electrophysiological activity, with a peak during the night when the moths display greater foraging behavior (Hoballah et al., 2005; Fenske et al., 2018). Therefore, it can be hypothesized that in the moth the central and the peripheral clocks interact to coordinate the organismal locomotor and odor perception circadian activities.

In the study of the interactions between plants and Lepidoptera there is also evidence indicating that an in phase synchronization between the two organismal circadian clocks have likely promoted a clock-controlled mechanism enhancing the plants resistance to caterpillar diurnal attacks. One such example derived from a study analyzing the relationship between the circadian clocks of the generalist herbivore T. ni and the model plant Arabidopsis thaliana (Goodspeed et al., 2012; Figure 4B). Trichoplusia ni is commonly known as the cabbage looper since larvae preferentially feed on crucifers but successfully feed on over 160 plant hosts. Arabidopsis thaliana possesses a well-characterized circadian system (Barak et al., 2000; Robertson, 2000), and can host cabbage looper larvae. When fed on A. thaliana, T. ni larvae showed a circadian feeding behavior, with a higher feeding activity during the end of the day and lower during the night, in both LD and DD conditions (Goodspeed et al., 2012; Table 1). However, it was demonstrated that T. ni growth was enhanced when the larval feeding rhythm was entrained to free-run out of phase with respect to A. thaliana circadian rhythmicity, and reduced when the caterpillar free-running rhythm was in phase with that of A. thaliana’s clock (Goodspeed et al., 2012; Figure 4B). The variable larval growth rate was linked to the interaction between the clock-controlled larval feeding behavior and the plant clock-mediated production of jasmonic acid, a hormone triggering plant herbivore defense (Reymond et al., 2000; Jander et al., 2001; Kessler and Baldwin, 2002; Howe and Jander, 2008), which shows a peak of accumulation in the middle of the day (Goodspeed et al., 2012). In fact, when caterpillars were fed on arrhythmic or jasmonate-defective A. thaliana mutants, their weight increment was independent of the phase of circadian feeding behavior (Goodspeed et al., 2012). These data suggest a protective effect of the circadian system in A. thaliana, as the circadian clock controlled phase patterns of jasmonate accumulation are consistent with an anticipated preparation to the peak of the circadian caterpillar feeding behavior, occurring at the end of the day (Goodspeed et al., 2012).

On the other hand, different works have indicated that the lepidopteran counterparts might have evolved circadian-controlled strategies for avoiding plant defense mechanisms. For example, the absence of any rhythmic daily profile in feeding behavior detected in the tobacco hornworm M. sexta when reared on an artificial diet has been suggested as a strategy to limit the negative effects of the diurnal defense mechanisms typical of its natural host Nicotiana attenuata (Reynolds et al., 1986; Kim et al., 2011; Jander, 2012). However, further experiments are required, since in some species, the rhythmicity in larval feeding behavior can be influenced by several environmental factors, including the type of diet. This is the case of the tobacco budworm Heliothis virescens (superfamily Noctuoidea), whose feeding behavior was arrhythmic when maintained on an artificial diet in LD laboratory conditions but rhythmic when reared on a corn-based diet in a greenhouse, with a LD and temperature cycling environment (Niepoth et al., 2018).

A large body of evidence indicating the presence of circadian-controlled mechanisms for escaping plant defense strategies derived from a series of studies performed on nocturnal Spodoptera spp., pest insects with a wide host range, including several plant species of economic importance. In particular, S. exigua, S. littoralis, and S. litura have shown rhythmic feeding behavior, with a peak at night, when maintained on artificial diet in LD conditions (Kim and Hong, 2015; Suszczynska et al., 2017; Zhang et al., 2021). In S. littoralis and S. litura this rhythmicity was shown to persist in DD, suggesting that feeding behavior is a true circadian rhythm in the larvae of both species (Suszczynska et al., 2017; Zhang et al., 2021; Table 1). Spodoptera litura larvae showed a similar circadian profile in their locomotor behavior, with a peak of activity during the night in both LD and DD conditions (Zhang et al., 2021; Table 1; Figure 4C). Interestingly, transcriptomic analyses on S. litura larval midguts and fat bodies in LD conditions showed daily rhythmicity in the transcriptional levels of detoxification genes with a peak during the daytime, when larvae were most inactive and their digestion activity was at its highest levels (Zhang et al., 2021; Figure 4C). Although, the same analyses have not been performed in DD conditions, the importance of the circadian clock in the control of detoxification processes was suggested by transient KD experiments indicating that different core clock elements (SlituClk, SlituBmal1, and SlituCwo) are involved in modulating the transcription of several detoxification genes, with SlituCLK and SlituBMAL1 acting as promoting factors and SlituCWO as an inhibitor (Zhang et al., 2021). A molecular link between the S. litura circadian system and detoxification rhythm was further suggested by in vitro luciferase assays in S. litura cell lines, indicating that intact E-boxes in the promoters of representative detoxification genes are important for the regulation of their expression, with SlituCLK, SlituBMAL1 acting as transcriptional promoters and SlituPER and SlituCWO as inhibitors (Zhang et al., 2021). Therefore, it might be speculated that in S. litura larvae the circadian system exerts a protective effect by maintaining a clear temporal separation between night feeding and daytime digestion/detoxification processes (Zhang et al., 2021).

It is important to note that the S. litura studies open the way for possible applications in agricultural pest control, as Zhang et al. (2021) demonstrated in S. litura larvae that topical treatments with insecticides were more efficient in inducing mortality when applied during nighttime than daytime. These findings are particularly relevant considering that crop management is evolving rapidly towards “precision agriculture” that supports management decisions for profitable and sustainable agricultural production. Automated systems and drones may also permit the distribution of pesticides when they are most effective against their target species. However, as species-specific differences are expected, preliminary studies of the larval circadian system in target pest Lepidoptera will enable specific and effective “chronobiological treatments,” limiting the spread of toxic insecticides in the environment.

To date, the circadian clock has been examined in a relatively small number of species compared to the multitude of extant Lepidoptera. Nevertheless, the studies performed so far indicate the ecological relevance of the lepidopteran circadian system, as it influences the fitness of a single organism and in turn impacts the biodiversity of an ecosystem. It is easy to predict that the technological advances in Next Generation Omic technologies, as well as genome editing methodologies will facilitate the study and manipulation of model and non-model species. When applied to lepidopteran chronobiology, these methodologies will expand our current knowledge on the role of the circadian clocks in these organisms when considered in their natural habitat. Similar approaches will also be useful for the systematic study of species with economic importance such as B. mori, to understand whether manipulating the circadian clock can improve both rearing conditions and/or silk production efficiency. It is worthy to mention that Bmper KD lines exhibited a reduction in developmental time which did not affect silk productivity parameters (Sandrelli et al., 2007). Thus, the employment of genome editing techniques which generated Bmper and Bmtim KO B. mori mutants (Ikeda et al., 2019; Nartey et al., 2020) for producing a battery of silkworm lines carrying mutations in different clock genes might permit an efficient evaluation of these aspects in B. mori.

FS and DB: manuscript organization. FS, SC, DB, and AS: writing and editing. All authors contributed to the article and approved the submitted version.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 765937 (CINCHRON).

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.